Validation of a Liquid-Liquid Extraction Method to Study the Temporal Production of D-Series Resolvins by Head Kidney Cells from Atlantic Salmon (Salmon salar) Exposed to Docosahexaenoic Acid.

A simple and rapid method for the extraction of D-series resolvins (RvD1, RvD2, RvD3, RvD4, RvD5) released into Leibovitz's L-15 complete medium by head kidney cells from Atlantic salmon and the further determination of liquid chromatography triple quadrupole mass spectrometry is proposed. A three-level factorial design was proposed to select the optimal concentrations of internal standards that were used in the evaluation of the performance parameters, such as linear range (0.1-50 ng mL-1), limits of detection and quantification (0.05 and 0.1 ng mL-1, respectively), and recovery values ranging from 96.9 to 99.8%. The optimized method was used to determine the stimulated production of resolvins by head kidney cells exposed to docosahexaenoic acid, and the results indicated that it is possible that the production was controlled by circadian responses.

Dietary chlorpyrifos-methyl exposure impair transcription of immune-, detoxification- and redox signaling genes in leukocytes isolated from cod (Gadus morhua).

Inclusion of new environmental toxicants increase with the amount of plant ingredients substituting marine proteins and oils in feed for farmed Atlantic salmon (Salma salar). Agricultural pesticides like chlorpyrifos-methyl, present in commercial salmon feeds, may affect salmon immune and detoxification responses. Atlantic cod (Gadus morhua), surrounding the net pens, grazing on feces and uneaten pellets may be affected accordingly. The aim of this study was to analyze transcription responses in Atlantic cod head kidney tissue and isolated leukocytes following dietary chlorpyrifos-methyl inclusions and possible interactions with proinflammatory signals. Head kidney tissues and leukocytes were isolated from cod fed diets contaminated with chlorpyrifos-methyl (0.5 mg/kg, 2.4 mg/kg, 23.2 mg/kg) for 30 days. The isolated leukocytes were further challenged with bacteria (lipopolysaccharide (LPS), virus (polyinosinic acid:polycytidylic acid (PIC) mimic and l-arginine, an immuno-modulating amino acid, in vitro. The LPS-induced transcription of the interleukin genes il-1ß, il-6, il-8 increased in leukocytes isolated from cod fed chlorpyrifos-methyl 23.2 mg/kg, compared to cod fed the control diet, indicating increased inflammation. Transcriptional levels of carnitine palmitoyl transferase (cpt1a), aryl hydrogen receptor (ahr) and catalase (cat) were all reduced by dietary inclusions of chlorpyrifos-methyl in the leukocytes. The findings suggests that dietary chlorpyrifos-methyl exposure impair inflammation, detoxification and redox signaling in cod leukocytes.

RNA form baker's yeast cultured with and without lipopolysaccharide (LPS) modulates gene transcription in an intestinal epithelial cell model, RTgutGC from rainbow trout (Oncorhynchus mykiss).

The objective of this study was to evaluate if the intestinal RTgutGC cell line could be suitable for research on dietary ingredients and their function as modulators of inflammation during lipopolysaccharide (LPS) induced stress. The RTgutGC cells cultured together with RNA from baker's yeast, reached confluency after 72 h. The cells were grown in either compete L-15 (CM) or nutrient deprived L-15 (DM). Then, the RTgutGC cells were exposed to LPS or RNA from baker's yeast, either alone, or in combination, in CM or DM. All cultures were harvested following LPS challenge for 48 h and 72 h. LPS induced transcription of Interleukin 1ß (IL-1ß), Interleukin -8 (IL-8), Toll like receptor 3 (TLR3), interferon regulating factor 3 (irf3), Nuclear factor Ä¸ß (NFĸß), one of the multidrug transporters, ABCC2, and glutamine synthase 1 (GLS01) in RTgutGC cells at one or both sampling points (48 h and/or 72 h post LPS challenge). RNA from baker's yeast in culture alone, (cultured 120 h and 144 h with RTgutGC cells and harvested at the respective LPS sampling points) induced transcription of INF1, TNFα and ticam/trif, not induced by LPS. In addition, RNA from baker's yeast affected IL-1ß, TLR3, irf3 and NFĸß, comparable to the responses triggered by LPS. RNA from baker's yeast alone did not affect ABCC2 or GLS01 transcriptions in this set up. So, LPS and RNA from baker's yeast affects distinct but also common gene transcripts in this intestinal cell line. Culturing RTgutGC cells in DM, adding a combination of LPS and RNA from baker's yeast, reduced IL-1ß transcription compared to cells grown in CM, 48 h and 72 h post LPS challenge. Also, in RTgutGC cells, grown in DM, the LPS induced transcription of ABCC2 declined, measured 48 h post LPS challenge. Possibly indicating that optimal transcription of IL-1ß and ABBC2 in RTgutGC cells, cultured over time, requires access of adequate nutrients under stressful condition. RNA from baker's yeast induced INF1 transcription in the RTgutGC cells, regardless if the medium was complete or deprived of nutrients. However, culturing RTgutGC cells in DM enriched with RNA from baker's yeast for a longer period of time (120 h, 144 h), seemed beneficial for INF1 transcription.

