RTgill-W1 Cells Research Articles

How relevant are sterols in the mode of action of prymnesins?

Prymnesins, produced by the haptophyte Prymnesium parvum, are considered responsible for fish kills when this species blooms. Although their toxic mechanism is not fully understood, membrane disruptive properties have been ascribed to A-type prymnesins. Currently it is suggested that pore-formation is the underlying cause of cell disruption. Here the hypothesis that A-, B-, and C-type prymnesins interact with sterols in order to create pores was tested. Prymnesin mixtures containing various analogs of the same type were applied in hemolysis and cytotoxicity assays using Atlantic salmon Salmo salar erythrocytes or rainbow trout RTgill-W1 cells. The hemolytic potency of the prymnesin types reflected their cytotoxic potential, with approximate concentrations reaching 50 % hemolysis (HC50) of 4 nM (A-type), 54 nM (C-type), and 600 nM (B-type). Variabilities in prymnesin profiles were shown to influence potency. Prymnesin-A (3 Cl) + 2 pentose + hexose was likely responsible for the strong toxicity of A-type samples. Co-incubation with cholesterol and epi-cholesterol pre-hemolysis reduced the potential by about 50 % irrespective of sterol concentration, suggesting interactions with sterols. However, this effect was not observed in RTgill-W1 toxicity. Treatment of RTgill-W1 cells with 10 µM lovastatin or 10 µM methyl-β-cyclodextrin-cholesterol modified cholesterol levels by 20-30 %. Regardless, prymnesin cytotoxicity remained unaltered in the modified cells. SPR data showed that B-type prymnesins likely bound with a single exponential decay while A-types seemed to have a more complex binding. Overall, interaction with cholesterol appeared to play only a partial role in the cytotoxic mechanism of pore-formation. It is suggested that prymnesins initially interact with cholesterol and stabilize pores through a subsequent, still unknown mechanism possibly including other membrane lipids or proteins.

Mixtures of legacy and replacement perfluorosulphonic acids (PFSAs) demonstrate ratio-, concentration- and endpoint-dependent synergistic interactions in vitro

The extensive use of poly- and per-fluoroalkyl substances (PFASs) has les to their widespread presence in the environment, raising concerns about potential toxicity. While certain PFASs of concern have been phased-out or banned, new PFASs continue to be produced. Two such substances are perfluoroethylcyclohexane sulphonate (PFECHS) and perfluorobutane sulphamide (FBSA), replacements of perfluoroctanesulphonic acid (PFOS) that have recently been detected in multiple environmental media around the globe. Despite PFASs generally occurring in the environment as mixtures, few data are available outlining the effects of PFAS mixtures. Therefore, this research investigated the interaction potential of binary and ternary mixtures of emerging and legacy PFASs. The immortalized rainbow trout gill cell line (RTgill-W1) was chosen as the experimental model to investigate two apical endpoints: cytotoxicity and phospholipidosis. RTgill-W1 cells were exposed for 24 h to each compound to obtain endpoint-specific effect concentrations (LCx; ECx). These values were then applied to formulate mixture predictions following the Loewes Additivity and Steel and Peckham methods. Based on cytotoxicity, relative potencies of individual compounds were: PFOS > PFECHS > FSBA. PFOS and PFECHS had nearly identical effects on phospholipidosis, while FSBA did not have any effects. Most mixtures had a synergistic effect on cytotoxicity, but the effect was both dose- and ratio-dependent. PFOS and PFECHS were additive at lower concentrations (LC10) and synergistic at higher concentrations (LC50; 3:1, 1:1, and 1:3). PFECHS and FSBA mixtures were synergistic at all doses and ratios (3:1, 1:1, 1:3), while FBSA and PFOS were mainly synergistic at higher concentrations and at ratios favouring PFOS (1:1, 1:3). Tertiary combinations were mainly synergistic. For phospholipidosis, mixtures were strictly additive. These results are strongly suggestive of synergism between emerging PFAS replacements and highlight that independent apical mechanisms of different PFASs could combine to induce unexpected toxicity. Considering that emerging replacements are continuing to increase in concentration in the environment, such mixture scenarios are also likely to continue to increase in probability.

