Trout Liver Research Articles

Regulation by Dietary Carbohydrates of Intermediary Metabolism in Liver and Muscle of Two Isogenic Lines of Rainbow Trout.

Rainbow trout (Oncorhynchus mykiss) is recognized as a typical “glucose-intolerant” fish, and the limits of dietary carbohydrate utilization have been investigated for many years. In this study, the objective was to test the molecular effects of dietary carbohydrates on intermediary metabolism in two major metabolic tissues, liver and muscle. Another objective was also to study if the response to carbohydrate intake depended on the genetic background. We fed two isogenic lines of rainbow trout (named A22h and N38h) with high carbohydrate diet (carbohydrate, 22.9%) or low carbohydrate diet (carbohydrate, 3.6%) for 12 weeks. Carbohydrates were associated with higher feed utilization owned by the well-known protein-sparing effect, with better fish growth performance. However, atypical regulation of glycolysis and gluconeogenesis in liver and absence of hk and glut4 induction in muscle, were also observed. Regarding the effects of carbohydrates on other metabolism, we observed an increased, at a molecular level, of hepatic cholesterol biosynthesis, fatty acid oxidation and mitochondrial energy metabolism. Genetic variability (revealed by the differences between the two isogenic lines) was observed for some metabolic genes especially for those involved in the EPA and DHA biosynthetic capacity. Finally, our study demonstrates that dietary carbohydrate not only affect glucose metabolism but also strongly impact the lipid and energy metabolism in liver and muscle of trout.

Circulating miRNA measurements are reflective of cholesterol-based changes in rainbow trout (Oncorhynchus mykiss).

MicroRNAs (miRNAs) are a class of small non-coding RNAs which are known to posttranscriptionally regulate the expression of most genes in both animals and plants. Meanwhile, studies have shown that numbers of miRNAs are present in body fluids including the plasma. Despite the mode of action of these circulating miRNAs still remains unknown, they have been found to be promising biomarkers for disease diagnosis, prognosis and response to treatment. In order to evaluate the potential of miRNAs as non-invasive biomarkers in aquaculture, a time-course experiment was implemented to investigate the postprandial regulation of miRNAs levels in liver and plasma as well as the hepatic expression of genes involved in cholesterol metabolism. We showed that miR-1, miR-33a, miR-122, miR-128 and miR-223 were expressed in the liver of rainbow trout and present at detectable level in the plasma. We also demonstrated that hepatic expression of miR-1, miR-122 and miR-128 were regulated by feed intake and reached their highest levels 12 hours after the meal. Interestingly, we observed that circulating levels of miR-128 and miR-223 are subjected to postprandial regulations similar to that observed in their hepatic counterparts. Statistical correlations were observed between liver and plasma for miR-128 and miR-223 and between hepatic and circulating miR-122, miR-128 and miR-223 and expression of genes related to cholesterol synthesis and efflux or glucose phosphorylation. These results demonstrated that circulating miR-122, miR-128 and miR-223 are potential biomarkers of cholesterol metabolism in rainbow trout.

Effects of chitosan edible coatings enriched with sumac on the quality and the shelf life of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) fillets

AbstractEffects of chitosan coating combined with sumac on the quality of rainbow trout (Oncorhynchus mykiss) fillets stored in refrigerated condition (4°C) for 12 days were evaluated. The treatments in this study were—T1, control; T2, chitosan: 2% w/v; T3, chitosan: 2% w/v and sumac: 1% w/v; and T4, chitosan: 2% w/v and sumac: 2% w/v. The shelf life of the samples were determined periodically using physicochemical (pH, peroxide value [PV], thiobarbituric acid [TBA], and total volatile basic nitrogen [TVB‐N]), microbiological (total viable counts [TVCs] and psychrotrophic bacteria), and sensory analyzes. During the storage period, T3 and T4 groups showed lower pH, PV, TBA, and TVB‐N values than T1 and T2 groups. The sensory results are in line with chemical and microbiological results. Treatment with 2% chitosan + 1% sumac and 2% chitosan + 2% sumac increased the shelf life of rainbow trout fillets by 6 days as compared to the control samples.Practical applicationsTo avoid the adverse effects of chemical preservatives and to answer natural and reliable product demands of the consumers, plant extracts and edible coatings are applied to increase the shelf life and to prevent the spoilage of fresh fish. The results of this study reported the efficacy of chitosan edible coating combined with sumac as a new natural preservative for improving the shelf life of rainbow trout fillets. The findings revealed that sumac extract combined with chitosan coating can be an alternative method to extend the shelf life, to preserve the quality and safety of fish, as a result of their good antioxidant and antimicrobial activity.

