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Gills are unique structures involved in respiration and osmoregulation in piscinids as well as in many aquatic invertebrates. The availability of the trout-derived gill cell line, RTgill-W1, is beginning to make impacts in fish health and toxicology. These cells are available from the American Type Culture Collection as ATCC CRL 2523. The cells hav...
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... the 12 gill cell lines reported to date (Table 2), none have been described that were initiated from neoplastic or cancerous tissues and only the FG-9307 gill cell line has been reported to undergo spontaneous neoplastic transfor- mation in vitro ( Guo et al. 2003). Nevertheless, literature on the use of fish gill cell lines have been scarce and most work performed to date involved two gill cell lines: RTgill- W1 derived from gill explants of adult rainbow trout (Oncorhynchus mykiss) ( Bols et al. 1994), is representative of gills from freshwater species; whereas, FG-9307, derived from flounder (Paralichthys olivaceus) (Tong et al. 1997), is representative of gills from marine fish. ...
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... Fish cell lines have been widely used for the study of biology and disease in salmon [20]. One of the most useful cell lines used in salmon for immunological studies is SHK-1, which has contributed to the study of viruses [21], bacteria [22], parasites [23], and iron metabolism [24], among others. ...
In this study, we investigated the function of a gene associated with iron metabolism using CRISPR-Cas9 and RNA sequencing in SHK-1 salmon cells. Our objective was to understand how different guide RNA (gRNA) sequences against the transferrin gene tf could influence gene expression and cellular processes related to iron uptake. RNA-Seq analysis was performed to evaluate the transcriptomic effects of two distinct gRNA targets with high knock-out (KO) efficiencies for the targeted tf gene in the SHK-1 genome. Our results showed no significant differential expression in transferrin-related transcripts between wild-type and CRISPR-edited cells; however, there were major differences between their transcriptomes, indicating complex transcriptional regulation changes. Enrichment analysis highlighted specific processes and molecular functions, including those related to the nucleus, cytoplasm, and protein binding. Notably, different sgRNAs targeting tf might result in different mutations at DNA levels in SHK-1 salmon cells.
... Fish gill epithelial cells are an appropriate in vitro alternative for toxicity testing since the gill is the first organ to be exposed and absorb toxicants [9]. This in vitro model serves as a replacement to whole animals in toxicity testing of pollutants in water [25]. ...
Lates calcarifer (Bloch) is a potential candidate fish species for culture in marine and brackishwater. A continuous gill cell line was derived from L. calcarifer by the explant culture method and has been passaged for 132 times, in Leibovitz’s L-15 medium containing 10% fetal bovine serum (FBS) at 28 °C. The cells showed a rate of recovery between 90 and 95% after being successfully cryopreserved at various passage levels and formed monolayer in 2–3 days without any morphological changes. Immunophenotypic analysis of the SBG cell line revealed that they are of epithelial origin. Polymerase chain reaction assay using mitochondrial 12S rRNA primer specific to L. calcarifer was used to confirm the authenticity of the established gill cell line origin from seabass. The transfection efficiency was evaluated in Seabass Gill (SBG) cell line using pEGFP-N1 and Lipofectamine™ 3000. Transfection efficiency was found to be between 13 and 16%. The cytotoxicity of three different metal detecting probes was evaluated by MTT and Alamar blue assays to determine safe concentration. The result revealed that SBG cell line can be applied for recognition of metals using probes. The current study established, for the first time, a gill-derived cell line (SBG) from Lates calcarifer and its application for the detection of intracellular indium, mercury, and lutetium ions by specific fluorescent probes.
... Importantly, RTgill-W1 cells lack the characteristics of pavement cells and ionocytes, for they are apparently derived from undifferentiated gill precursor stem cells (Bols et al. 1994;Lee et al. 2009). The ion regulation of this cell line must thereby differ, at least to a certain degree, from the physiological functions described for rainbow trout gill cells. ...
... Specifically, because Na + and Cl − regulation is often strongly interspersed (Hwang et al. 2011;Marshall 2002). Seeing how gills are vital organs for osmoregulation, the effect PRMs have on the ionoregulation is either a result of PRM toxicity or integral to the toxic mode of action or PRMs (Hwang et al. 2011;Lee et al. 2009). Further studies are needed to better understand this correlation. ...