Differential production of prostaglandins and prostacyclins by liver and head kidney cells from Atlantic salmon challenged with arachidonic and eicosapentaenoic acids.

Polyunsaturated fatty acids such as arachidonic and eicosapentaenoic acids are the precursors of eicosanoid metabolites (e.g prostaglandins and prostacyclins) which regulate inflammatory and immune response processes in fish organs. The present research studies the differential production of PGI2, PGI3, PGE2 and PGE3 by primary liver and head kidney cells isolated from salmon and challenged with single or combined ARA and/or EPA. There was a significant increase in the production of PGE2 and PGI3 in both types of cells after exposure to single and combined fatty acids. Increased production of PGE3 was only detected in liver cells after exposure to ARA+EPA. The levels of PGI2 in liver cells were significantly increased after exposure to all the tested fatty acid systems, while the production levels in head kidney cells were only significant after exposure to ARA or ARA+EPA, but not to EPA, where the production was non-significantly decreased compared to the control cells. In general, liver cells synthetized higher prostaglandin levels than prostacyclins, and the opposite was observed in head kidney cells, which synthetized highly remarkable amounts of prostacyclin compared to liver cells. The overall production for both types of cells and various fatty acid systems were characterized by a high proportion of the omega-3 fatty acid metabolites (PGE3+PGI3) compared to the omega-6 counterpart (PGE2+PGI2). Some potential production mechanisms are proposed and comprehensively discussed. The results of the present research are the first to deliver the differential production of prostacyclins and prostaglandins by liver and head kidney cells from salmon, thereby paving the way for understanding the significance of these prostanoids in fish physiology and disease.

Resveratrol inhibited LPS induced transcription of immune genes and secretion of eicosanoids in Atlantic salmon (Salmo salar), comparing mono-, co- and a novel triple cell culture model of head kidney leukocytes, liver cells and visceral adipocyte tissue.

The aim was to study the effect of resveratrol on the interplay of inflammatory signals using three different cell models; a metabolic organ (liver), an endocrine organ (visceral adipose tissue, VAT) and an immune organ (head kidney leukocytes, HKL) following lipopolysaccharide challenge (LPS). Atlantic salmon HKL, liver cells and VAT were isolated from the same fish (n = 5). Each cell type was cultured either as mono-cultures, as co-cultures between HKL-liver cells, liver cells-VAT and HKL-VAT. Triple -cultures included all three tissues. In all cultures of HKL, LPS induced transcription of IL-1ß, cox2, tnfα, IL-12, ccattß and Ahr were significantly inhibited by resveratrol (100, 200 µM). Likewise, in all cultures of liver cells, the LPS induced expression of IL-1ß was inhibited by resveratrol (100 and 200 µM). HKL, both mono-cultures and triple-cultures and VAT cocultured with liver cells, showed LPS induced cox2 transcription that was inhibited by resveratrol (100 and 200 µM). In contrast, VAT cultured as triple cultures, resveratrol 200 µM particularly, in the presence of LPS, seemed to increase the expression of IL-1ß and ccattß. Resveratrol did not significantly affect lox5 expression in any culture. HKL and VAT are the main producers of PGE2 in response to inflammatory stimuli. VAT showed high endogenous production of eicosanoids, particularly LTB4 and LTB5. Resveratrol inhibited bot LPS induced and endogenous eicosanoid production. Possible targets of resveratrol, Sirt1 and pAMPK were affected differently in the different cells and tissue studied.