Cytotoxicity of 19 Pesticides in Rainbow Trout Gill, Liver, and Intestinal Cell Lines.

The rainbow trout gill cell line (RTgill-W1), via test guideline 249 of the Organisation for Economic Co-operation and Development, has been established as a promising New Approach Methodology, although to advance confidence in the method more case studies are needed that: 1) expand our understanding of applicability domains (chemicals with diverse properties); 2) increase methodological throughput (96-well format); and 3) demonstrate biological relevance (in vitro to in vivo comparisons; gill vs. other cells). Accordingly, the objective of our study was to characterize the cytotoxicity of 19 pesticides against RTgill-W1 cells, and also liver (RTL-W1) and gut epithelial (RTgutGC) cell lines, and then to compare the in vitro and in vivo data. Of the 19 pesticides tested, 11, 9, and 8 were cytotoxic to the RTgill-W1, RTL-W1, and RTgutGC cells, respectively. Six pesticides (carbaryl, chlorothalonil, chlorpyrifos, dimethenamid-P, metolachlor, and S-metolachlor) were cytotoxic to all three cell lines. Aminomethylphosphonic acid, chlorantraniliprole, dicamba, diquat, imazethapyr, and permethrin exhibited cell-line-specific toxicity. No cytotoxic responses were observed for three herbicides (atrazine, glyphosate, and metribuzin) and four insecticides (clothianidin, diazinon, imidacloprid, and thiamethoxam). When cytotoxicity was measured, there was a strong correlation (rs = 0.9, p < 0.0001) between in vitro median effect concentration (EC50) values (based on predicted concentrations using the In Vitro Mass Balance Model Equilibrium Partitioning (IV-MBM EQP) Ver. 2.1) derived from RTgill-W1 and RTL-W1 cells with in vivo median lethal concentration (LC50) values from 96-h acute toxicity studies with trout. In all 28 cases, the in vitro EC50 was within 18-fold of the in vivo LC50. These data help increase our understanding of the ecotoxicological domains of applicability for in vitro studies using cultured rainbow trout cells, while also demonstrating that these assays performed well in a 96-well format and have promise to yield data of biological relevance. Environ Toxicol Chem 2024;00:1-13. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Cytotoxicity of Prymnesium parvum extracts and prymnesin analogs on epithelial fish gill cells RTgill-W1 and the human colon cell line HCEC-1CT.

Harmful algal blooms kill fish populations worldwide, as exemplified by the haptophyte microalga Prymnesiumparvum. The suspected causative agents are prymnesins, categorized as A-, B-, and C-types based on backbone carbon atoms. Impacts of P.parvum extracts and purified prymnesins were tested on the epithelial rainbow trout fish gill cell line RTgill-W1 and on the human colon epithelial cells HCEC-1CT. Cytotoxic potencies ranked A > C > B-type with concentrations spanning from low (A- and C-type) to middle (B-type) nM ranges. Although RTgill-W1 cells were about twofold more sensitive than HCEC-1CT, the cytotoxicity of prymnesins is not limited to fish gills. Both cell lines responded rapidly to prymnesins; with EC50 values for B-types in RTgill-W1 cells of 110 ± 11nM and 41.5 ± 0.6nM after incubations times of 3 and 24h. Results of fluorescence imaging and measured lytic effects suggest plasma membrane interactions. Postulating an osmotic imbalance as mechanisms of toxicity, incubations with prymnesins in media lacking either Cl-, Na+, or Ca2+ were performed. Cl- removal reduced morphometric rearrangements observed in RTgill-W1 and cytotoxicity in HCEC-1CT cells. Ca2+-free medium in RTgill-W1 cells exacerbated effects on the cell nuclei. Prymnesin composition of different P.parvum strains showed that analog composition within one type scarcely influenced the cytotoxic potential, while analog type potentially dictate potency. Overall, A-type prymnesins were the most potent ones in both cell lines followed by the C-types, and lastly B-types. Disturbance of Ca2+ and Cl- ionoregulation may be integral to prymnesin toxicity.

