Intestinal cellular localization of PCNA protein and CYP1A mRNA in Atlantic salmon Salmo salar L. exposed to a model toxicant

<p>Abstract</p> <p>Background</p> <p>The aim of the study was to examine the intestinal cellular localization of proliferating cell nuclear antigen (PCNA) and cytochrome P450 A1 (CYP1A) expression in Atlantic salmon <it>Salmo salar </it>L. exposed to a model toxicant. The stress response was induced by intraperitoneal injection of four salmon with a single dose (50 mg/kg) of the CYP1A inducer β-naphthoflavone (BNF) and intestinal tissue (mid and distal intestine; MI and DI) was sampled seven days later. Samples for histology and gene transcription analysis were collected from four exposed fish and four control fish. PCNA was assessed by immunohistochemistry, CYP1A mRNA was studied by <it>in situ </it>hybridization (ISH) and finally the transcription of five genes was quantified by real-time quantitative RT-PCR (real-time RT-PCR); two detoxifying genes (CYP1A and glutathione S-transferase; GST), a stress marker gene (heat shock protein 70; HSP70), PCNA and a gene marker of apoptosis (caspase 6A).</p> <p>Results</p> <p>PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI) of both experimental groups. At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group. The real-time RT-PCR data, which showed an increased mRNA expression both in the MI and DI of 139- and 62-fold, respectively, confirmed the increased cellular CYP1A mRNA levels detected using ISH. HSP70 expression was also up-regulated in the exposed fish. The other examined genes did not show any differential regulation in the experimental fish group.</p> <p>Conclusion</p> <p>This study showed that CYP1A mRNA had a specific intestinal cellular transcription pattern in Atlantic salmon exposed to BNF. At the cellular level CYP1A mRNA expression was always observed at or around the cell nucleus close to the basolateral cell membrane and at the tissue level CYP1A mRNA expression was most frequently observed in the basal and apex area of the intestinal folds. Taken together, a link between the intestinal detoxification system (CYP1A) and cell renewal system (PCNA) is indicated with these two processes being inversely correlated in BNF exposed fish.</p

Characterization of an Atlantic cod (Gadus morhua) embryonic stem cell cDNA library

<p>Abstract</p> <p>Background</p> <p>The Atlantic cod is an ecologically and economically important North Atlantic fish species and also an emerging aquaculture species. To study gene expression in Atlantic cod embryonic stem (ES) cells, our goal was to generate and analyze expressed sequence tags (ESTs) from an ES cell cDNA library of mRNA consisting of approximately 3,900 ESTs.</p> <p>Results</p> <p>We sequenced 3,935 EST clones using a directional cDNA library made from pooled ES cells harvested at the blastula stage. Quality filtering of these ESTs allowed identification of 2,719 high-quality sequences with an average length of 442 bp containing 368 contigs and 1,276 singletons (1,644 unique sequences). BLASTX searches produced 889 significant (E-value < 10^-3) hits, of which 698 (42.5%) were annotated with Gene Ontology terms (E-value < 10^-6). The number of unknown unique sequences was 946 (57.5%). All the high-quality EST sequences have been deposited in GenBank (GenBank: 2,719 sequences in UniGene library dbEST id: 22,021). Gene discovery and annotations are presented and discussed.</p> <p>Conclusion</p> <p>This set of ESTs represents one of the first attempts to describe mRNA in ES cells from a marine cold-water fish species, and provides a basis for gene expression studies of Atlantic cod ES cells.</p

Selection of reference genes for qRT-PCR examination of wild populations of Atlantic cod Gadus morhua

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Do anesthetics and sampling strategies affect transcription analysis of fish tissues?