Evaluation of antioxidant properties of Chlorella vulgaris and Spirulina platensis and their application in order to extend the shelf life of rainbow trout (Oncorhynchus mykiss) fillets during refrigerated storage

The present study was undertaken to evaluate the antioxidant effect of alcoholic extract of Chlorella vulgaris and Spirulina platensis on extending the shelf life of rainbow trout (Oncorhynchus mykiss) fillet in 4 ± 1 °C. The experiments were performed on 5 samples (1 blank and 4 treatments) and in triplicate. The results from DPPH and FRAP assays represents the higher antioxidant activity of Chlorella (42.96% and 0.33) at concentration of 200 μg/ml and Spirulina (62.46% and 0.41; 77.12% and 0.46) at concentrations of 400 and 600 μg/ml, respectively. The results from performed chemical tests compared to the blank sample showed that samples treated with algae extracts were able to control the increase in these parameters. Among the treated samples, maximum control of peroxide value, thiobarbituric acid and free fatty acid, respectively pertained to the treatments containing 0.1% of Spirulina (3.14, 2.93 and 2.25) and Chlorella (3.21, 3.06 and 2.28) extracts. The results of sensory evaluation showed that except the sample containing 0.05% of Chlorella, in other samples containing the algae extract, less negative effects on color, odor, texture and taste were observed. Consequently, Chlorella and Spirulina extracts can delay lipid oxidation process and maintain or improve sensory properties of refrigerated fish.

Identification of a piscine reovirus-related pathogen in proliferative darkening syndrome (PDS) infected brown trout (Salmo trutta fario) using a next-generation technology detection pipeline.

The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) with a mortality rate of near 100% in pre-alpine rivers of central Europe. So far the etiology and causation of this disease is still unclear. The objective of this study was to identify the cause of PDS using a next-generation technology detection pipeline. Following the hypothesis that PDS is caused by an infectious agent, brown trout specimens were exposed to water from a heavily affected pre-alpine river with annual occurrence of the disease. Specimens were sampled over the entire time period from potential infection through death. Transcriptomic analysis (microarray) and RT-qPCR of brown trout liver tissue evidenced strong gene expression response of immune-associated genes. Messenger RNA of specimens with synchronous immune expression profiles were ultra-deep sequenced using next-generation sequencing technology (NGS). Bioinformatic processing of generated reads and gap-filling Sanger re-sequencing of the identified pathogen genome revealed strong evidence that a piscine-related reovirus is the causative organism of PDS. The identified pathogen is phylogenetically closely related to the family of piscine reoviruses (PRV) which are considered as the causation of different fish diseases in Atlantic and Pacific salmonid species such as Salmo salar and Onchorhynchus kisutch. This study also highlights that the approach of first screening immune responses along a timeline in order to identify synchronously affected stages in different specimens which subsequently were ultra-deep sequenced is an effective approach in pathogen detection. In particular, the identification of specimens with synchronous molecular immune response patterns combined with NGS sequencing and gap-filling re-sequencing resulted in the successful pathogen detection of PDS.

RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss)

BackgroundIn the past years, our laboratory successfully generated transgenic rainbow trout bearing cecropin P1 transgene. These fish exhibited resistant characteristic to infection by Aeromonas salmonicida, Infectious Hematopoietic Necrosis Virus (IHNV) and Ceratomyxa shasta (a parasitic pathogen). Previously, treating rainbow trout macrophage cells (RTS-11) with cecropin B, pleurocidin and CF17, respectively, resulted in elevated expression of two pro-inflammatory genes, e.g. cyclooxygenase-2 (cox-2) and interleukin-1β (il-1β). In addition, a profiling of global gene expression by 44 k salmonid microarray analysis was conducted, and the results showed that immune relevant processes have been perturbed in cecopin P1 transgenic rainbow trout. Therefore, we hypothesized that cecropin P1 may not only eliminate pathogens directly, but also modulate the host immune systems, leading to increased resistance against pathogen infections. To confirm this hypothesis, we performed de novo mRNA deep sequencing (RNA-Seq) to analyze the transcriptomic expression profiles in three immune competent tissues of cecropin P1 transgenic rainbow trout.ResultsDe novo sequencing of mRNA of the rainbow trout spleen, liver and kidney tissues were conducted by second-generation Illumina system, followed by Trinity assembly. Tissue specific unigenes were obtained, and annotated according to the Gene Ontology (GO) and the Nucleotide Basic Local Alignment Search Tool (BLAST). Over 2000 differentially expressed genes (DEGs) were determined by normalized ratio of Reads Per Kilobase of transcript per million mapped reads (RPKM) among the transgenic and non-transgenic fish in a tissue specific manner, and there were 82 DEGs in common among the three tissues. In addition, the enrichment analysis according to Gene Ontology Biological Process (GO:BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) based pathway analysis associated with innate/adaptive immunity of fish were also performed to illustrate the altered immune-related functions in each tissue.ConclusionsAccording to the RNA-Seq data, the correlations between alteration of gene expression profiles and the functional perturbations of the host immune processes were revealed. In comparison with the results of cDNA microarray analysis conducted by Lo et al., the overall results supported our hypothesis that the gene product of cecropin P1 transgene may not only directly eliminate pathogens, but also modulate the host immune system. Results of this study present valuable genetic information for Oncorhynchus mykiss, and will benefit future studies on the immunology of this fish species.

Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1

In vitro experimental systems based on continuous piscine cell lines can be used as an alternative to animal tests for obtaining qualitative and quantitative information on the possible fate and effect of chemicals in fish. However, their capability to reproduce complex metabolic processes and toxic responses as they occur in vivo is limited due to the lack of organ-specific tissue architecture and functions. Here we introduce a three-dimensional (3D) in vitro experimental system based on spheroidal aggregate cultures (spheroids) of the continuous rainbow trout liver cell line RTL-W1 and provide a first description of their structural and functional properties including growth, viability/longevity, metabolic activity, ultrastructure and cytochrome P450 1A (CYP1A) expression determined by bright-field, multi-photon fluorescence and transmission electron microscopy as well as RT-qPCR analysis. Our results show that RTL-W1 cells in 3D spheroids (ø ~ 150 µm) (including those in the interior) were viable, metabolically active and had higher basal and β-naphthoflavone-induced CYP1A expression levels than conventional 2D cell cultures. Furthermore, they displayed ultrastructural characteristics similar to differentiated hepatocytes. The available evidence suggests that 3D RTL-W1 spheroids may have enhanced hepatotypic functions and be a superior in vitro model to assess hepatic biotransformation, bioaccumulation and chronic toxicity compared to conventional cell monolayer cultures.

Exploring the interactions between polyunsaturated fatty acids and cadmium in rainbow trout liver cells: a genetic and proteomic study