The Effect of Omega-3 and Omega-6 Polyunsaturated Fatty Acids on the Production of Cyclooxygenase and Lipoxygenase Metabolites by Human Umbilical Vein Endothelial Cells.

Although the correlation between polyunsaturated fatty acids (PUFA) and the production of pro- and anti-inflammatory metabolites is well documented, little is known about the simultaneous effect of different PUFA on the production of cyclooxygenase and lipoxygenase metabolites. The present research examines the association between different omega-3 (ω-3) and omega-6 (ω-6) PUFA and the release of four cyclooxygenase and six lipoxygenase metabolites in cell medium by human umbilical vein endothelial cells (HUVEC). The different combinations of ω-3 and ω-6 PUFA were prepared according to a full 24 factorial design that enables studying not only the main effects but also the different interactions between fatty acids. In addition, interactions diagrams and principal component analysis were useful tools for interpreting higher order interactions. To the best of our knowledge, this is the first report addressing the combined effect of ω-3 and ω-6 PUFA on the signaling of prostaglandins, prostacyclins, leukotrienes and resolvins by HUVEC.

Effect of dietary replacement of fish meal with insect meal on in vitro bacterial and viral induced gene response in Atlantic salmon (Salmo salar) head kidney leukocytes.

With the fast growth of today's aquaculture industry, the demand for aquafeeds is expanding dramatically. Insects, which are part of the natural diet of salmonids, could represent a sustainable ingredient for aquaculture feed. The aim of the current study was to test how a partial or total replacement of dietary fishmeal with insect meal affect gene responses involved in inflammation, the eicosanoid pathway and stress response in Atlantic salmon (Salmo salar L.) in isolated head kidney leukocytes after exposure to bacterial or viral mimic. Insect meal (IM) was produced from black soldier fly (BSF, Hermetia illucens) larvae. Seawater Atlantic salmon were fed three different diets for 8 weeks; a control diet (IM0, protein from fishmeal and plant based ingredients (25:75) and lipid from fish oil and vegetable oil (33:66); and two insect-meal containing diets, IM66 and IM100, where 66 and 100% of the fishmeal protein was replaced with IM, respectively. Leukocytes were isolated from the head kidney of fish (n = 6) from each of the three dietary groups. Isolated leukocytes were seeded into culture wells and added either a bacterial mimic (lipopolysaccharide, LPS) or a viral mimic (polyinosinic acid: polycytidylic acid, poly I: C) to induce an inflammatory response. Controls (Ctl) without LPS and poly I: C were included. The transcription of interleukins IL-1ß, IL-8, IL-10 and TNF-α were elevated in LPS treated leukocytes isolated from salmon fed the three dietary groups (IM0, IM66 and IM100). The inflammatory-related gene expression in head kidney cells were, however, not affected by the pre-fed substitution of fish meal with IM in the diet of salmon. Gene transcriptions of PTGDS and PTGES were neither affected by LPS, poly I: C or the experimental diets fed prior to cell isolation, while salmon fed with IM showed a lower expression of LOX5. The gene expression of TLR22 and C/EBP-ß were down-regulated by the LPS treatment in the cells isolated from salmon fed insect-based diets (IM66 and IM100) compared to fish fed the IM0. Similarly, the leukocytes challenged with LPS and isolated from fish fed with IM66 and IM100 down-regulated the expression of Mn-SOD, GPx1, HSP27 and HSP70 compared to salmon fed IM0. In general, these results suggested that replacement of fishmeal with IM in the diets of Atlantic salmon had no effect on the transcription of pro-inflammatory genes in the head kidney cells. There was, however, an effect of dietary IM on the transcription of antioxidant and stress related genes in the leukocytes.

Surplus arginine reduced lipopolysaccharide induced transcription of proinflammatory genes in Atlantic salmon head kidney cells.

