17α-Ethynylestradiol and Levonorgestrel Exposure of Rainbow Trout RTL-W1 Cells at 18 °C and 21 °C Mainly Reveals Thermal Tolerance, Absence of Estrogenic Effects, and Progestin-Induced Upregulation of Detoxification Genes.

Fish are exposed to increased water temperatures and aquatic pollutants, including endocrine-disrupting compounds (EDCs). Although each stressor can disturb fish liver metabolism independently, combined effects may exist. To unveil the molecular mechanisms behind the effects of EDCs and temperature, fish liver cell lines are potential models needing better characterisation. Accordingly, we exposed the rainbow trout RTL-W1 cells (72 h), at 18 °C and 21 °C, to ethynylestradiol (EE2), levonorgestrel (LNG), and a mixture of both hormones (MIX) at 10 µM. The gene expression of a selection of targets related to detoxification (CYP1A, CYP3A27, GST, UGT, CAT, and MRP2), estrogen exposure (ERα, VtgA), lipid metabolism (FAS, FABP1, FATP1), and temperature stress (HSP70b) was analysed by RT-qPCR. GST expression was higher after LNG exposure at 21 °C than at 18 °C. LNG further enhanced the expression of CAT, while both LNG and MIX increased the expressions of CYP3A27 and MRP2. In contrast, FAS expression only increased in MIX, compared to the control. ERα, VtgA, UGT, CYP1A, HSP70b, FABP1, and FATP1 expressions were not influenced by the temperature or the tested EDCs. The RTL-W1 model was unresponsive to EE2 alone, sensitive to LNG (in detoxification pathway genes), and mainly insensitive to the temperature range but had the potential to unveil specific interactions.

Transfection, cytotoxicity, and cell cycle studies on the two newly developed and characterized humpback grouper (Cromileptes altivelis) fin cell lines.

The development and characterization of two novel humpback grouper (Cromileptes altivelis) fin cell lines are described in this study. The CA1F3Ex and CA1F4Tr cell lines were developed by explant and trypsinization methods, respectively, in Leibovitz's L15 (L-15) medium supplemented with 20% FBS (fetal bovine serum) and subcultured over 150 times. Cell lines exhibited high stability, as evidenced by the high revival rate (85-95%) and good attachment while seeding after one year of cryostorage. They displayed good seeding (91%) and plating efficiencies (15-25%). The optimum temperature for growth was recorded at 28˚C. Serum requirement decreased with increased passage and lowered to 2% FBS beyond 30-35 passages. However, higher serum concentration (2-20%) caused a concurrent increase in cell growth. Both the cell lines were fibroblast-type, and immunotyping results showed strong reactivity towards the fibroblast marker. Chromosome analysis of these cell lines revealed aneuploidy, and the authenticity was confirmed by mitochondrial Cytochrome C Oxidase Subunit I (COI) genotyping analysis. Cell cycle studies were performed utilizing the flow cytometric technique. CA1F3Ex and CA1F4Tr cell lines showed high transfection efficiency with pEGFP-N1 plasmid using Lipofectamine and cytotoxicity towards heavy metals (Hg and Cd) was also studied. Hence, these continuous cell lines could be employed as in vitro models for aquatic toxicological and genetic manipulation studies.

Non-microcystin extracellular metabolites of Microcystis aeruginosa impair viability and reproductive gene expression in rainbow trout cell lines.

Microcystis aeruginosa is a ubiquitous freshwater cyanobacterium best known for producing hepatotoxic microcystins; however, this common bloom-forming species also produces myriad biologically active and potentially deleterious other metabolites. Our understanding of the effects of these non-microcystin metabolites on fish is limited. In this study, we evaluated cytotoxicity of extracellular metabolites harvested from both microcystin-producing (MC+) and non-producing (MC-) strains of M. aeruginosa on rainbow trout (Oncorhynchus mykiss) cell lines derived from tissues of the brain, pituitary, heart, gonads, gills, skin, liver, and milt. We also examined the influence of M. aeruginosa exudates (MaE) on the expression of critical reproduction-related genes using the same cell lines. We found that exudates of the MC- M. aeruginosa strain significantly reduced viability in RTBrain, RTgill-W1, and RT-milt5 cell lines and induced significant cellular stress and/or injury in six of the eight cell lines-highlighting potential target tissues of cyanobacterial cytotoxic effects. Observed sublethal consequences of Microcystis bloom exposure occurred with both MC+ and MC- strains' exudates and significantly altered expression of developmental and sex steroidogenic genes. Collectively, our results emphasize the contributions of non-MC metabolites to toxicity of Microcystis-dominated algal blooms and the need to integrate the full diversity of M. aeruginosa compounds-beyond microcystins-into ecotoxicological risk assessments.