Alternatives to Fish Acute Whole Effluent Toxicity (WET) Testing: Predictability of RTgill-W1 Cells and Fathead Minnow Embryos with Actual Wastewater Samples.

Toxicity assays using fish cells and embryos continue to gain momentum as a more ethical and informative alternative to fish acute toxicity testing. The goal of our study was to test the accuracy of RTgill-W1 cells and the fathead minnow (Pimephales promelas) embryos to predict actual whole effluent toxicity (WET) in the fathead minnow larvae. The three models were compared concurrently using samples of various origins and treatment types. Additionally, the toxicity of reference toxicants (Cd, Cu, NH3-N, 3,4-dichloraniline, and benzalkonium chloride) spiked into a nontoxic wastewater was compared. The toxicity of reference toxicants was tested in isosmotic and hypoosmotic exposure media in RTgill-W1 cells. Of the 28 wastewater samples, 14 induced a toxic response in fish larvae. Embryos predicted 11 of the 14 wastewater samples toxic to the larvae, whereas RTgill-W1 cells predicted the toxicity of all 14 toxic samples to the larvae. In addition, embryos and RTgill-W1 cells predicted toxicity in two and six additional samples, respectively, that were nontoxic to larvae. Exposures in hypoosmotic medium significantly increased sensitivity of RTgill-W1 cells to all reference toxicants, excluding benzalkonium chloride, compared to exposures in isosmotic medium and showed toxicity levels similar to that in larvae. Thus, hypoosmotic exposure medium should be considered for aquatic toxicity testing applications. Overall, both gill cell and embryo models predicted toxicity in the majority of wastewater samples toxic to larvae and demonstrated their applicability for regulatory WET testing.

Characterization of RTgill-W1 cells epithelial development on transwell inserts: Evaluation of osmotic and toxic challenges

RTgill-W1 cells cannot be directly exposed to freshwater (FW) or seawater (SW) due to osmotic stress. Adjustments of exposure solutions are needed, but these might reduce the bioavailability and toxicity of pollutants. To facilitate cell polarization and allow direct exposure of water samples, cells were cultured on transwell inserts. Monolayer formation was measured by trans-epithelial electrical resistance (TEER) and an apparent permeability (Papp) assay. At 14 days both TEER and Papp indicated the lowest permeability. Cell viability showed that cells can tolerate apical FW with complete medium (L-15/FBS) in the basolateral compartment but SW reduced cell viability. However, when reference toxicants, silver nitrate and sodium dodecyl benzene sulfonate, were added no toxicity was detected. Increased osmolality in the apical side and presence of proteins indicated diffusion from the basolateral to the apical side. Thus, reduced toxicity was likely caused by complexation with media salts and amino acids. A protein and amino acid free exposure medium (L-15/ex) was applied in the basolateral compartment. However, FW exposures with basolateral L-15/ex resulted in reduced cell viability. To reduce osmotic stress, mannitol was added to apical FW maintaining basolateral L-15/ex which improved cell viability and allowed detection of silver toxicity. Finally, RTgill-W1 cells did not show normal tight junction protein (ZO-1) immunocytochemical staining, which fits with the formation of a leaky epithelium. Overall, culturing of RTgill-W1 cells on transwell inserts allowed direct exposure to mannitol FW medium but showed a reduced sensitivity to toxicants. Thus, exposure on flat bottom wells is recommended for routine toxicity testing.