<p>Abstract</p> <p>Background</p> <p>The aim of the current examination was to evaluate if sedation and anesthetic treatment techniques affect the quality of RNA extracted from liver, gill, head kidney and brain tissues in Atlantic salmon <it>Salmo salar </it>L. Blood parameters were measured and tissue specimens sampled in six groups of fish; one control group (0 minutes), two groups kept in pure seawater in 90 liter tanks for 30 and 120 minutes, two groups treated with the anesthetic isoeugenol for 30 and 120 minutes, and one group kept in pure seawater for 105 minutes and then anaesthetized with metacaine for 15 minutes. RNA quality was assessed with the NanoDrop ND-1000 spectrophotometer (260/280 and 260/230 nm ratios) and with the Agilent Bioanalyzer (28S/18S ratio and RIN data) in samples either preserved in liquefied nitrogen (N₂) or in RNA<it>later</it>. In addition, the transcriptional levels of two fast-responding genes were quantified in gill and brain tissues.</p> <p>Results</p> <p>The results show that physiological stress during sampling does not affect the quality of RNA extracted from fish specimens. However, prolonged sedation (2 hours) resulted in a metabolic alkalosis that again affected the transcriptional levels of genes involved in ionoregulation and respiration. In gills, <it>Na</it>⁺-<it>K</it>⁺-<it>ATPase α1b </it>was significantly downregulated and <it>hypoxia inducible factor 1 </it>(<it>HIF1</it>) significantly upregulated after two hours of treatment with isoeugenol, suggesting that this commonly used sedative affects osmo-regulation and respiration in the fish. The results also suggest that for tissue preservation in general it is better to flash-freeze fish specimens in liquefied N₂than to use RNA<it>later</it>.</p> <p>Conclusion</p> <p>Prolonged sedation may affect the transcription of fast-responding genes in tissues of fish. Two hours of sedation with isoeugenol resulted in downregulation of the <it>Na</it>⁺-<it>K</it>⁺-<it>ATPase α1b </it>gene and upregulation of the <it>HIF1 </it>gene in gills of Atlantic salmon. The quality of RNA extracted from tissue specimens, however, was not affected by sedation treatment. Flash-freezing of tissue specimens seems to be the preferred preservation technique, when sampling fish tissue specimens for RNA extraction.</p

Spatial transcription of CYP1A in fish liver

<p>Abstract</p> <p>Background</p> <p>The aim of this work was to study how evenly detoxifying genes are transcribed spatially in liver tissue of fish. Ten Atlantic salmon <it>Salmo salar </it>were intraperitoneally injected with 50 mg/kg of the strong CYP1A inducer β-naphthoflavone and liver tissue harvested seven days later. The liver from 10 control and 10 exposed fish were split into eight sections, RNA extracted and three reference (β-actin, elongation factor 1A_B(EF1A_B)) and two detoxifying genes (CYP1A and GST) quantified with real-time RT-PCR. The cellular localization of the EF1A_Band CYP1A mRNA in the liver of control and β-naphthoflavone treated fish was then determined by <it>in situ </it>hybridization (ISH) using EF1A_Band CYP1A biotinylated oligonucleotide probes.</p> <p>Results</p> <p>The study shows that genes encoding phase I and phase II conjugating enzymes are unevenly transcribed in different parts of the liver of Atlantic salmon seven days after a single-dose of β-naphthoflavone exposure. Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ. The ISH data suggest that CYP1A transcription happens mainly in hepatocyte cells in the liver, and that hepatocytes in the vicinity of blood vessels respond stronger to β-naphthoflavone than cells further away from the blood supply.</p> <p>Conclusion</p> <p>Overall, the qRT-PCR and ISH results reported here suggest that gene expression analysis should be performed on as pure cell populations as possible. If bulk tissue samples are to be used, one should always check how evenly the target genes are expressed in tissue sections and organs in every study.</p

Ontogeny-Specific Skeletal Deformities in Atlantic Haddock Caused by Larval Oil Exposure

Bone deformities are one of the main effects of crude oil exposure in marine fish larvae. Craniofacial and jaw deformities, if severe enough, may restrict feeding and ultimately kill the developing larvae. This study aimed to examine the impact of dispersed crude oil on bone development in Atlantic haddock (Melanogrammus aeglefinus) larvae, a fish species spawning in areas approached for oil and gas exploration in the North Atlantic Ocean. Atlantic haddock larvae were exposed to low (60 μg oil/L), high (600 μg oil/L), or pulsed (0–600, average 60 μg oil/L over time) dispersed crude oil from 0 to 18 days post hatch (dph). Endpoints included survival and growth, bone integrity, and transcriptional parameters, which were assessed during (0–18 dph) and after exposure until the fish reached 8 months of age (243 dph). The results showed that the larvae in the high treatment group had reduction in growth at 2–19, 44, 134, and 243 dph. Craniofacial abnormalities were most severe at 8 and 19 dph. These deformities were not present at 44 dph, possibly because the larvae with deformed jaws failed to feed properly and died. Higher prevalence of spinal deformities was observed in haddocks that survived for 243 dph. Three genes encoding proteins critical for osteoblast function, sp7, postn, and col10a1, were downregulated in the high treatment group larvae. We discuss possible mechanisms of action in the developing larvae after oil exposure. In conclusion, this study shows that larval exposure to oil can potentially have long-term effects on growth and bone integrity in Atlantic haddock.publishedVersio

Genome-wide transcription analysis of histidine-related cataract in Atlantic salmon (Salmo salar L)