In aquaculture production, studies of salmon health and interaction between pathogens and nutrition are of high importance. This study aimed to compare genes and pathways involved in salmon head kidney cells and liver cells, isolated from the same fish, towards polyinosinic acid: polycytidylic acid (poly I:C) and lipopolysaccharide (LPS), with and without addition of surplus arginine. Selected transcriptional responses of genes involved in inflammation, polyamine synthesis, oxidation and apoptosis were elucidated. For the genes related to inflammation, viperin, Mx and Toll like receptor 3 (TLR3), transcription were significantly upregulated by poly I:C in head kidney cells, while viperin was upregulated in liver cells. Surplus arginine did not affect poly I:C induced responses with the exception of reducing poly I:C induced Mx transcription in head kidney cells. Gene transcription of Interleukin 1ß (IL-1ß), Interleukin-8 (IL-8) and cyclooxygenase 2 (Cox2) were elevated during LPS treatment in all liver and head kidney cell cultures. In addition, LPS induced significantly, CD83 transcription in liver cells and TNF-α transcription in head kidney cells. Surplus arginine significantly reduced IL-8, Cox2 and TNF-α transcription in head kidney cells. LPS upregulated arginase in head kidney cells while poly I:C upregulated S-adenosyl methionine decarboxylase (SAMdc) transcription in liver cells. This suggests that LPS and poly I:C modulates genes involved in polyamine synthesis. In addition, in head kidney cells, surplus arginine, when cultured together with LPS, increased the transcription of ornithine decarboxylase (ODC) the limiting enzyme of polyamine synthesis. The genes involved with oxidation and apoptosis were not affect by any of the treatments in liver cells, while LPS decreased caspase 3 transcription in head kidney cells. In liver cells, protein expression of catalase was reduced by surplus arginine alone and when challenged with poly I:C. Both liver cells and head kidney cells isolated from the same individual fish responded to LPS and poly I:C, depending on the gene analyzed. Additionally, arginine could modulate transcription of pro-inflammatory genes induced by LPS in salmon immune cells, thus affecting salmon immunity.

A comparative study: Difference in omega-6/omega-3 balance and saturated fat in diets for Atlantic salmon (Salmo salar) affect immune-, fat metabolism-, oxidative and apoptotic-gene expression, and eicosanoid secretion in head kidney leukocytes.

The aim of this study was to compare how different dietary vegetable oil n-6/n-3 ratios affect gene responses involved in inflammation, signaling pathways, fatty acid synthesis and oxidation, oxidation and apoptosis as well as eicosanoid production in salmon head kidney tissues and isolated head kidney leukocytes. Salmon smolts (200 g) were fed four different diets where the main lipid components were palm oil (n-6/n-3 ratio = 0.7), rapeseed oil (n-6/n-3 ratio = 0.9), and soybean oil (n-6/n-3 ratio = 2.4) and a high soybean oil diet with an n-6/n-3 ratio = 4. Both head kidney tissue and leukocytes isolated from head kidneys were sampled from the four diets, but from different fish. Leukocytes isolated from the head kidneys were seeded into culture wells and added lipopolysaccharide (LPS) to induce inflammatory responses. Controls without LPS were included. Head kidney leukocytes and the tissues should have the same phenotype reflecting the different diets. Interleukin 1ß (IL-1ß) transcription was elevated in head kidney tissue and especially in LPS treated leukocytes isolated from soybean oil (n-6/n-3 = 2.4) fed salmon, which confirmed the suitability of the in vitro model in this experiment. Leukocytes, treated with LPS, and isolated from salmon fed the soybean oil diet (n-6/n-3 = 2.4) also upregulated tumor necrosis factor alpha (tnf-α), cyclooxygenase (cox2), prostaglandin D and E synthase (ptgds, ptges), fatty acyl synthase (fas), 5 and 6 desaturases (5des, 6 des) and a fatty acid translocase protein (cd36) when compared to the other diets. The results suggest that diets with a specific n-6/n-3 ratio influence the transcription of pro-inflammatory genes and may be cross-linked to transcription of selected fatty acid metabolism genes. Salmon fed the palm oil diet (n-6/n-3 = 0.7) showed a lower expression of inflammatory genes. Instead, peroxisome proliferator activated receptor ß1 (pparß1), acyl coenzyme A (aco), apoptosis regulator (bax) and superoxide dismutase (sod) were upregulated in leukocytes in vitro, while head kidney tissue transcription of a dendritic marker (cd83) was lower than measured in tissues from fish fed the other diets. The concentration of LTB4 (10-20 ng/mL) were relatively constant in leukocyte supernatants, all diets. Head kidney leukocytes from soybean oil (n-6/n-3 = 2.4) fed fish produced LPS induced PGE2 (mean 0.5 ng/mL) while leukocytes isolated from palm oil diet (n-6/n-3 = 0.7) secreted very high amounts of LTB5 (50-70 ng/mL). In addition, equal amounts of LPS induced PGE2 and PGE3 (mean 0, 5 ng/mL) were produced, indicating that the n-6/n-3 ratio of this saturated fatty acid may have a specific impact on eicosanoid production in the head kidney of salmon.