Cecropin P1 antimicrobial peptide modulates differential expression of immune relevant genes in rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1

Accumulated evidence indicates that antimicrobial peptides modulate immune activities in fish. In this study, we profiled the differential expression patterns of representative immune relevant genes in an epithelial-like cell line of rainbow trout gill, RTgill-W1, in response to exposure of cecropin P1 antimicrobial peptide. RTgill-W1 cells were treated with synthetic cecropin P1 over time (0, 2, 4 and 24 h) with or without the present of lipopolysaccharide (LPS) or polyinosinic polycytidylic acid (PolyI:C). The relative abundances of each mRNA were measured by real-time quantitative PCR. The dose-response study revealed significant perturbations of mRNA levels of genes related to pro-inflammation, acute phase, surface proteins and transcription factors at 30 μM of cecropin P1. In addition, cecropin P1 altered the differential expression patterns that were induced by LPS or PolyI:C, at different time points in RTgill-W1. Overall, our results indicate that cecropin P1 exhibits pro-inflammation activity, modulate cell-cell interaction and cytokine signal transduction in rainbow trout gill cell, and may suggest a potential application of this peptide as an immune adjuvant for disease control in aquaculture.

Differential response of RTGUTGC and RTGILL-W1 rainbow trout epithelial cell lines to viral stimulation.

Mucosal surfaces constitute the main route of entry of pathogens into the host. In fish, these mucosal tissues include, among others, the gastrointestinal tract, the gills and the skin. However, knowledge about the mechanisms of regulation of immunity in these tissues is still scarce, being essential to generate a solid base that allows the development of prevention strategies against these infectious agents. In this work, we have used the RTgutGC and RTgill-W1 epithelial-like cell lines, derived from the gastrointestinal tract and the gill of rainbow trout (Oncorhynchus mykiss), respectively, to investigate the transcriptional response of mucosal epithelial cells to a viral mimic, the dsRNA poly I:C, as well as to two important viral rainbow trout pathogens, namely viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Additionally, we have established how the exposure to poly I:C affected the susceptibility of RTgutGC and RTgill-W1 cells to both viruses. Our results reveal important differences in the way these two cell lines respond to viral stimuli, providing interesting information on these cell lines that have emerged in the past years as useful tools to study mucosal responses in fish.

TRPV1 Receptor Identification in Rainbow Trout (Oncorhynchus mykiss) and Evaluation of the Effects Produced by Ocimum basilicum Super Critical Fluid Extract

Transient receptor potential vanilloid type 1 (TRPV1) has been investigated in humans and mammals; in recent years, some researchers have focused on this receptor in fishes. The present study aimed to identify TRPV1 receptors in cultures of RT-gill W1 cells and in the organs of rainbow trout (Oncorhynchus mykiss), in addition to evaluating the possible modulation induced by super critical fluid extract of basil (Ocimum basilicum), named F1-BEO. In vitro evaluation consisted of cell cultures and immunocytochemistry assays. During in vivo experimental sessions, eighty trout were divided into five groups that received a fish diet supplemented with 0, 0.5, 1, 2 and 3% w/w F1-BEO. Forty trout were euthanized after 15 and 30 days; organs were collected and processed according to the immunohistochemistry technique. Receptor expression was quantitatively measured using Image Pro Plus software. TRPV1 was identified in RT-gill W1 cells and in all organs, with a higher positivity in the muscle layers of the stomach, intestine and kidneys. F1-BEO induced an increased expression of TRPV1 in the stomach while a lower expression was appreciated in the bowel. No morphological alterations have been highlighted in the liver or kidneys. Further investigation will be necessary to evaluate the functionality of this receptor in rainbow trout.