Evaluation of tire tread particle toxicity to fish using rainbow trout cell lines.

Tire and road wear particles (TRWP) resulting from tire abrasion while driving raise concerns due to their potential contribution to aquatic toxicity. Our study aimed to assess cryogenically milled tire tread (CMTT) particle toxicity, used as a proxy for TRWP, and associated chemicals to fish using two Rainbow Trout (Oncorhynchus mykiss) cell lines representing the gill (RTgill-W1) and the intestinal (RTgutGC) epithelium. CMTT toxicity was evaluated through several exposure pathways, including direct contact, leaching, and digestion, while also assessing the impact of particle aging. Following OECD TG249, cell viability was assessed after 24 h acute exposure using a multiple-endpoint assay indicative of cell metabolic activity, membrane integrity and lysosome integrity. In vitro EC50 values for the fish cell lines exceeded river TRWP concentrations (2.02 g/L and 4.65 g/L for RTgill-W1 and RTgutGC cell lines, respectively), and were similar to in vivo LC50 values estimated at 6 g/L. Although toxicity was mainly driven by the leaching of tire-associated chemicals, the presence of the particles contributed to the overall toxicity by inducing a continuous leaching, highlighting the importance of considering combined exposure scenarios. Aging and digestion conditions were also found to mediate CMTT toxicity. Thermooxidation resulted in a decreased chemical leaching and toxicity, while in vitro digestion under mimicked gastrointestinal conditions increased leaching and toxicity. Specific chemicals, especially Zn, 2-mercaptobenzothiazole, 1,3-diphenylguanidine, and N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) were identified as contributors to the overall toxicity. Although 6PPD-quinone was detected in CMTT digestate, cytotoxicity assays with RTgill-W1 and RTgutGC cell lines showed no toxicity up to 6 mg/L, supporting the notion of a specific mode of action of this chemical. This study provides insights into the toxicological mechanisms induced by tire particles and their associated chemicals and can help in the evaluation of potential risks to aquatic life associated with TRWP.

Upregulation of oxidative stress by triphenyl phosphate (TPhP) exposure causes antioxidant insult and apoptotic process in Epithelioma papulosum cyprini (EPC) cells.

Triphenyl phosphate (TPhP) is the predominant compound of organophosphate flame retardants (OPFRs), which can elicit a toxicological effect on physiological response and tissue development of fish. In this study, we investigated the effect of TPhP exposure on cell viability, antioxidant capacities, and apoptosis in EPC cells. Current study revealed that TPhP exposure could decrease cell viability and promote intracellular oxidative stress in EPC cells. In addition, high-dose TPhP exposure could facilitate antioxidant insults and cause mitochondrial collapse in a dose-dependent manner, along with increased gene expressions involved in apoptosis and unfolded protein response (UPR). These results indicated that reactive oxygen species (ROS)-induced cytotoxic stress and cell death were involved in antioxidant insults and apoptotic activation in TPhP-exposed fish cells.

Detrimental effects of individual versus combined exposure to tetrabromobisphenol A and polystyrene nanoplastics in fish cell lines.

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.

Comparative cytotoxicity induced by parabens and their halogenated byproducts in human and fish cell lines.