ß-naphthoflavone interferes with cyp1c1, cox2 and IL-8 gene transcription and leukotriene B4 secretion in Atlantic cod (Gadus morhua) head kidney cells during inflammation.

The objective of this study was to evaluate how ß-naphthoflavone interacts with lipopolysaccharide (LPS) and polyinosinic acid: polycytidylic acid (poly I: C) induced innate immune parameters as well as phase I and phase II detoxification enzymes in head kidney cells isolated from Atlantic cod. ß-naphthoflavone is a pure agonist of aryl hydrocarbon receptor (AhR) while LPS and poly I: C are not. ß-naphthoflavone was added to head kidney leukocytes alone or together with LPS or poly I: C and the responses were evaluated in terms of protein and gene expression. The results showed that ß-naphthoflavone (25 nM), with and without LPS, significantly induced cytochrome P450 (cyp1c) transcription in cod head kidney cells. ß-naphthoflavone (100 nM) in the presence of the virus mimic, poly I: C, also increased cyp1c1transcription. LPS induced cyp1c1, cyclooxygenase 2 (cox2), interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and interleukin 8 (IL-8) transcription, genes that were not affected by the tested ß-naphthoflavone concentrations alone. However, ß-naphthoflavone (25 and 50 nM) strengthened LPS induced cox2 and IL-8 transcription. Cod head kidney cells exposed to ß-naphthoflavone concentrations ranging from 25 to 100 nM, with and without LPS or poly I: C, expressed AhR protein. LPS or ß-naphthoflavone (5-50 nM) significantly induced leukotriene B4 (LTB4) secretion compared to control. In conclusion, this study suggests that ß-naphthoflavone could interfere with LPS induced immune cell signaling in cod head kidney cells.

Hydrolyzed fish proteins modulates both inflammatory and antioxidant gene expression as well as protein expression in a co culture model of liver and head kidney cells isolated from Atlantic salmon (Salmo salar).

Hydrolyzed fish proteins (H-pro) contain high concentrations of free amino acids and low molecular peptides that potentially may benefit fish health. The following study aimed to test whether the water-soluble phase of H-pro could attenuate lipopolysaccharide (LPS) provoked inflammation in liver cells and head kidney cells isolated from Atlantic salmon. Cells were grown as mono cultures or co cultures to assess possible crosstalk between immune cells and metabolic cells during treatments. Cells were added media with or without H-pro for 2 days before LPS exposure and harvested 24 h post LPS exposure. Respective cells without H-pro and LPS were used as controls. H-pro alone could affect expression of proteins directly as H-pro increased catalase protein expression in head kidney- and liver cells, regardless of culturing methods and LPS treatment. Leukotriene B4 (LTB4) production was also increased by H-pro in head kidney cells co cultured with liver cells. H-pro increased LPS induced interleukin 1ß (IL-1ß) transcription in liver cells co cultured with head kidney cells. All cultures of head kidney cells showed a significant increase in IL-1ß transcription when treated with H-pro + LPS. H-pro decreased caspase-3 transcription in liver cells cultured co cultured with head kidney cells. Peroxisome proliferator activated receptor α (PPAR α) was upregulated, regardless of treatment, in liver cells co cultured with head kidney cells clearly showing that culturing method alone affected gene transcription. H-pro alone and together with LPS as an inflammation inducer, affect both antioxidant and inflammatory responses.

Combining eicosapentaenoic acid, decosahexaenoic acid and arachidonic acid, using a fully crossed design, affect gene expression and eicosanoid secretion in salmon head kidney cells in vitro.

