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.
•3D Atlantic salmon hepatocyte cell culture was superior to 2D culture.•Glyphosate exposure (500 μM) altered lipid metabolism in liver and kidney cells.•Glyphosate caused phospholipid remodelling and affected membrane stability of cells.•3D hepatocyte-kidney co-culture model is valuable in risk assessment.
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.
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•Organophosphorus pesticides have been detected in fish feed.•Toxicity of chlorpyrifos was studied in Atlantic salmon.•Lipid metabolism endpoints were studied in liver cells.•Chlorpyrifos exposure leads to accumulation of monoacylglycerol.•Supplementation of EPA and ARA did not prevent accumulation of monoacylglycerol.
•Dietary inorganic or organic selenium levels ∼1–2mgkg−1 do not cause toxic responses.•Inorganic Se, but not organic Se, affects the S-Adenosylmethionine (SAM) pathway.•Dietary organic and inorganic ...Se causes oxidative stress and altered lipid metabolism.
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.
Commercially produced Atlantic cod (
Gadus morhua L.) larvae are fed rotifers, while in the wild cod larvae feed on copepods. Rotifers contain up to 30-fold less selenium (Se) than copepods, and may ...contain insufficient Se to meet cod larvae requirements. In this study, rotifers were fed Se-enriched yeast (Se
+
rotifers) to obtain copepod levels of Se (3
mg
kg
−
1
dry weight (DW)), and the effects of feeding the Se
+
rotifers to cod larvae was investigated and compared to cod larvae fed control rotifers containing 0.7
mg
Se
kg
−
1
DW. No differences were observed in growth or survival between groups at the end of the rotifer-feeding period (29
days post hatch (dph)), or in the rate of skeletal deformities at 120
dph. Cod larvae fed Se
+
rotifers had increased whole-body levels of Se, and expression and activity of Se-dependent glutathione peroxidases during the rotifer-feeding period (3 to 29
dph) compared to controls. Meanwhile no effect of the treatment was seen in the activity or mRNA expression of the deiodinases. There was no difference in thyroid hormone levels at 29
dph, but the T
3/T
4 ratio was increased in the Se
+
larvae. Thyroid follicles of Se
+
larvae had increased total volume, colloid volume and ratios of epithelium to colloid compared to controls. The results demonstrate that Se-dependent enzyme mRNA expression and activities in cod are retained according to a selenoprotein hierarchy as found in other vertebrates, and suggest that rotifers need extra supplementation of Se to meet cod larvae requirements.
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.
•Precision-cut liver slices from Atlantic cod were investigated.•Cod liver slices are viable in culture for several days.•Cod liver slices respond to environmental contaminants.•Liver slices are a ...promising in vitro system for toxicological studies of cod liver.
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 8mm in diameter and 250μm 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 72h in culture. The liver slices were also exposed to two well-known model environmental contaminants, β-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.
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.
The mRNA levels of three antioxidant genes, Cu/Zn superoxide dismutase (SOD), catalase (CAT) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px), were quantified with real-time qRT-PCR in ...liver of Atlantic salmon
Salmo salar exposed to 80% (normoxia), 105% and 130% O
2 saturation for 54 days. The salmon were then translocated and exposed to 90% and 130% O
2 saturation for additional 72 days during smoltification. TBARS and vitamin E levels in liver and the levels of oxidized glutathione (GSSG), total glutathione (GSH) and the resulting oxidative stress index (OSI) in blood were quantified as traditional oxidative stress markers. No significant mean normalized expression (MNE) differences of SOD, CAT or GSH-Px were found in liver after hyperoxia exposure at the two sampling times. Significantly decreased OSI was found in smolt exposed to 130% O
2 saturation after 126 days (
n
=
18,
P
<
0.0001), indicating hyperoxia-induced oxidative stress. No effects were seen on growth, or on the levels of thiobarbituric reactive substances (TBARS) and vitamin E in liver after the exposure experiment. Overall, the mRNA expression of SOD, CAT and GSH-Px in liver related poorly with the hyperoxic exposure regimes, and more knowledge are needed before the expressed levels of these antioxidant genes can be applied as biomarkers of hyperoxia in Atlantic salmon.
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 (∼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.
Elevated levels of chlorpyrifos‑methyl have been detected in plant-based Atlantic salmon feeds. To evaluate the potential negative effects of long-term and continuous dietary exposure to ...chlorpyrifos‑methyl 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 chlorpyrifos‑methyl 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 chlorpyrifos‑methyl, 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 chlorpyrifos‑methyl 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 chlorpyrifos‑methyl 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.
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