Lake Trout Liver Research Articles

Analysis of the lake trout heart, blood, liver, and brain proteome using evolutionary proteomics

IntroductionSalvelinus namaycush (lake trout) is a top‐predator fish in the Great Lakes region. The Great Lakes Fish Monitoring and Surveillance Program (GLFMSP) use lake trout as a bioindicator of chemical stress by monitoring the concentrations of persistent, bioaccumulative and toxic (PBT) chemicals in their bodies. Studies have shown that elevated concentrations of PBTs can cause changes in transcribed genes, translated mRNAs, proteins produced, and post‐translational modifications in aquatic species. Though lake trout is used to monitor chemical concentrations for over 50 years, there is very little information available on the proteome of this species. A well‐developed protein database for the lake trout would help elucidate the effects of PBT chemicals on this species providing a direct health metric for the Great Lakes ecosystem.MethodsIn this study, heart, blood, brain, and liver samples from lake trout were analyzed by SDS‐PAGE, followed by in‐gel trypsin digestion and analysis by nanoLC‐MS/MS. The raw data was searched against different NCBI and UniProtKB databases in Mascot Daemon and the output was analyzed by Scaffold 4.3 software. Databases used include Actinopterygii, Salmonidae, Salvelinus, as well as the highly studied species Oncorhynchus mykissand Danio rerio.Preliminary DataPreviously, our group has reported a large number of proteins for lake trout liver, heart and blood (Dupree et al., Proteomics, 2019, PMID: 31578773 and Dupree et al., Proteomics, 2021, PMID: 34676671). In lake trout liver we identified 4194 proteins using the NCBI databases and 3811 potential protein hits in the UniProtKB databases. In the blood and heart we identified 838 and 580 proteins, respectively, using NCBI databases and 1180 and 561 potential protein hits for the heart and blood, respectively in BR2. A lake trout brain proteomics analysis completed using an in‐house SDS‐PAGE, followed by in gel digestion and nanoLC‐MS/MS analysis. The Mascot database search is underway. Once protein hits are cataloged, BLAST and Scaffold‐based comparisons of the liver, heart, blood and brain will be performed. Additional search of the data against the other fish relatives will also be completed. Through this process, evolutionary relationships for the lake trout species will also be explored. The current study will add to our understanding of the lake trout proteome that will be used to understand the biochemical/physiological effects of legacy PBT chemicals in the Great Lakes ecosystem.Novel AspectUse of four types of lake trout tissue together to develop a protein database for the species.

Proteomic analysis of the lake trout (Salvelinus namaycush) heart and blood: The beginning of a comprehensive lake trout protein database.

Lake trout (Salvelinus namaycush) are a top-predator species in the Laurentian Great Lakes that are often used as bioindicators of chemical stressors in the ecosystem. Although many studies are done using these fish to determine concentrations of stressors like legacy persistent, bioaccumulative and toxic chemicals, there are currently no proteomic studies on the biological effects these stressors have on the ecosystem. This lack of proteomic studies on Great Lakes lake trout is because there is currently no complete, comprehensive protein database for this species. Here, we employed proteomics approaches to develop a lake trout protein database that could aid in future research on this fish, in particular exposomics and adductomics. The current study utilized heart tissue and blood from two lake trout. Our previous work using lake trout liver revealed 4194 potential protein hits in the NCBI databases and 3811 potential protein hits in the UniProtKB databases. In the current study, using the NCBI databases we identified 838 proteins for the heart and 580 proteins for the blood tissues in the biological replicate 1 (BR1) and 1180 potential protein hits for the heart and 561 potential protein hits for the blood in BR2. Similar results were obtained using the UniProtKB databases. This study builds on our previous work by continuing to build the first comprehensive lake trout protein database and provides insight into protein homology through evolutionary relationships. This data is available via the PRIDE partner repository with the dataset identifier PXD023970.

Proteomic Analysis of the Lake Trout (Salvelinus namaycush) Liver Identifies Proteins from Evolutionarily Close and -Distant Fish Relatives.

Lake trout are used as bioindicators for toxics exposure in the Great Lakes ecosystem. Here the first lake trout (Salvelinus namaycush) liver proteomics study is performed and searched against specific databases: (NCBI and UniProtKB) Salvelinus, Salmonidae, Actinopterygii, and Oncorhynchus mykiss, and the more distant relative, Danio rerio. In the biological replicate 1 (BR1), technical replicate 1 (TR1), (BR1TR1), a large number of lake trout liver proteins are not in the Salvelinus protein database, suggesting that lake trout liver proteins have homology to some proteins from the Salmonidae family and Actinopterygii class, and to Oncorhynchus mykiss and Danio rerio, two more highly studied fish. In the NCBI search, 4194 proteins are identified: 3069 proteins in Actinopterygii, 1617 in Salmonidae, 68 in Salvelinus, 568 in Oncorhynchus mykiss, and 946 in Danio rerio protein databases. Similar results are observed in the UniProtKB searches of BR1RT1, as well as in a technical replicate (BR1TR2), and then in a second biological replicate experiment, with two technical replicates (BR2TR1 and BR2TR2). This study opens the possibility of identifying evolutionary relationships (i.e., adaptive mutations) between various groups (i.e., zebrafish, rainbow trout, Salmonidae, Salvelinus and lake trout) through evolutionary proteomics. Data are available via the PRIDE Q2 (PXD011924).