Future feed for farmed fish are based on untraditional feed ingredients, which will change nutrient profiles compared to traditional feed based on marine ingredients. To understand the impact of oils from different sources on fish health, n-6 and n-3 polyunsaturated fatty acids (PUFAs) were added to salmon head kidney cells, in a fully crossed design, to monitor their individual and combined effects on gene expression. Exposing salmon head kidney cells to single fatty acids, arachidonic acid (AA) or decosahexaenoic acid (DHA), resulted in down-regulation of cell signaling pathway genes and specific fatty acid metabolism genes as well as reduced prostaglandin E2 (PGE2) secretion. Eicosapentaenoic acid (EPA) had no impact on gene transcription in this study, but reduced the cell secretion of PGE2. The combined effect of AA + EPA resulted in up-regulation of eicosanoid pathway genes and the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α), Bclx (an inducer of apoptosis) and fatty acid translocase (CD36) as well as increased cell secretion of PGE2 into the media. Adding single fatty acids to salmon head kidney cells decreased inflammation markers in this model. The combination AA + EPA acted differently than the rest of the fatty acid combinations by increasing the inflammation markers in these cells. The concentration of fatty acid used in this experiment did not induce any lipid peroxidation responses.

Methionine deficiency does not increase polyamine turnover through depletion of hepatic S-adenosylmethionine in juvenile Atlantic salmon.

During the last few decades, plant protein ingredients such as soya proteins have replaced fishmeal in the diets of aquacultured species. This may affect the requirement and metabolism of methionine as soya contains less methionine compared with fishmeal. To assess whether methionine limitation affects decarboxylated S-adenosylmethionine availability and polyamine status, in the present study, juvenile Atlantic salmon were fed a methionine-deficient plant protein-based diet or the same diet supplemented with dl-methionine for 8 weeks. The test diets were compared with a fishmeal-based control diet to assess their effects on the growth performance of fish. Methionine limitation reduced growth and protein accretion, but when fish were fed the dl-methionine-supplemented diet their growth and protein accretion equalled those of fish fed the fishmeal-based control diet. Methionine limitation reduced free methionine concentrations in the plasma and muscle, while those in the liver were not affected. S-adenosylmethionine (SAM) concentrations were higher in the liver of fish fed the methionine-deficient diet, while S-adenosylhomocysteine concentrations were not affected. Putrescine concentrations were higher and spermine concentrations were lower in the liver of fish fed the methionine-deficient diet, while the gene expression of SAM decarboxylase (SAMdc) and the rate-limiting enzyme of polyamine synthesis ornithine decarboxylase (ODC) was not affected. Polyamine turnover, as assessed by spermine/spermidine acetyltransferase (SSAT) abundance, activity and gene expression, was not affected by treatment. However, the gene expression of the cytokine TNF-α increased in fish fed the methionine-deficient diet, indicative of stressful conditions in the liver. Even though taurine concentrations in the liver were not affected by treatment, methionine and taurine concentrations in muscle decreased due to methionine deficiency. Concomitantly, liver phospholipid and cholesterol concentrations were reduced, while NEFA concentrations were elevated. In conclusion, methionine deficiency did not increase polyamine turnover through depletion of hepatic SAM, as assessed by SSAT activity and abundance.

Aryl hydrocarbon receptor protein and Cyp1A1 gene induction by LPS and phenanthrene in Atlantic cod (Gadus morhua) head kidney cells.

The objective of this study was to evaluate interactions between environmental toxicants and cod immune cells during inflammation. Phenanthrene is abundant in plant oils (rapeseed, palm, and soya oil) as compared to fish oils, and consequently constitute an undesirable element in plant replacement diets in aquaculture. Phenanthrene was added to head kidney cell cultures, alone or together with LPS (lipopolysaccharide) or poly I: C (polyinosinic acid: polycytidylic acid), and the responses were evaluated in terms of protein and gene expression. The results showed that LPS, poly I: C or phenanthrene, added to the cultures separately, induced aryl hydrocarbon receptor (AhR) protein expression. Phenanthrene treatment in combination with LPS induced AhR protein expression and Cyp1A1 gene transcription, which not was observed combining poly I: C and phenanthrene. Phenanthrene exposure up regulated the transcription of common stress and detoxification enzymes like catalase, caspase 3 and glutathione S-transferase alfa 3 subunit B (GSTAB3), while LPS exposure alone or combined with phenanthrene down regulated GSTAB3 and catalase in cod leukocytes. It seems clear that immune regulation and phenanthrene induced signaling pathways interact; transcriptional down regulation of detoxification and antioxidant enzymes by LPS could indicate that combating bacterial infections is the number one priority in these cells, and that AhR and Cyp1A1 is somehow involved in this signaling cascade. LPS seems to affect the mitogen activated protein kinases (MAPKs) pathways (P-p38 and ERK1/2) thus modulating the AhR protein and Cyp1A1 gene transcription, while phenanthrene possibly activates AhR by ligand binding.