Harmful algal blooms kill fish populations worldwide, as exemplified by the haptophyte microalga Prymnesium parvum. 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 ± 11 nM and 41.5 ± 0.6 nM after incubations times of 3 and 24 h. 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.
... mykiss), and RTgill-W1, with which, in addition to RTG-2, specific toxicity reactions for individual chemicals (e.g. Clemons et al. 1996;Behrens et al. 2001;Bols et al. 2005;Lee et al. 2009) or for toxicity screening of complex environmental samples such as wastewater or sediment extracts can be measured (e.g. Castaño et al. 1994;Brack et al. 2000;Dayeh et al. 2002;Kramer et al. 2009). ...
In mammalian research, the implementation of the 3Rs is ubiquitous. However, the adaptation of procedures for experimental work on fish seems less extensive in comparison, even though fish model organisms are common in a broad range of research fields already. To strengthen animal welfare in experimental research, we provide an overview of current research results, including studies on the nociception of fishes. Furthermore, we describe the potential of implementing the 3Rs in fish experimental research. In the context of "Reduction", we show alternative research methods to lethal sampling. Considering "Refinement", we point out possibilities to improve fish handling and indicate that adaptations to the individual species ecology are necessary. Under the aspect of "Replacement", we describe the high potential of cell cultures that can be obtained from fish tissue and give an overview of the already extensive use in ecotoxicology and virology. In addition, we illustrate that cell cultures could also be increasingly used for basic research.
Graphical abstract
... Taking into account these previousstudies performed using in vitro gill models and considering that the RT-gills cell line presents different physiological and osmoregulatory responses after cortisol stimulus (e.g., increased transepithelial resistance and reduced passive ion fluxes) (Scott et al., 2004;Lee et al., 2009), we propose a potential participation of surface and cytoplasmic GRs and MRs in the early osmoregulatory responses of isolated gill cells of rainbow trout. However, more experiments are necessary to address this hypothesis. ...
Cortisol is a key stress-related hormone involved in the physiological adjustments of fish. In gills, cortisol contributes to acclimatization to changes in environmental salinity, promoting both ion uptake or salt excretion. Cortisol exerts its biological effects through its interaction with specific intracellular glucocorticoid (GR) and mineralocorticoid (MR) receptors. Additionally, the further identification of GR and MR on the surface of different tissues, together with the existence of cortisol-mediated effects observed using membrane-impermeable analogs (e.g., cortisol-BSA), supports the existence of membrane-initiated cortisol actions in fish. Nevertheless, the impact of this alternative cortisol mechanism in relevant tissues for fish salinity acclimation, such as gill, is unknown. In this work, we sought to explore the contribution of rapid membrane-initiated cortisol on GR and MR regulation in rainbow trout (Oncorhynchus mykiss) gills using in vivo and in vitro approaches. Juvenile rainbow trout intraperitoneally injected with cortisol or cortisol-BSA showed increased gr2 but no gr1 or mr mRNA levels in gills after one hour of treatment. This result was further confirmed using RT-gills-W1 cell lines stimulated with both versions of cortisol. Interestingly, after three and six hours of cortisol or cortisol-BSA treatment, there were no changes in the mRNA levels of any corticosteroid receptor in RT-gills-W1 cells. Finally, using immunofluorescence analysis, we identified GR and MR in rainbow trout gill cells localized on the cell surface. Considering the in vivo and in vitro results of this work, we suggest that membrane-initiated cortisol action contributes to the early expression of gr2 in rainbow trout gills during salinity acclimation.
... This cell line exhibits positive features, including fast growth capacity at room temperature, forming tight monolayer and enduring hypo-and hyper-osmotic conditions. Therefore, RTgill-W1 serves as a great model to in vitro studies about fish toxicology, gill function and infectious diseases [12]. In the current study, we demonstrated the differential expression patterns of gene of interest (GOI) from cecropin P1 treated RTgill-W1 cells with or without the presence of lipopolysaccharide (LPS) or polyinosinic polycytidylic acid (PolyI:C). ...
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.