Characterization of Rtgill-W1 Cells Epithelial Development on Transwell Inserts: Evaluation of Osmotic and Toxic Challenges

Characterization of Rtgill-W1 Cells Epithelial Development on Transwell Inserts: Evaluation of Osmotic and Toxic Challenges

The gill epithelial cell lines RTgill-W1, from Rainbow trout and ASG-10, from Atlantic salmon, exert different toxicity profiles towards rotenone

In order to ensure the proper use and interpretation of results from laboratory test systems, it is important to know the characteristics of your test system. Here we compare mitochondria and the handling of reactive oxygen species (ROS) in two gill epithelial cell lines, the well-known RTgill-W1 cell line from Rainbow trout and the newly established ASG-10 cell line from Atlantic salmon. Rotenone was used to trigger ROS production. Rotenone reduced metabolic activity and induced cell death in both cell lines, with RTgill-W1 far more sensitive than ASG-10. In untreated cells, the mitochondria appear to be more fragmented in RTgill-W1 cells compared to ASG-10 cells. Furthermore, rotenone induced mitochondrial fragmentation, reduced mitochondria membrane potential (Δψm) and increased ROS generation in both cell lines. Glutathione (GSH) and catalase is important to maintain the cellular oxidative balance by eliminating hydrogen peroxide (H2O2). In response to rotenone, both GSH and catalase depletion were observed in the RTgill-W1 cells. In contrast, no changes were found in the GSH levels in ASG-10, while the catalase activity was increased. In summary, the two salmonid gill cell lines have different tolerance towards ROS, probably caused by differences in mitochondrial status as well as in GSH and catalase activities. This should be taken into consideration with the selection of experimental model and interpretation of results.Graphical abstract

Fish Embryo Acute Toxicity Testing and the RTgill-W1 Cell Line as In Vitro Models for Whole-Effluent Toxicity (WET) Testing: An In Vitro/In Vivo Comparison of Chemicals Relevant for WET Testing.

The fathead minnow (Pimephales promelas) fish embryo acute toxicity (FET) test was compared to the fish gill cells (RTgill-W1) in vitro assay and to the fish larvae acute toxicity test to evaluate their sensitivity for whole-effluent toxicity (WET) testing. The toxicity of 12 chemicals relevant for WET testing was compared as proof of principle. The concentrations lethal to 50% of a population (LC50) of embryos were compared to those in fish larvae and to the 50% effect concentration (EC50) in RTgill-W1 cells from previous literature. Along with traditional FET endpoints (coagulation, somite development, tail detachment, and heartbeat), cardiotoxicity was evaluated for WET applicability. Heart rate was measured at LC20 and LC50 values of six subselected chemicals (Cd, Cu, Ni, ammonia, 3,4-dichloraniline, and benzalkonium chloride). In addition, the toxicity of Cd and Ni was evaluated in RTgill-W1 cells exposed in a hypoosmotic medium to evaluate the effect that osmolarity may have on metal toxicity. A significant correlation was found between the FET and larvae LC50 values but not between the RTgill-W1 EC50 and FET LC50 values. Although sensitivity to Ni and Cd was found to increase with hypoosmotic conditions for FET and RTgill-W1 cells, a correlation was only found with removal of Ni from the analysis. Hypoosmotic conditions increased sensitivity with a significant correlation between RTgill-W1 cells and larvae. Cardiotoxicity was shown in three of the five subselected chemicals (Cd, Cu, and 3,4-dichloroaniline). Overall, both in vitro alternative models have shown good predictability of toxicity in fish in vivo for WET chemicals of interest. Environ Toxicol Chem 2022;41:2721-2731. © 2022 SETAC.

Toxicity of lanthanides on various fish cell lines.