The impact of exogenous ω-6 and ω-3 polyunsaturated fatty acids on the induced production of pro- and anti-inflammatory prostaglandins and leukotrienes in Atlantic salmon head kidney cells using a full factorial design and LC-MS/MS.

The production of prostaglandins (PGE2, PGE3) and leukotrienes (LTB4, LTB5) in salmon head kidney cell cultures, exposed to different combinations of 20:4ω-6, 20:5ω-3 and 22:6ω-3 polyunsaturated fatty acids (PUFAs), was evaluated by means of a two level factorial design and LC-MS/MS. The method was selective for the pro- and anti-inflammatory analytes and their corresponding stable-isotope labelled internal standards. The regression models were linear over the concentration range 0.5-150 ng/ml with limits of detection of 0.25 ng/ml and quantification of 0.40 ng/ml for the analysed metabolites. The recovery ranged from 78 to 107% for prostaglandins and 73 to 115% for leukotrienes. The analysis of the samples exposed to different combinations of PUFAs revealed that the presence of single ω-3 PUFAs brought an enhancement of the metabolites from the lipooxygenase pathway, specially LTB4, and a reduction of the metabolites from the cyclooxygenase pathway (PGE2 and PGE3), while the two-term interactions generated the opposite effect (high concentration of prostaglandins and low concentrations of leukotrienes). To our knowledge, this is the first implementation of a fully crossed design for investigating the impact of ω-6 and ω-3 PUFAs on the production of eicosanoids not only through their individual but also through their combined effects on Atlantic salmon head kidney cells.

Arginine supplementation and exposure time affects polyamine and glucose metabolism in primary liver cells isolated from Atlantic salmon.

Arginine has been demonstrated to enhance glucose and lipid oxidation in mammals through activation of polyamine turnover. We aimed to investigate how arginine affects energy utilization through polyamine metabolism and whether this effect is time dependent. Primary liver cells were isolated from Atlantic salmon (2.2 kg body weight) fed diets containing 25.5 (low arginine, LA) or 36.1 (high arginine, HA) g arginine/kg dry matter for 12 weeks, to investigate the effect of long-term arginine supplementation. The cells were cultured for 24 h in L-15 medium to which either alpha-difluoromethylornithine (DFMO) or N (1),N (11)-diethylnorspermine (DENSPM) was added. Analysis of the medium by nuclear magnetic resonance revealed significant differences between the two dietary groups as well as between cells exposed to DFMO and DENSPM, with decreased glucose, fumarate and lactate concentrations in media of the HA cells. Liver cells from fish fed the HA diet had higher spermidine/spermine-N1-acetyltransferase protein abundance and lower adenosine triphosphate concentration as compared to the LA-fed fish, while gene expression was not affected by either diet or treatment. Primary liver cells isolated from salmon fed a commercial diet and cultured in L-15 media with or without arginine supplementation (1.82 or 3.63 mM) for 48 h, representing short-term effect of arginine supplementation, showed differential expression of genes for apoptosis and polyamine synthesis due to arginine supplementation or inhibition by DFMO. Overall, arginine concentration and exposure time affected energy metabolism and gene regulation more than inhibition or activation of key enzymes of polyamine metabolism, suggesting a polyamine-independent influence of arginine on cellular energy metabolism and survival.

A co culture approach show that polyamine turnover is affected during inflammation in Atlantic salmon immune and liver cells and that arginine and LPS exerts opposite effects on p38MAPK signaling.