Parabens are a group of para-hydroxybenzoic acid (p-HBA) esters widely used in pharmaceutical industries. Their safety is well documented in mammalian models, but little is known about their toxicity in non-mammal species. In addition, chlorinated and brominated parabens resulting from wastewater treatment have been identified in effluents. In the present study, we explored the cytotoxic effects (EC50) of five parabens: methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BuP), and benzylparaben (BeP); the primary metabolite, 4-hydroxybenzoic acid (4-HBA), and three of the wastewater chlorinated/brominated byproducts on fish and human cell lines. In general, higher cytotoxicity was observed with increased paraben chain length. The tested compounds induced toxicity in the order of 4-HBA < MP < EP < PP < BuP < BeP. The halogenated byproducts led to higher toxicity with the addition of second chlorine. The longer chain-parabens (BuP and BeP) caused a concentration-dependent decrease in cell viability in fish cell lines. Intriguingly, the main paraben metabolite, 4-HBA, proved to be more toxic to fish hepatocytes than human hepatocytes by 100-fold. Our study demonstrated that the cytotoxicity of some of these compounds appears to be tissue-dependent. These observations provide valuable information for early cellular responses in human and non-mammalian models upon exposure to paraben congeners.

Development, scrutiny, and modulation of transient reporter gene assays of the xenobiotic metabolism pathway in zebrafish hepatocytes.

The "toxicology in the twenty-first century" paradigm shift demands the development of alternative in vitro test systems. Especially in the field of ecotoxicology, coverage of aquatic species-specific assays is relatively scarce. Transient reporter gene assays could be a quick, economical, and reliable bridging technology. However, the user should be aware of potential pitfalls that are influenced by reporter vector geometry. Here, we report the development of an AhR-responsive transient reporter-gene assay in the permanent zebrafish hepatocytes cell line (ZFL). Additionally, we disclose how viral, constitutive promoters within reporter-gene assay cassettes induce squelching of the primary signal. To counter this, we designed a novel normalization vector, bearing an endogenous zebrafish-derived genomic promoter (zfEF1aPro), which rescues the squelching-delimited system, thus, giving new insights into the modulation of transient reporter systems under xenobiotic stress. Finally, we uncovered how the ubiquitously used ligand BNF promiscuously activates multiple toxicity pathways of the xenobiotic metabolism and cellular stress response in an orchestral manner, presumably leading to a concentration-related inhibition of the AhR/ARNT/XRE-toxicity pathway and non-monotonous concentration-response curves. We named such a multi-level inhibitory mechanism that might mask effects as "maisonette squelching." A transient reporter gene assay in zebrafish cell lines utilizing endogenous regulatory gene elements shows increased in vitro toxicity testing performance. Synthetic and constitutive promotors interfere with signal transduction ("squelching") and might increase cellular stress (cytotoxicity). The squelching phenomenon might occur on multiple levels (toxicity pathway crosstalk and normalization vector), leading to a complete silencing of the reporter signal.

Cytotoxicity inhibition of catechol's type molecules by grafting on TiO2 and Fe2O3 nanoparticles surface.

The potential toxicity deriving from the interaction between chemicals and manufactured nanoparticles (NPs) represents an emerging threat to the environment and human health. Several studies have focused on the risks and (eco)toxicity of manufactured NPs as a consequence of their extensive use in recent years, however, there is still a limited understanding of the combined effects caused by manufactured NPs in the presence of other environmental contaminants. This is particularly relevant to aquatic environments, where many types of pollutants are inevitably released and can be involved in many kinds of reactions. In this context, the interaction between catecholate type ligands and two different nanomaterials, namely TiO2 and Fe2O3 NPs, was investigated by performing cytotoxicity assays with the topminnow fish hepatoma cell line (PLHC-1) using: i) the original organic molecules, ii) pristine NPs alone, and iii) modified NPs obtained by grafting the ligands on the NPs surface. Cytotoxic effects were explored at three different levels, specifically on cellular metabolism, membrane integrity and lysosomal activity. The outcomes from these assays showed cytotoxicity only for the free catechol type ligands, while in general no significant decrease in cell viability was observed for pristine NPs, as well as for the modified NPs, regardless the initial cytotoxicity level of the organic ligands These results suggest that the binding of catechols on the NPs' surface inhibited their cytotoxicity, indicating that TiO2 and Fe2O3 NPs may act as sorbents of these contaminants, thus reducing their possible detrimental effects.

Susceptibilities of ten fish cell lines to infection with Tilapia lake virus.