Developing Well-Annotated Species-Specific Protein Databases Using Comparative Proteogenomics.

Proteomics is a mass spectrometry-based discipline that aims to analyze proteomes and their functions. Many proteomic studies require well-developed protein databases for reference. However, most proteomes are not well-annotated, aside from model organisms. Techniques like six-frame translation, ab initio gene prediction, and EST databases can aid in maximizing the amount of proteins identified in proteomics experiments, however, each of these has its downfalls. Proteogenomics is a term used to describe the union of proteomics, genomics and transcriptomics to assist in the identification of peptides which would help build better annotated proteome databases. Here, current proteomic and proteogenomic methods will be reviewed, and an example of a comparative proteomics method using lake trout liver samples will be described.

Sea lampreys elicit strong transcriptomic responses in the lake trout liver during parasitism.

BackgroundThe sea lamprey (Petromyzon marinus) is a jawless vertebrate that parasitizes fish as an adult and, with overfishing, was responsible for the decline in lake trout (Salvelinus namaycush) populations in the Great Lakes. While laboratory studies have looked at the rates of wounding on various fish hosts, there have been few investigations on the physiological effects of lamprey wounding on the host. In the current study, two morphotypes of lake trout, leans and siscowets, were parasitized in the laboratory by sea lampreys and the liver transcriptomes of parasitized and nonparasitized fish were analyzed by RNA-seq (DESeq2 and edgeR) to determine which genes and gene pathways (Ingenuity Pathway Analysis) were altered by lamprey parasitism.ResultsOverall, genes encoding molecules involved in catalytic (e.g., enzymatic) and binding activities (factors and regulators) predominated the regulated gene lists. In siscowets, the top upregulated gene was growth arrest and DNA-damage-inducible protein and for leans it was interleukin-18-binding protein. In leans, the most significantly downregulated gene was UDP-glucuronosyltransferase 2A2 - DESeq2 or phosphotriesterase related - edgeR. For siscowets, the top downregulated gene was C-C motif chemokine 19 - DESeq2 or GTP-binding protein Rhes - edgeR. Gene pathways associated with inflammatory-related responses or factors (cytokines, chemokines, oxidative stress, apoptosis) were regulated following parasitism in both morphotypes. However, pathways related to energy metabolism (glycolysis, gluconeogenesis, lipolysis, lipogenesis) were also regulated. These pathways or the intensity or direction (up/downregulation) of regulation were different between leans and siscowets. Finally, one of the most significantly downregulated pathways in both leans and siscowets was the kynurenine (tryptophan degradation) pathway.ConclusionsThe results indicate a strong transcriptional response in the lake trout to lamprey parasitism that entails genes involved in the regulation of inflammation and cellular damage. Responses to energy utilization as well as hydromineral balance also occurred indicating an adjustment in the host to energy demands and osmotic imbalances during parasitism. Given the role of the kynurenine pathway in promoting immunotolerance in mammals, the downregulation observed in this pathway during parasitism may signify an attempt by the host to inhibit any feedback suppression of the immune response to the lamprey.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2959-9) contains supplementary material, which is available to authorized users.

Effects of xenobiotics and steroids on renal and hepatic estrogen metabolism in lake trout

Experiments were conducted to (1) elucidate the biochemical pathways of E2 metabolism in the lake trout ( Salvelinus namaycush) kidney and liver, and (2) test the hypothesis that specific xenobiotics and endogenous steroids inhibit E2 metabolism by these tissues. Kidney and liver tissue fragments from immature lake trout were incubated in vitro in the presence of radiolabelled E2 plus various xenobiotics or steroids. E2 metabolites were identified by liquid chromatography/mass spectroscopy, and quantified by liquid scintillation spectroscopy. A major metabolite produced by both tissues was an unidentified hydroxylated estrogen metabolite (E2-OH) with a molecular mass of 288 that was not estriol (16-OH-E2), but possibly 7α-OH-E2 or 2-OH-E2 (catecholestrogen). Both tissues also produced estradiol-17-glucuronide (E2-17-G), estradiol-17-sulfate (E2-17-S), and estradiol-3-glucuronide (E2-3-G). Compared to the kidney, the liver produced half the amount of conjugated metabolites, but twofold more E2-OH. The following xenobiotics (at a concentration of 100 μM) inhibited the production of water-soluble (i.e., conjugated) E2 metabolites by both the kidney and liver: 4,4′-(OH) 2-3,3′,5,5′- tetrachlorobiphenyl (4,4′-OH-TCB), bisphenol A (BPA), tetrabromobisphenol A (TB-BPA), tetrachlorobisphenol A (TC-BPA), tribromophenol (TBP), trichlorophenol (TCP), and pentachlorophenol (PCP). The alkylphenols, 4- n-nonylphenol (NP), and 4-octylphenol (OP), and 2,2′,4,4′-tetrabromodiphenyl ether (TBDE) had no significant effect on E2 metabolism by either tissue. Testosterone and 17α,20β-dihydroxy-4-pregnen-3-one inhibited the production of conjugated E2 metabolites by both the kidney and liver. Cortisol and 11-ketotestosterone inhibited E2 metabolism by the liver only. The median inhibitory concentrations (IC 50) for 4,4′-OH-TCB ranged from 7–32 μM in the kidney and 0.6–1.6 μM in the liver. For BPA, IC 50’s ranged from 40–108 μM in the kidney and 11–18 μM in the liver. Low doses (0.1 μM) of 4,4′-OH-TCB and BPA significantly increased estrogen metabolism in the kidney. The results suggest that certain estrogenic xenobiotics and endogenous steroids may inhibit the phase II conjugation of E2 by the kidney and liver of lake trout, and some of the known biological effects of these compounds are likely mediated, at least partially, by this mechanism of action.