The growing use of Lanthanides in new technologies has increased their anthropogenic releases into the aquatic environment over the last decades. However, knowledge on their ecotoxicological impacts is still incomplete, especially with regard to biological effects of Lanthanides mixtures and the possible regular variation in toxicity along the Lanthanides series. The present study evaluated the individual toxicity of all Lanthanides and the toxicity of mixtures of three of them, namely Neodymium (Nd3+), Gadolinium (Gd3+), and Ytterbium (Yb3+) on Danio rerio fibroblast-like cells (ZF4). Individual and mixtures toxicity of Neodymium (Nd3+) and Ytterbium (Yb3+) were also assessed on Danio rerio hepatic cells (ZFL) and Oncorhynchus mykiss epithelial cells (RTgill-W1). The measured Lanthanide concentrations were close to the nominal ones in the culture media of ZF4, ZFL, and RTgill-W1 cells (85-99%). A toxic impact was observed on the three fish cell lines exposed to all Lanthanides tested individually. RTgill-W1 appeared as the less sensitive cells, compared to the two others. Four Lanthanides, Erbium (Er3+), Thulium (Tm3+), Ytterbium (Yb3+) and Lutetium (Lu3+) showed a higher toxicity than the others on ZF4 cells but no correlation could be established between the toxicity of Lanthanides and the order of the elements within the Lanthanides series. Exposures to binary mixtures highlighted the presence of synergistic effects on cell viability for all cell lines.

Innate response of rainbow trout gill epithelial (RTgill-W1) cell line to ultraviolet-inactivated VHSV and FliC and rhabdovirus infection

Gills reportedly play a crucial role in induction of an antiviral immune response in fish. We investigated the expression of innate response genes in the rainbow trout gill epithelial cell line RTgill-W1 36 h after pretreatment with ultraviolet-inactivated viral hemorrhagic septicemia virus (UV-VHSV), flagellin C protein from Edwardsiella tarda (FliC), VHSV and SVCV using an Agilent 4 × 44k cGRASP salmonid microarray. RTgill-W1 cells pretreated with UV-VHSV, triggered an independent gene expression profile from those treated with a recombinant flagellin C protein from Edwardsiella tarda. In addition, exposure of RTgill-W1 cells to live viruses spring viremia of carp virus and viral hemorrhagic septicemia virus induced a less robust transcriptional change of 24 and 22 gene probes, respectively, when compared to 123 genes for UV-VHSV. Further the pretreatment of RTgill-W1 cells with (UV-VHSV) significantly reduced VHSV genome copy number at 6 d post infection (dpi) relative to the FliC-treated and untreated control. A quantitative PCR was used to study the transcriptional modulation of a set of 25 innate immune-related genes highlighted by the microarray data and a panel of 7 established antiviral genes in the protected cells. Notably, the expression of ifn1, ifn2, mx1 and mx3 were expressed more in untreated cells than in UV-VHSV-treated cells where virus replication was inhibited. The results from this study shed light on the mechanisms and pathways used by teleost gill epithelium innate immunity in combating viral and bacterial infection.

Applications of the RTgill-W1 Cell Line for Acute Whole-Effluent Toxicity Testing: In Vitro-In Vivo Correlation and Optimization of Exposure Conditions.

The cell line RTgill-W1 was evaluated as an in vitro alternative model for acute fish whole-effluent toxicity (WET) testing. We determined the 50% effective concentration (EC50) that reduces the viability of RTgill-W1 cells for selected toxicants commonly found in effluent samples and correlated those values with the respective 50% lethal concentration (LC50) of freshwater (fathead minnow, Pimephales promelas) and marine (sheepshead minnow, Cyprinodon variegatus) fish species obtained from the literature. Excluding low water-soluble organics and the volatile sodium hypochlorite, significant correlations were measured for metal, metalloids, ammonia, and higher water-soluble organics between in vitro EC50 values and in vivo LC50 values for both species. Typically, toxicity studies with RTgill-W1 cells are conducted by adding salts to the exposure medium, which may affect the bioavailability of toxicants. Osmotic tolerance of RTgill-W1 cells was found between 150 and 450 mOsm/kg, which were set as the hypoosmotic and hyperosmotic limits. A subset of the toxicants were then reexamined in hypoosmotic and hyperosmotic media. Copper toxicity decreased in hyperosmotic medium, and nickel toxicity increased in hypoosmotic and hyperosmotic media. Linear alkylbenzene sulfonate toxicity was not affected by the medium osmolality. Overall, RTgill-W1 cells have shown potential for applications in measuring metal, metalloids, ammonia, and water-soluble organic chemicals in acute WET tests, as well as complementing current toxicity identification and reduction evaluation strategies. In the present study, RTgill-W1 cells have been established as a valid animal alternative for WET testing, and we show that through manipulation of medium osmotic ranges, sensitivity to nickel was enhanced. Environ Toxicol Chem 2021;40:1050-1061. © 2020 SETAC.