Tilapia lake virus disease (TiLVD) caused by Tilapia lake virus (TiLV) is a great threat to the global tilapia culture industry. Effective prevention and control strategies have not been developed due to limited basic research of pathogenesis of TiLVD. Cell lines from different fish species have been found to be permissive to TiLV infection. In the current study, we comprehensively analyzed TiLV susceptibilities to 10 permanent growing fish cell lines. We found that the highest viral titers were generated onto TiB cells originated from the tilapia species Oreochromis mossambicus, MSF from the largemouth bass Micropterus salmoides, CAMK from the hybrid snakehead Channa argus × Channa maculata and SS derived from the perch species Siniperca chuatsi. Viral copy numbers from these four cell lines ranged from 4 × 107 copies/µL to 4.6 × 108 copies/µL. Confocal immunofluorescent microscopy also indicated that all 10 cell lines can support varying degrees of viral infection and replication. TiLV particles can be observed in cells from randomly selected three fish species using electron microscope. This study will assist in research and development of prevention and control of TiLVD.

Differential Nervous Necrosis Virus (NNV) Replication in Five Putative Susceptible Cell Lines.

Viral encephalopathy and retinopathy caused by nervous necrosis virus (NNV), is one of the most threatening viral diseases affecting marine fish worldwide. In vitro propagation of NNV strains is essential for the design of effective control measures. In the present study we analysed both the susceptibility and the permissiveness of five fish cell lines (E-11, GF-1, SAF-1, DLB-1, and SaB-1) to three NNV strains (one RGNNV, one SJNNV, and one reassortant RGNNV/SJNNV). E-11 and DLB-1 were demonstrated to be highly susceptible to NNV strains, with average adsorption efficiency (AE) values higher than 90%. SAF-1 also showed high susceptibility (AE 88%), whereas GF-1 can be regarded as moderately susceptible (AE around 50%). On the contrary, SaB-1 can be considered a poorly susceptible cell line (AE values below 20%). E-11 and GF-1 cell lines provided the highest production rates for RGNNV and RG/SJ (around 103) and both cell lines can be regarded as fully permissive for these viral types. However, the SJNNV production rate in GF-1 was only 17.8 and therefore this cell line should be considered semi-permissive for this genotype. In SAF-1 cells, moderate viral replication was recorded but differences in intracellular and extracellular production suggest that viral progeny was not efficiently released. In DLB-1 and SaB-1 the final viral titres obtained in E-11 were lower than those of the inoculum. However, RNA1 synthesis values seem to indicate that RGNNV replication in DLB-1 and SAF-1 could have been underestimated, probably due to a poor adaptation of the virus grown in these cell lines to E-11. Based on all these results, E-11 seems to be the most appropriate cell for in vitro culture of RGNNV, SJNNV, and reassortant strains.

Development of 3D cultures of zebrafish liver and embryo cell lines: a comparison of different spheroid formation methods.

Fish cell spheroids are promising 3D culture models for vertebrate replacement in ecotoxicology. However, new alternative ecotoxicological methods must be adapted for applications in industry and for regulatory purposes; such methods must be cost-effective, simple to manipulate and provide rapid results. Therefore, we compared the effectiveness of the traditional hanging drop (HD), orbital shaking (OS), and HD combined with OS (HD+OS) methods on the formation of zebrafish cell line spheroids (ZFL and ZEM2S). Time in HD (3-5 days) and different 96-well plates [flat-bottom or ultra-low attachment of round-bottom (ULA-plates)] in OS were evaluated. Easy handling, rapid spheroid formation, uniform-sized spheroids, and circularity were assessed to identify the best spheroid protocol. Traditional HD alone did not result in ZFL spheroid formation, whereas HD (5 days)+OS did. When using the OS, spheroids only formed on the ULA-plate. Both HD+OS and OS were reproducible in size (177.50 ± 2.81 µm and 225.62 ± 19.20 µm, respectively) and circularity (0.83 ± 0.02 and 0.80 ± 0.01, respectively) of ZFL spheroids. Nevertheless, HD+OS required a considerable time to completely form spheroids (10 days) and intensive handling, whereas the OS was fast (5 days of incubation) and simple. OS also yielded reproducible ZEM2S spheroids in 1 day (226.23 ± 0.57 µm diameter and 0.80 ± 0.01 circularity). In conclusion, OS in ULA-plate is an effective and simple spheroid protocol for high-throughput ecotoxicity testing. This study contributes to identify a fast, reproducible, and simple protocol of single piscine spheroid formation in 96-well plates and supports the application of fish 3D model in industry and academia.