Polyunsaturated fatty acids (PUFAs) have key biological roles in fish cells. We recently showed that the phospholipid composition of rainbow trout liver cells (RTL-W1 cell line) modulates their tolerance to an acute cadmium (Cd) challenge. Here, we investigated (i) the extent to which PUFAs and Cd impact fatty acid homeostasis and metabolism in these cells and (ii) possible mechanisms by which specific PUFAs may confer cytoprotection against Cd. First, RTL-W1 cells were cultivated for one week in growth media spiked with 50 μmol L−1 of either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), linoleic acid (LA, 18:2n-6) or arachidonic acid (AA, 20:4n-6) in order to modulate their fatty acid profile. Then, the cells were challenged with Cd (0, 50 or 100 μmol L−1) for 24 h prior to assaying viability, fatty acid profile, intracellular Cd content, proteomic landscape and expression levels of genes involved in fatty acid metabolism, synthesis of PUFA-derived signalling molecules and stress response. We observed that the fatty acid supply and, to a lesser extent, the exposure to Cd influenced cellular fatty acid homeostasis and metabolism. The cellular fatty acid composition of fish liver cells modulated their tolerance to an acute Cd challenge. Enrichments in ALA, EPA, and, to a lesser extent, AA conferred cytoprotection while enrichment in LA had no impact on cell viability. The present study ruled out the possibility that cytoprotection reflects a decreased Cd burden. Our results rather suggest that the PUFA-derived cytoprotection against Cd occurs through a reduction of the oxidative stress induced by Cd and a differential induction of the eicosanoid cascade, with a possible role of peroxiredoxin and glutaredoxin (antioxidant enzymes) as well as cytosolic phospholipase A2 (enzyme initiating the eicosanoid cascade).

Identification and characterization of microRNAs in the liver of rainbow trout in response to heat stress by high-throughput sequencing

MicroRNAs (miRNAs) play key roles in the stress response in animals. The rainbow trout is a widely cultivated cold-water fish species that usually encounters heat stress, which affects its growth and may cause diseases. However, there is limited information on the miRNAs involved in heat stress in this species. To identify the heat-responsive miRNAs at the genome level in rainbow trout, we conducted high-throughput sequencing of 6 small RNA libraries from the liver tissues of rainbow trout specimens under control and heat-treated conditions. A total of 363 conserved miRNAs and 638 novel miRNAs were identified, among which 39 (21 conserved and 18 novel) miRNAs were differentially expressed in response to heat stress. Eight important miRNAs were further validated using quantitative real-time PCR. GO enrichment and KEGG pathway analyses of the potential target genes of the DE miRNAs, revealed DNA replication and protein folding and unfolding were affected by heat stress. Several key pathways involved in the heat stress response were characterized, such as protein processing in the endoplasmic reticulum, NOD-like receptor signaling pathway, and progesterone-mediated oocyte maturation. In addition, 20 important functional interaction pairs among the 65 negatively correlated miRNA-mRNA pairs were identified using integrated miRNA-mRNA analysis and miRNA prediction algorithms, which including potential important miRNAs and target genes such as miR-301a, miR-301d, let-7c, novel_1181 and HSP90BA, HSP90BB, HSP40, PDIA3. This study showed that the identified miRNAs probably play important roles in the heat stress response and lay a foundation for further studies on the thermal adaptation mechanisms in rainbow trout.

Age- and sex-dependent variation in the activity of antioxidant enzymes in the brown trout (Salmo trutta)

Oxidative stress is defined as the imbalance between pro-oxidant and antioxidant molecules in favor of the former and it represents one of the main driving forces of aging. To counteract the harmful effects of oxidative stress, organisms evolved a complex antioxidant system. According to the free radical theory of aging, while the production of reactive oxygen species (ROS) increases with age, the antioxidant defenses decline. Although this relationship has been investigated in diverse vertebrate taxa, the information in fish is scant and inconsistent, particularly for wild populations. Thus, the aim of the present study was the investigation of age- and sex-related changes of the antioxidant enzymes activity in free-living individuals of the brown trout (Salmo trutta). We measured the activity of the main enzymes involved in antioxidant protection, namely superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), as well as lipid peroxidation, in the gills and the liver dissected from brown trout (1+- to 5+-year-old). A significant age-dependent variation in the activity of antioxidant enzymes was noted, with the exception of CAT. GPx activity followed a significant increasing trend with age in both the organs, while SOD decreased in the liver. Increased GST activity was found in the gills only. Lipid peroxidation levels significantly decreased with age in both the organs. SOD and CAT showed sex-dependent differences in the liver of brown trout, with males showing lower enzymatic activity than females. Our data contribute to improve the knowledge on the relationship between antioxidant enzyme activity, aging, and sex in fish.

Oil Sands Derived Naphthenic Acids Are Oxidative Uncouplers and Impair Electron Transport in Isolated Mitochondria.