Purpose: Elevated levels of dietary histidine have previously been shown to prevent or mitigate cataract formation in farmed Atlantic salmon (Salmo salar L). The aim of this study was to shed light on the mechanisms by which histidine acts. Applying microarray analysis to the lens transcriptome, we screened for differentially expressed genes in search for a model explaining cataract development in Atlantic salmon and possible markers for early cataract diagnosis. Methods: Adult Atlantic salmon (1.7 kg) were fed three standard commercial salmon diets only differing in the histidine content (9, 13, and 17 g histidine/kg diet) for four months. Individual cataract scores for both eyes were assessed by slitlamp biomicroscopy. Lens N-acetyl histidine contents were measured by high performance liquid chromatography (HPLC). Total RNA extracted from whole lenses was analyzed using the GRASP 16K salmonid microarray. The microarray data were analyzed using J-Express Pro 2.7 and validated by quantitative real-time polymerase chain reaction (qRT–PCR). Results: Fish developed cataracts with different severity in response to dietary histidine levels. Lens N-acetyl histidine contents reflected the dietary histidine levels and were negatively correlated to cataract scores. Significance analysis of microarrays (SAM) revealed 248 significantly up-regulated transcripts and 266 significantly down-regulated transcripts in fish that were fed a low level of histidine compared to fish fed a higher histidine level. Among the differentially expressed transcripts were metallothionein A and B as well as transcripts involved in lipid metabolism, carbohydrate metabolism, regulation of ion homeostasis, and protein degradation. Hierarchical clustering and correspondence analysis plot confirmed differences in gene expression between the feeding groups. The differentially expressed genes could be categorized as “early” and “late” responsive according to their expression pattern relative to progression in cataract formation. Conclusions: Dietary histidine regimes affected cataract formation and lens gene expression in adult Atlantic salmon. Regulated transcripts selected from the results of this genome-wide transcription analysis might be used as possible biological markers for cataract development in Atlantic salmon

Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon

BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1A(A )and EF1A(B)) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1A(B)>EF1A(A)>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1A(B)>EF1A(A)>S20>β-actin>18S rRNA>GAPDH. CONCLUSION: Overall, this work suggests that the EF1A(A )and EF1A(B )genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon

A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts

To avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon.info:eu-repo/semantics/publishedVersio

Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Goksoyr, S. O., Goldstone, J., Lille-Langoy, R., Lock, E.-J., Olsvik, P. A., Goksoyr, A., & Karlsen, O. A. Polycyclic aromatic hydrocarbons modulate the activity of Atlantic cod (Gadus morhua) vitamin D receptor paralogs in vitro. Aquatic Toxicology, 238, (2021): 105914, https://doi.org/10.1016/j.aquatox.2021.105914.Vitamin D receptor (VDR) mediates the biological function of the steroid hormone calcitriol, which is the metabolically active version of vitamin D. Calcitriol is important for a wide array of physiological functions, including calcium and phosphate homeostasis. In contrast to mammals, which harbor one VDR encoding gene, teleosts possess two orthologous vdr genes encoding Vdr alpha (Vdra) and Vdr beta (Vdrb). Genome mining identified the vdra and vdrb paralogs in the Atlantic cod (Gadus morhua) genome, which were further characterized regarding their phylogeny, tissue-specific expression, and transactivational properties induced by calcitriol. In addition, a selected set of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, fluorene, pyrene, chrysene, benzo[a]pyrene (BaP), and 7-methylbenzo[a]pyrene, were assessed for their ability to modulate the transcriptional activity of gmVdra and gmVdrb in vitro. Both gmVdra and gmVdrb were activated by calcitriol with similar potencies, but gmVdra produced significantly higher maximal fold activation. Notably, none of the tested PAHs showed agonistic properties towards the Atlantic cod Vdrs. However, binary exposures of calcitriol together with phenanthrene, fluorene, or pyrene, antagonized the activation of gmVdra, while chrysene and BaP significantly potentiated the calcitriol-mediated activity of both receptors. Homology modeling, solvent mapping, and docking analyses complemented the experimental data, and revealed a putative secondary binding site in addition to the canonical ligand-binding pocket (LBP). Calcitriol was predicted to interact with both binding sites, whereas PAHs docked primarily to the LBP. Importantly, our in vitro data suggest that PAHs can interact with the paralogous gmVdrs and interfere with their transcriptional activities, and thus potentially modulate the vitamin D signaling pathway and contribute to adverse effects of crude oil and PAH exposures on cardiac development and bone deformities in fish.This study was funded by the Research Council of Norway through the ”iCod 2.0: Integrative environmental genomics of Atlantic cod” project (project no. 244564) and the ”dCod 1.0: decoding systems toxicology of Atlantic cod” project (Center for Digital Life Norway project no. 248840)