Polarized Trout Epithelial Cells Regulate Transepithelial Electrical Resistance, Gene Expression, and the Phosphoproteome in Response to Viral Infection.

The burden of disease is a major challenge in aquaculture production. The fish gill characterized with a large surface area and short route to the bloodstream is a major environmental interface and a significant portal of entry for pathogens. To investigate gill responses to viral infection the salmonid gill cell line RTgill-W1 was stimulated with synthetic dsRNA and the salmonid alphavirus subtype 2 (SAV-2). Epithelial integrity in polarized cells can be measured as transepithelial electrical resistance (TEER) which is defined as the electrical resistance across a cell monolayer. TEER is a widely accepted quantitative measure of cellular integrity of a cell monolayer. TEER increased immediately after stimulation with the synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I:C)). In parallel, tight junction and gene expression of innate immune activation markers was modulated in response to poly(I:C). The SAV-2 virus was found to replicate at a low level in RTgill-W1 cells where TEER was disturbed at an early stage of infection, however, gene expression related to tight junction regulation was not modulated. A strong poly(I:C)-driven antiviral response was observed including increases of Rig-like receptors (RLRs) and interferon stimulating genes (ISGs) mRNAs. At the level of signal transduction, poly(I:C) stimulation was accompanied by the phosphorylation of 671 proteins, of which 390 were activated solely in response to the presence of poly(I:C). According to motif analysis, kinases in this group included MAPKs, Ca2+/calmodulin-dependent kinase (CaMK) and cAMP-dependent protein kinase (PKA), all reported to be activated in response to viral infection in mammals. Results also highlighted an activation of the cytoskeletal organization that could be mediated by members of the integrin family. While further work is needed to validate these results, our data indicate that salmonid gill epithelia has the ability to mount a significant response to viral infection which might be important in disease progression. In vitro cell culture can facilitate both a deeper understanding of the anti-viral response in fish and open novel therapeutic avenues for fish health management in aquaculture.

Prymnesium parvum differentially triggers sublethal fish antioxidant responses in vitro among salinity and nutrient conditions

Significant fish kills have been attributed to Prymnesium parvum in coastal and inland waters around the world. However, specific mechanisms responsible for adverse outcomes resulting from this harmful algal bloom (HAB) species remain unclear, though the gill has previously been identified as an important target organ. In the present study, an in vitro approach was used to examine cytotoxicity and antioxidant responses in fish liver (Hepa-E1 and PLHC-1) and gill (G1B and RTgill-W1) cell lines, following exposure to P. parvum grown at different salinities and nutrient concentrations, which can influence the magnitude of acute toxicity. Cultures from high salinity compromised survival of hepatic cell lines exposed to high dilutions, whereas no significant cytotoxicity was observed for gill cell lines. With respect to control groups, catalase showed significant activity in both gill cell lines, especially RTgill-W1, following exposure to high salinity cultures. High levels of superoxide dismutase were measured in Hepa-E1 cells exposed to all experimental treatment combinations and in RTgill-W1 cells following exposure to high salinity conditions, with respect to non-exposed cells Glutathione peroxidase activity was also detected at significant levels in Hepa-E1 cells after exposure to cultures from high salinity and the low salinity X low nutrients. Slight GPx increases were only observed in PLHC-1 and G1B exposed to P. parvum grown at high salinity. These results suggest that: 1. specific combinations of salinity and nutrient levels may contribute to production and potency of P. parvum toxins resulting in sub-lethal effects, and 2. sub-lethal responses are more prominent than cytotoxicity, and that oxidative stress may be a significant adverse effect of toxins produced by P. parvum.