Naphthenic acids (NAs) are predominant compounds in oil sands influenced waters. These acids cause numerous acute and chronic effects in fishes. However, the mechanism of toxicity underlying these effects has not been fully elucidated. Due to their carboxylic acid moiety and the reported disruption of cellular bioenergetics by similar structures, we hypothesized that NAs would uncouple mitochondrial respiration with the resultant production of reactive oxygen species (ROS). Naphthenic acids were extracted and purified from 17-year-old oil sands tailings waters yielding an extract of 99% carboxylic acids with 90% fitting the classical O2-NA definition. Mitochondria were isolated from rainbow trout liver and exposed to different concentrations of NAs. Mitochondrial respiration, membrane potential, and ROS emission were measured using the Oroboros fluorespirometry system. Additionally, mitochondrial ROS emission and membrane potential were evaluated with real-time flow cytometry. Results showed NAs uncoupled oxidative phosphorylation, inhibited respiration, and increased ROS emission. The effective concentration (EC50) and inhibition concentration (IC50) values for the end points measured ranged from 21.0 to 157.8 mg/L, concentrations similar to tailings waters. For the same end points, EC10/IC10 values ranged from 11.8 to 66.7 mg/L, approaching concentrations found in the environment. These data unveil mechanisms underlying effects of NAs that may contribute to adverse effects on organisms in the environment.

Assessing off-target cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol using novel lake sturgeon cell lines

Lampricides are currently being applied to streams and rivers to control the population of sea lamprey, an invasive species, in the Great Lakes. The most commonly used lampricide agent used in the field is 3-trifluoromethyl-4-nitrophenol (TFM), which targets larval sea lamprey in lamprey-infested rivers and streams. The specificity of TFM is due to the relative inability of sea lamprey to detoxify the agent relative to non-target fishes. There is increasing concern, however, about non-target effects on fishes, particularly threatened populations of juvenile lake sturgeon (LS; Acipenser fulvescens). There is therefore a need to develop models to better define lake sturgeon's response to TFM. Here we report the establishment of five LS cell lines derived from the liver, gill, skin and intestinal tract of juvenile LS and some of their cellular characteristics. All LS cell lines grew well at 25 °C in Leibovitz's (L)− 15 medium supplemented with 10% FBS. All cell lines demonstrated high senescence-associated β–galactosidase activity and varying levels of Periodic acid Schiff-positive polysaccharides, indicating substantial production of glycoproteins and mucosubstances by the cells. Comparative toxicity of TFM in the five LS cell lines was assessed by two fluorescent cell viability dyes, Alamar Blue and CFDA-AM, in conditions with and without serum and at 24 or 72 h exposure. Deduced EC50 values were compared between the cell lines and to the reported in vivo LC50s. Tissues sensitive to the effects of TFM in vivo correlated with cell lines from the same tissues being most sensitive to TFM in vitro. EC50 values for the LSliver-e cells was significantly lower than the EC50 for the rainbow trout (RBT) liver cells RTL-W1, reaffirming the in vivo observation that LS was generally more TFM-sensitive than rainbow trout. Our data suggests that whole-fish sensitivity of LS to TFM is likely attributable to sensitivity at the cellular level. Thus, LS cell lines, as well as those of RBT, can be used to screen and evaluate the toxicity of the next generation of lampricides on non-target fish such as lake sturgeon.

Intra- and interspecific densities shape life-history traits in a salmonid population.

Population dynamics can be regulated through intra- and interspecific density dependence. In species with close ecological requirements, interspecific competition for resources may add to intraspecific density, or even exceed its effect; it may impact single or multiple traits. However, the relative impact of intra- and interspecific densities on demographic parameters has been rarely empirically assessed. We analyzed 18years of capture-mark-recapture data from brown trout (Salmo trutta) coexisting with Atlantic salmon (Salmo salar) during the juvenile freshwater phase in the Oir River (France) to estimate the relative effects of intra- and interspecific density on trout early life. In trout, a species with optional migration, we estimated the migration probability of young-of-the-year trout out of their natal site, survival probability during the first winter, as well as body size, in relation to both intra- and interspecific density. Trout density correlated negatively with body size and with winter survival in resident trout but not with trout migration. Salmon density correlated positively with trout migration, but no impact was detected on trout body size or survival. Our study highlighted contrasting effects of intra- and interspecific density on trout early life, and the need to account for both factors when studying population dynamics in coexisting species. In particular, by affecting trout migration decision, salmon density may drive trout life history.