This study assess which pathways and molecular processes are affected by exposing salmon head kidney cells or liver cells to arginine supplementation above the established requirements for growth support. In addition to the conventional mono cultures of liver and head kidney cells, co cultures of the two cell types were included in the experimental set up. Responses due to elevated levels of arginine were measured during inflammatory (lipopolysaccharide/LPS) and non -inflammatory conditions. LPS up regulated the genes involved in polyamine turnover; ODC (ornithine decarboxylase), SSAT (spermidine/spermine-N1-acetyltransferase) and SAMdc (S-adenosyl methionine decarboxylase) in head kidney cells when co cultured with liver cells. Regardless of treatment, liver cells in co culture up regulated ODC and down regulated SSAT when compared to liver mono cultures. This suggests that polyamines have anti-inflammatory properties and that both salmon liver cells and immune cells seem to be involved in this process. The transcription of C/EBP ß/CCAAT, increased during inflammation in all cultures except for liver mono cultures. The observed up regulation of this gene may be linked to glucose transport due to the highly variable glucose concentrations found in the cell media. PPARα transcription was also increased in liver cells when receiving signals from head kidney cells. Gene transcription of Interleukin 1ß (IL-1ß), Interleukin-8 (IL-8), cyclooxygenase 2 (COX2) and CD83 were elevated during LPS treatment in all the head kidney cell cultures while arginine supplementation reduced IL-1ß and IL-8 transcription in liver cells co cultured with head kidney cells. This is probably connected to p38MAPK signaling as arginine seem to affect p38MAPK signaling contrary to the LPS induced p38MAPK signaling, suggesting anti-inflammatory effects of arginine/arginine metabolites. This paper shows that co culturing these two cell types reveals the connection between metabolism and inflammation, suggesting different pathways and candidate biomarkers to be further explored.

Dietary arginine affects energy metabolism through polyamine turnover in juvenile Atlantic salmon (Salmo salar).

In the present study, quadruplicate groups of juvenile Atlantic salmon (Salmo salar) were fed plant protein-based diets with increasing arginine inclusions (range 28·8-37·4 g/kg DM) to investigate whether arginine supplementation affects growth and lipid accumulation through an elevated polyamine turnover. Dietary lysine was held at a constant concentration, just below the requirement. All other amino acids were balanced and equal in the diets. Arginine supplementation increased protein and fat accretion, without affecting the hepatosomatic or visceralsomatic indices. Dietary arginine correlated with putrescine in the liver (R 0·78, P= 0·01) and with ornithine in the muscle, liver and plasma (P= 0·0002, 0·003 and 0·0002, respectively). The mRNA of ornithine decarboxylase, the enzyme producing putrescine, was up-regulated in the white adipose tissue of fish fed the high-arginine inclusion compared with those fed the low-arginine diet. Concomitantly, spermidine/spermine-(N1)-acetyltransferase, the rate-limiting enzyme for polyamine turnover that consumes acetyl-CoA, showed an increased activity in the liver of fish fed the arginine-supplemented diets. In addition, lower acetyl-CoA concentrations were observed in the liver of fish fed the high-arginine diet, while ATP, which is used in the process of synthesising spermidine and spermine, did not show a similar trend. Gene expression of the rate-limiting enzyme for ß-oxidation of long-chain fatty acids, carnitine palmitoyl transferase-1, was up-regulated in the liver of fish fed the high-arginine diet. Taken together, the data support that increased dietary arginine activates polyamine turnover and ß-oxidation in the liver of juvenile Atlantic salmon and may act to improve the metabolic status of the fish.

Cytokine gene expression and prostaglandin production in head kidney leukocytes isolated from Atlantic cod (Gadus morhua) added different levels of arachidonic acid and eicosapentaenoic acid.

Primary head kidney leukocytes from Atlantic cod were isolated to evaluate the use of arachidonic acid and eicosapentaenoic acid by cyclooxygenases and the production of prostaglandins E2 and E3. The expression of cyclooxygenase genes and selected interleukin genes like Interleukin 1ß, Interleukin 6, interleukin 8 and interleukin 10 were monitored. Increasing concentrations of eicosapentaenoic acid and arachidonic acid in equal amounts increased cyclooxygenase2 transcription as well as cell secretion of prostaglandin E2. Even though the ratio of the two fatty acids was 1:1, the ratio between prostaglandin E2 and E3 was 50:1. The addition of arachidonic acid alone increased prostaglandin E2 secretion but did not induce cyclooxygenase2 transcription. However, when the concentration of eicosapentaenoic acid was increased, maintaining arachidonic acid constant, both prostaglandin E3 and prostaglandin E2 production was induced and the prostaglandin E2 production was higher than in cell cultures only added arachidonic acid. An up-regulation of cyclooxygenase2 transcription was also observed. The addition of the two fatty acids also affected the immune response by alteration of leukocytic cytokines gene expression. According to our results the Cyclooxygenase in cod seem to prefer arachidonic acid as substrate. Therefore, we suggest that the shift from marine oils (rich in n-3 fatty acids) to plant oils (higher in n-6 fatty acids) in the diet of commercially reared Atlantic cod could have negative effects on the whole organism through the increase in the production of prostaglandins belonging to those derived from n-6 fatty acids.