Repeatability and Reproducibility of the RTgill-W1 Cell Line Assay for Predicting Fish Acute Toxicity.

Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout (Oncorhynchus mykiss) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC50) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings.

Autophagy-related genes in rainbow trout Oncorhynchus mykiss (Walbaum) gill epithelial cells and their role in nutrient restriction.

Autophagy is primarily an adaptive response to provide nutrients and energy following exposure to stress and starvation but can also regulate muscle mass and impact infectious disease susceptibility. Expression of 10 autophagy-related (Atg) genes in rainbow trout was monitored throughout the autophagosome formation cycle. The Atg gene sequences of rainbow trout were compared to other species to identify highly conserved regions and to generate primers. Phylogeny trees created with rainbow trout and 14 other species demonstrate that rainbow trout Atg gene sequences have greatest similarity to Atlantic salmon and other fish species. RTgill-W1 cells were subjected to nutrient restriction and compared to cells in normal nutrient conditions using quantitative reverse transcriptase polymerase chain reaction to assess changes in Atg gene expression. Nutrient restriction had a direct impact on Atg gene expression, with atg4, atg9, atg12, lc3, gabarap and becn1 undergoing the greatest differential expression (p<0.05), most dramatically on Day 3. This was corroborated by Western blot detection of LC3, which also showed a peak of autophagy activity at Day 3 post-nutrient restriction. Atg gene expression revealed autophagy flux in RTgill-W1, as well as, those genes that were most significantly altered by nutrient restriction.

Cellular uptake and intracellular localization of poly (acrylic acid) nanoparticles in a rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1

The ever-growing production of engineered nanoparticles (NPs) for use in many agricultural, commercial, consumer, and industrial applications will lead to their accidental or intentional release into the environment. Potential routes of environmental exposure include manufacturing or transport spills, disposal of NP‐containing products down the drain and/or in landfills, as well as direct usage on agricultural land. Therefore, NPs will inevitably contaminate aquatic environments and interact with resident organisms. However, there is limited information regarding the mechanisms that regulate NP transport into fish from the environment. Thus, our primary objective was to elucidate the mechanism(s) underlying cellular uptake and intracellular fate of 3–9nm poly (acrylic acid) NPs loaded with the fluorescent dye Nile red using a rainbow trout (Oncorhynchus mykiss) gill epithelial cell line (RTgill‐W1). In vitro measurements with NP‐treated RTgill-W1 cells were carried out using a combination of laser scanning confocal microscopy, flow cytometry, fluorescent biomarkers (transferrin, cholera toxin B subunit, and dextran), endocytosis inhibitors (chlorpromazine, genistein, and wortmannin), and stains (4′, 6‐diamidino-2-phenylindole, Hoechst 33342, CellMask Deep Red, and LysoTracker Yellow). Clathrin-mediated endocytosis (CME), caveolae‐mediated endocytosis and macropinocytosis pathways were active in RTgill‐W1 cells, and these pathways were exploited by the non-cytotoxic NPs to enter these cells. We have demonstrated that NP uptake by RTgill‐W1 cells was impeded when clathrin-coated pit formation was blocked by chlorpromazine. Furthermore, colocalization analysis revealed a moderate positive relationship between NPs and LysoTracker Yellow-positive lysosomal compartments indicating that CME was the dominant operative mechanism involved in NP internalization by RTgill-W1 cells. Overall, our results clearly show that fish gill epithelial cells internalized NPs via energy‐dependent endocytotic processes. This study enhances our understanding of complex NP‐cell interactions and the results obtained in vitro imply a potential risk to aquatic organisms.