Fine-scale population structure in lake trout (Salvelinus namaycush) influenced by life history variation in the Husky Lakes drainage basin, Northwest Territories, Canada

Partial anadromy is common within salmonid populations, where resident and anadromous individuals interbreed and overlap in habitat use during portions of life. Deviation to this definition occurs within the Husky Lakes drainage basin (HLDB), Northwest Territories, where freshwater resident, semi-anadromous, and brackish-water resident lake trout (Salvelinus namaycush) life history types are documented. In this study, microsatellite DNA variation was assayed to evaluate genetic structuring among life history types from the HLDB and adjacent lower Mackenzie River system. Significant differentiation was resolved among most locations and life histories (global FST= 0.192). Brackish-water residents were differentiated from all locations and life histories, including sympatric semi-anadromous individuals, providing evidence for genetically fixed strategies. Also, this provides the first evidence of breeding partial migration in salmonids using brackish-water environments, where brackish-water residents and semi-anadromous migrants interact during the nonbreeding season, but the latter migrate elsewhere to spawn. Alternatively, the lack of genetic differentiation between semi-anadromous and Sitidgi Lake residents suggests conditional mating tactics may also influence partial anadromy. This work provides novel insights into partial anadromy in Arctic salmonids and expands our knowledge of biodiversity in this region.

СЕЗОННАЯ ДИНАМИКА СОСТАВА ЖИРНЫХ КИСЛОТ В ТКАНЯХ РАДУЖНОЙ ФОРЕЛИ PARASALMOMYKISS (WALBAUM, 1792), КУЛЬТИВИРУЕМОЙ НА РАЗЛИЧНЫХ КОМБИКОРМАХ

A comparative study of the composition of fatty acids in muscles, liver and internal fat in rainbow trout Parasalmo mykiss (Walbaum, 1792) grown on various diets in the period from April to July was effected. Differences in content of dietary fatty acids in diet 1 and diet 2 were found. A significantly lower level of long-chain polyunsaturated fatty acids (including physiologically significant eicosapentaenoic 20: 5(n-3) and docosapentaenoic 22: 6(n-3) acids) was detected in diet № 2, compared to diet № 1. As a result, deficiency of dietary eicosapentaenoic and docosapentaenoic acids leads to a slow rate of fishes growth. A significant influence of the trophic factor on the distribution of fatty acids in muscles and internal fat of fish is shown, because lipids in rainbow trout accumulate in these tissues. The degree of accumulation of different dietary fatty acids in muscles and internal fat of trout was depended on fatty acids that prevailed in the diet on which the rainbow trout was grown. A high degree of influence of the trophic factor on the level of polyunsaturated fatty acids of families n-9 and n-4 was found, the content of which in trout tissues was about 1%. Composition of fatty acids in trout liver, as the most metabolically active organ, is less conditioned by the influence of the factors studied. The degree of assimilation of docosahexaenoic 22: 6 (n-3) acid in rainbow trout probably depended on the level of eicosapentaenoic 20: 5 (n-3) acid in the diet. It has been stated that changing the level of fatty acids in muscles and internal fat of trout during seasonal changes is associated with an increase in the frequency of feeding of fishes in the summer.