... Our results indicate that, despite the similar trend, RTgill-W1 cell line was the most sensitive to TBBPA exposure, as confirmed by the EC 50 values for both AB and CFDA-AM. This outstanding performance and sensitivity of RTgill-W1 cell line in cytotoxicity studies is well documented (Lee et al., 2009;Tanneberger et al., 2013;Yue et al., 2015) and further supported by their recent incorporation in a new OECD testing guideline for fish acute toxicity (OECD, 2021). The lower sensitivity of hepatic and intestine-derived cell lines could be related to the susceptibility of TBBPA to enzymatic detoxification mechanisms in fish (Nos et al., 2020;Shen et al., 2012). ...
The potential interactions between the diverse pollutants that can be released into the environment and the
resulting outcomes are a challenging issue that needs to be further examined. This in vitro study was aimed to
assess potential toxic effects caused by combined exposure to tetrabromobisphenol A, a flame retardant widely
used and frequently detected in aquatic matrices, and commercially available polystyrene nanoparticles as
reference material to evaluate nanoplastics risks. Our results, using freshwater fish cell lines and a set of relevant
cytotoxicity endpoints including cell viability, oxidative stress, and DNA damage, provide additional mechanistic
insights that could help to fully characterize the toxicity profiles of tetrabromobisphenol A and polystyrene
nanoparticles. Furthermore, we describe subtle changes in cell viability as well as the generation of oxidative
DNA damage after coexposure to subcytotoxic concentrations of the tested pollutants.
... Lines such as RTgill-W1, RTL-W1, RTG-2 and from rainbow trout (Oncorhynchus mykiss), CHSE-14 from chinook salmon (Oncorhynchus tshawytscha) or PLHC-1 from the clear fin livebearer (Poeciliopsis lucida) form a gross majority of applications in published ecotoxicity studies (Fent, 2001;Segner, 2004). The most widely used is the RTgill-W1 gill cell line characterized by slow proliferation capacity with monolayers that can be grown at ambient carbon dioxide levels from 6 to 20 • C for up to 78 weeks without any media changes (Lee et al., 2009;Bury et al., 2014;Fischer et al., 2019). It has been applied in a plethora of applications such as the assessment of toxicokinetic properties including bioaccumulation and biotransformation of chemicals, genotoxicity, cytotoxicity, immunotoxicity as well as analysis of mixtures of contaminates and complex environmental samples such as water effluents and sediment (Fent, 2001;Segner, 2004;Lee et al., 2009;Langan et al., 2017;Rehberger et al., 2018;Fischer et al., 2019;Kalman et al., 2019;Zurita et al., 2019;Bonomo et al., 2020). ...
... The most widely used is the RTgill-W1 gill cell line characterized by slow proliferation capacity with monolayers that can be grown at ambient carbon dioxide levels from 6 to 20 • C for up to 78 weeks without any media changes (Lee et al., 2009;Bury et al., 2014;Fischer et al., 2019). It has been applied in a plethora of applications such as the assessment of toxicokinetic properties including bioaccumulation and biotransformation of chemicals, genotoxicity, cytotoxicity, immunotoxicity as well as analysis of mixtures of contaminates and complex environmental samples such as water effluents and sediment (Fent, 2001;Segner, 2004;Lee et al., 2009;Langan et al., 2017;Rehberger et al., 2018;Fischer et al., 2019;Kalman et al., 2019;Zurita et al., 2019;Bonomo et al., 2020). In vitro cytotoxicity assays with fish cell lines have also been proposed as alternative strategies to reduce or even replace lethality test with adult fish (Segner, 2004;Schirmer, 2006;Knauer et al., 2007;Fischer et al., 2019;Rodrigues et al., 2020). ...
The rapidly increasing number of new production chemicals coupled with stringent implementation of global chemical management programs necessities a paradigm shift towards boarder uses of low-cost and high-throughput ecotoxicity testing strategies as well as deeper understanding of cellular and sub-cellular mechanisms of ecotoxicity that can be used in effective risk assessment. The latter will require automated acquisition of biological data, new capabilities for big data analysis as well as computational simulations capable of translating new data into in vivo relevance. However, very few efforts have been so far devoted into the development of automated bioanalytical systems in ecotoxicology. This is in stark contrast to standardized and high-throughput chemical screening and prioritization routines found in modern drug discovery pipelines. As a result, the high-throughput and high-content data acquisition in ecotoxicology is still in its infancy with limited examples focused on cell-free and cell-based assays.