Genistein and other isoflavones found in soybeans inhibit estrogen metabolism in salmonid fish

The study was conducted to test the hypothesis that genistein, an isoflavone found in high concentrations in soybeans, inhibits hepatic and renal estrogen metabolism in salmonid fish. Liver and kidney tissues of rainbow trout ( Oncorhyncus mykiss), Atlantic salmon ( Salmo salar), and lake trout ( Salvelinus namaycush) were incubated in vitro with radiolabelled estradiol-17β (E2) in the presence of various doses of genistein. The cultures were extracted to remove unmetabolized E2 and neutral E2 metabolites, and the production of water-soluble (conjugated) E2 metabolites was quantified by liquid scintillation counting. High doses (1 and 10 μM) of genistein inhibited E2 metabolism in the liver and kidney of Atlantic salmon and rainbow trout, and in the liver of lake trout. At 1 μM, daidzein, glycitein, and a mix of genistein/daidzein/glycitein at a ratio typically found in soybeans (1.3:1:0.2), all inhibited estrogen metabolism in the liver of rainbow trout, but had no effect on the kidney. The results suggest that the inhibition of hepatic and renal E2 metabolism may be, at least in part, a mechanism mediating the estrogenic effects of isoflavones in salmonid fish.

Development of Cell Cultures Derived from Lake Trout Liver and Kidney in a Hormone-Supplemented, Serum-Reduced Medium

Abstract Primary cultures of liver cells and kidney cells of lake trout Salvelinus namaycush were initiated in a hormone-supplemented, serum-reduced medium in plastic dishes. Cells plated on fibronectin-coated plastic attached with high efficiency (87–93%), in contrast to cells cultured on uncoated or polymer-coated plastic (< 8–52%). Primary cultures were obtained from young lake trout by trypsin-EDTA dissociation at 10–15°C for 40 min; the primary cultures were maintained in a growth medium (either medium 199 or Leibovitz's L-15 medium) supplemented with 2–3% fetal bovine serum, epidermal growth factor, bovine serum albumin, cholera toxin, insulin, transferrin, and selenium. Lake trout liver and kidney cultures currently contain both epithelioid and fibroblastic cells. The cells, cultivated at 18–20°C from primary culture, have been subcultured 32 times over a period of 6 months.

Branched-chain amino acid aminotransferase activity in the tissues of lake trout

1. 1. The enzyme branched-chain amino acid aminotransferase (BCAT) was found in five tissues of fingerling lake trout, Salvelinus namaycush, (listed in order of decreasing tissue specific activity): posterior kidney, skeletal muscle, gill, liver, and anterior kidney. 2. 2. This pattern is consistent with that found in other animals. 3. 3. The results of this study seem to indicate that BCAT in the liver of lake trout has a higher specific activity than that of the rat and that the specific activity is higher in both the liver and skeletal muscle than it is in these organs of the chick.

Subcellular distribution of mercury in liver of lake trout (Salvelinus namaycush).

Mercury was found primarily (80%) in the submicroscopic material (magnification x<7700) of environmentally exposed adult lake trout from Lake Michigan, USA.

Lactate dehydrogenases in poikilotherms: Definition of a complex isozyme system

1. 1. Five kinds of subunits (A—E) are involved in generating 14 isozymes of lactic dehydrogenase (LDH) in skeletal muscle of Salmo namaycush and S. fontinalis. One group of 5 LDHs, migrating near the origin at neutral pH, is derived from random assembly of D and E subunits into tetramers. Another essentially independent series of 9 LDHs, migrating anodally at neutral pH, are formed by a non-random assembly of subunits A, B and C to yield A 4, A 3B 1, A 2B 2, A 1B 3, B 4, B 3C 1, B 2C 2, B 1C 3 and C 4. 2. 2. Liver of lake trout contains 5 LDHs homologous to the A—B series in muscle, but only the homopolymer B 4 is abundant. Brook trout liver has the B—C series with only C 4 in abundance. Liver of S. gairdneri has only the A 4 homologue. 3. 3. The LDHs formed in dissociation-reassociation tests and in interspecies crosses are consistent with the 5-subunit model advanced.