Differences in fatty acid composition of gill and liver phospholipids between Steelhead trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) under declining temperatures

This study investigated the fatty acid (FA) composition of gill and liver phospholipids in Steelhead trout (Oncorhynchus mykiss) (average weight: 75.45 ± 6.27 g) and Atlantic salmon (Salmo salar) (average weight: 72.89 ± 3.12 g) as water temperature decreased from 16 °C to 12 °C, 8 °C, 6 °C, 4 °C, 2 °C, and 1 °C. At normal temperature (16 °C), both gill and liver phospholipids of Atlantic salmon had lower saturated fatty acid (SFA) and higher unsaturated (UFA) compositions than those of Steelhead trout. Atlantic salmon was more sensitive to the temperature decline than Steelhead trout at the early stage of the experiment. Polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid (MUFA) contents of Atlantic salmon liver phospholipids significantly increased as temperature decreased from 16 °C to 12 °C. However, both PUFA and MUFA contents decreased significantly at temperatures below 4 °C. In contrast, there was only a negligible decrease in MUFA content in Steelhead trout liver phospholipids at 1 °C. Starvation and cold stress influenced liver phospholipid FA composition in Atlantic salmon but not in Steelhead trout, suggesting that the latter can better adapt to low temperatures. Changes in gill phospholipid composition in response to temperature fluctuations were species-specific. When the ambient temperature was below 8 °C, SFA content of gill phospholipids decreased whereas PUFA content increased and MUFA content was unaltered in Atlantic salmon. Although the FA composition of gill phospholipids in Atlantic salmon was altered at 6 °C, it remained stable as temperature further decreased. These results indicate that the FA composition of gill phospholipids in Atlantic salmon is less influenced by starvation stress than that of liver phospholipids. Moreover, fewer changes in FA composition were observed for gill as compared to liver phospholipids in both species of fish. Our results can be useful for optimizing Atlantic salmon and Steelhead trout aquaculture systems.

H2O2 metabolism in liver and heart mitochondria: Low emitting-high scavenging and high emitting-low scavenging systems

Although mitochondria are presumed to emit and consume reactive oxygen species (ROS), the quantitative interplay between the two processes in ROS regulation is not well understood. Here, we probed the role of mitochondrial bioenergetics in H2O2 metabolism using rainbow trout liver and heart mitochondria. Both liver and heart mitochondria emitted H2O2 at rates that depended on their metabolic state, with the emission rates (free radical leak) constituting 0.8–2.9% and 0.2–2.5% of the respiration rate in liver and heart mitochondria, respectively. When presented with exogenous H2O2, liver and heart mitochondria consumed it by first order reactions with half-lives (s) of 117 and 210, and rate constants of 5.96 and 3.37 (× 10−3 s−1), respectively. The mitochondrial bioenergetic status greatly affected the rate of H2O2 consumption in heart but not liver mitochondria. Moreover, the activities and contribution of H2O2 scavenging systems varied between liver and heart mitochondria. The significance of the scavenging systems ranked by the magnitude (%) of inhibition of H2O2 removal after correcting for emission were, liver (un-energized and energized): catalase > glutathione (GSH) ≥ thioredoxin reductase (TrxR); un-energized heart mitochondria: catalase > TrxR > GSH and energized heart mitochondria: GSH > TrxR > catalase. Notably, depletion of GSH evoked a massive surge in H2O2 emission that grossly masked the contribution of this pathway to H2O2 scavenging in heart mitochondria. Irrespective of the organ of their origin, mitochondria behaved as H2O2 regulators that emitted or consumed it depending on the ambient H2O2 concentration, mitochondrial bioenergetic state and activity of the scavenging enzyme systems. Indeed, manipulation of mitochondrial bioenergetics and H2O2 scavenging systems caused mitochondria to switch from being net consumers to net emitters of H2O2. Overall, our data suggest that the low levels of H2O2 typically present in cells would favor emission of this metabolite but the scavenging systems would prevent its accumulation.

Cytosolic distributions of highly toxic metals Cd and Tl and several essential elements in the liver of brown trout (Salmo trutta L.) analyzed by size exclusion chromatography and inductively coupled plasma mass spectrometry