In this work we outline recent developments and emerging prospects of high-throughput bioanalytical approaches in ecotoxicology that reach beyond in vitro biotests. We discuss future importance of automated quantitative data acquisition for cell-free, cell-based as well as developments in phytotoxicity and in vivo biotests utilizing small aquatic model organisms. We also discuss recent innovations such as organs-on-a-chip technologies and existing challenges for emerging high-throughput ecotoxicity testing strategies. Lastly, we provide seminal examples of the small number of successful high-throughput implementations that have been employed in prioritization of chemicals and accelerated environmental risk assessment.
... Such approaches enable a deeper understanding of molecular mechanisms on cellular function [50]. There are several cell lines developed for salmonid fish, including intestinal derived (RTgutGC) [51], gill (RTgill-W1) [52], and the monocyte/macrophage cell line RTS11 [53], the use of fish cell lines is reviewed by Collet, Collins, and Lester, [54]. However, the phenotype of these cells is likely to have changed considerably from their original tissues and the choice of cell lines needs consideration for specific experiments, even the RTgutGC cells are unlikely to reflect the complex nature of the intestine with multiple cells phenotypes present. ...
Within aquaculture prebiotics are composed of complex carbohydrate molecules that cannot be digested by the fish directly but are metabolised by the microbial communities within the host gut, with the desire that “healthy” bacterial species are promoted with subsequently improved performance of the fish, there are likely some direct responses of intestinal cells to these dietary components. The sources and processing of prebiotics, which fall under the overarching theme of “functional feeds” are highly varied between species and types of prebiotics administered. How these feeds exert their effect, and the host responses are hard to determine, but new technologies and the development of high-throughput technologies (omics) are enabling the mechanisms and methods of action to be further understood. The recent advances in the availability of ‘omics’ technologies with the transition from single gene assays to microarray and RNA-seq in fish health have enabled novel functional ingredients to be analysed. This review will focus on recent studies on targeted gene expression and ‘omics’ technologies to characterize immune responses. Comparisons between the immunomodulatory effect of different prebiotics have been made and specific examples of how transcriptomics techniques have been used to identify immune responses to prebiotics are given.
... Lots of studies have reported that cell lines are mainly used in virology, environmental toxicology, cell biology, oncology, drug screening and development, genetics, and genomics (Lee et al. 2009;Taju et al. 2014). Particularly, fish cell lines have been widely used in many biological research fields by virtue of multiple advantages including fewer nutrients to sustain growth, the ability to proliferate over a wide range of temperatures, and the ability to adapt to simple culture methods in comparison with mammalian cell lines (Zhu et al. 2013). ...
Humpback grouper (Cromileptes altivelis), one kind of commercial fish with considerable economic value, has been recognized as a promising candidate for mariculture. In the wake of the development of aquaculture industry, the breeding density of C. altivelis has increased gradually, which gave rise to the occurrence of various pathogenic diseases. In our research, we established a new kidney cell line (designated as CAK) from humpback grouper and evaluated its susceptibility to bacteria and heavy metals. The results of our study showed that the optimal growth temperature was 26 °C, and optimal medium was L-15 supplemented with 20% fetal bovine serum (FBS). The sequencing of 18S rRNA gene indicated that CAK cell line was derived from C. altivelis. Chromosome analysis showed that the number of chromosome in CAK was 48. After being transfected of pEGFP-N3 plasmid, high transfection efficiency of CAK was observed, suggesting the potential to be used for the study of foreign functional genes. Moreover, the bacterial susceptibility results revealed that CAK cells were sensitive to Vibrio harveyi and Edwardsiella tarda, especially V. harveyi. Meanwhile, three heavy metals (Hg, Cu, and Cd) had toxic effects on the CAK cells with a dose-dependent manner. To sum up, the CAK cell line might be an ideal tool in vitro for analyzing the function of exogenous genes, bacterial susceptibility, and toxicity assay of heavy metals.