Cytosolic distributions of nonessential metals Cd and Tl and seven essential elements among compounds of different molecular masses were studied in the liver of brown trout (Salmo trutta) from the karstic Krka River in Croatia. Analyses were done by size exclusion high performance liquid chromatography and high resolution inductively coupled plasma mass spectrometry. Common feature of Cd and Tl, as highly toxic elements, was their distribution within only two narrow peaks. The increase of cytosolic Cd concentrations was reflected in marked increase of Cd elution within low molecular mass peak (maximum at ∼15 kDa), presumably containing metallothioneins (MTs), which indicated successful Cd detoxification in brown trout liver under studied exposure conditions. Contrary, the increase of cytosolic Tl concentrations was reflected in marked increase of Tl elution within high molecular mass peak (maximum at 140 kDa), which probably indicated incomplete Tl detoxification. Common feature of the majority of studied essential elements was their distribution within more peaks, often broad and not well resolved, which is consistent with their numerous physiological functions. Among observed associations of essential metals/nonmetal to proteins, the following could be singled out: Cu and Zn association to MTs, Fe association to storage protein ferritin, and Se association to compounds of very low molecular masses (<5 kDa). The obtained results present the first step towards identification of metal-binding compounds in hepatic cytosol of brown trout, and thus a significant contribution to better understanding of metal fate in the liver of that important bioindicator species.

Development of a new tool for the long term in vitro ecotoxicity testing of nanomaterials using a rainbow-trout cell line (RTL-W1)

The current wide use of manufactured nanomaterials (MNs) is leading to the release of nanoparticles (NPs) to water bodies. Aquatic organisms, including fish, are exposed to low concentrations of NPs for long periods of time being necessary to develop laboratory toxicity tests reflecting realistic conditions. Additionally, today there is a demand of in vitro assays respecting the 3Rs principle. Thus, the main aim of this work was to stablish an in vitro tool for the assessment of long-term NPs ecotoxicity. Considering the key role of liver in detoxification, a rainbow trout liver cell line, RTL-W1, was used. CuO NPs were chosen to validate this tool taking into account their important production level. Cells were exposed for 21 days to 25 or 100 μg CuO NPs/ml. Every seven days cells were split and one fourth of them transferred to a new plate with appropriate concentrations of NPs in culture medium. Lower concentrations of CuO NPs did not cause any deleterious effect, whereas higher concentrations led to significant mortality after 14 days and to the intracellular accumulation of Cu particles. Identical results were observed in cells exposed to CuSO4 at the same Cu concentrations. Therefore, the observed toxic effects might be mainly due to Cu2+ ions.

Transcriptional effects of phospholipid fatty acid profile on rainbow trout liver cells exposed to methylmercury

Lipids, and their constitutive fatty acids, are key nutrients for fish health as they provide energy, maintain cell structure, are precursors of signalling molecules and act as nuclear receptor ligands. These specific roles may be of crucial importance in a context of exposure to pollutants. We recently showed that the fatty acid profile of rainbow trout liver cell phospholipids modulates sensitivity to an acute methylmercury challenge. In order to investigate mechanisms of effects, we herein tested whether specific polyunsaturated fatty acids (PUFAs) may protect cells from methylmercury through decreasing intracellular mercury accumulation and/or enhancing cellular defences (e.g. via modulation of gene expression patterns). We also investigated the inverse relationship and assessed the impact of methylmercury on cellular fatty acid metabolism. To do so, the fatty acid composition of rainbow trout liver cell phospholipids was first modified by incubating them in a medium enriched in a specific PUFA from either the n-3 family (alpha-linolenic acid, ALA; eicosapentaenoic acid, EPA) or the n-6 family (linoleic acid, LA; arachidonic acid, AA). Cells were then exposed to methylmercury (0.15 or 0.50 μM) for 24 h and sampled thereafter for assessing phospholipid fatty acid profile, intracellular total mercury burden, and expression pattern of genes involved in fatty acid metabolism, synthesis of PUFA-derived signalling molecules and stress response. We observed that cells incorporated the given PUFA and some biotransformation products in their phospholipids. Methylmercury had few impacts on this cellular phospholipid composition. None of the PUFA enrichments affected the cellular mercury burden, suggesting that the previously observed cytoprotection conferred by ALA and EPA was not linked to a global decrease in cellular accumulation of mercury. Fatty acid enrichments and methylmercury exposure both modulated gene expression patterns. Genes involved in the synthesis of PUFA-derived signalling molecules, in stress response and the orphan cytochrome P450 20A1 were identified as possible sites of interaction between fatty acids and methylmercury in rainbow trout liver cells.