Effect of Lipopolysaccharide (LPS) stimulation on apoptotic process and oxidative stress in fibroblast cell of hybrid crucian carp compared with those of Carassius cuvieri and Carassius auratus red var.

Bacterial LPS is a heat-stable endotoxin and wall components of gram negative bacteria, which can exhibit a toxicological effect on physiology and biochemical activities of fish. In this study, we investigated the effect of LPS exposure on cell viability, oxidative stress, caspase activity and immune-related gene expressions in cultured fin cell lines of red crucian carp, white crucian carp and their hybrid offspring. LPS stimulation could reduce fish cell viability, whereas gene expression levels and promoter activities in inflammatory signals increased dramatically. Moreover, enhanced levels of intracellular oxidative stress and decreased levels of mitochondrial membrane potential (MMP) were observed in LPS-induced fish cells. N-Acetyl-L-cysteine (NAC) could alleviate LPS-stimulated reactive oxygen species (ROS) generation and caspase-3 activity in fish cells. These results suggested that ROS-mediated cytotoxic stress was involved in LPS-induced inflammation and mitochondrial damage in cultured fish cells.

Cytochrome oxidase gene sequencing reveals channel catfish ovary cell line is contaminated with brown bullhead cells.

The channel catfish (Ictalurus punctatus, Rafinesque) ovary (CCO) cell line is the standard cell line used for channel catfish diagnostics. Next-gen sequencing studies of a virus cultured in the CCO cells revealed mitochondrial sequences matching those of brown bullhead (Ameiurus nebulosus, Lesueur). Therefore, we systematically performed partial cytochrome oxidase 1 gene sequencing of several sources of the CCO cell line and all matched the brown bullhead and not the channel catfish.

Screening of Fish Cell Lines for Piscine Orthoreovirus-1 (PRV-1) Amplification: Identification of the Non-Supportive PRV-1 Invitrome.

Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells.

In vitro assessment of the antimicrobial efficacy of chitosan nanoparticles against major fish pathogens and their cytotoxicity to fish cell lines.

Nanotechnology is an emerging avenue employed in disease prevention and treatment. This study evaluated the antimicrobial efficacy of chitosan nanoparticles (CSNPs) against major bacterial and oomycete fish pathogens in comparison with chitosan suspension. Initially, the minimum inhibitory concentrations (MIC, MIC90 ) were determined and the per cent inhibition of bacterial growth was calculated. Subsequently, the minimum bactericidal concentrations (MBCs) were determined. The time-dependent disruptions of CSNP-treated pathogens were observed via transmission electron microscopy (TEM), and the effect of CSNPs on the viability of two fish cell lines was assessed. No antimicrobial effect was observed with chitosan, while CSNPs (105 nm) exhibited a dose-dependent and species-specific antimicrobial properties. They were bactericidal against seven bacterial isolates recording MBC values from 1 to 7 mg/ml, bacteriostatic against four further isolates recording MIC values from 0.125 to 5 mg/ml and fungistatic against oomycetes recording MIC90 values of 3 and 4 mg/ml. TEM micrographs showed the attachment of CSNPs to the pathogenic cell membranes disrupting their integrity. No significant cytotoxicity was observed using 1 mg/ml CSNPs, while low dose-dependent cytotoxicity was elicited by the higher doses. Therefore, it is anticipated that CSNPs are able to compete and reduce using antibiotics in aquaculture.

Extending the concept of predicting fish acute toxicity in vitro to the intestinal cell line RTgutGC.

Testing chemicals for fish acute toxicity is a legal requirement in many countries as part of environmental risk assessment. To reduce the numbers of fish used, substantial efforts have been focussed on alternative approaches. Prominently, the cell viability assay with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, has been recognized, owing to its high predictive power and robustness. Like gills, the intestine is considered a major site of chemical uptake and biotransformation but, in contrast to gills, is expected to be exposed to rather hydrophobic chemicals, which enter the fish via food. In the present study, we therefore aimed to extend the cell bioassay to the rainbow trout epithelial cell line from intestine, RTgutGC. Using 16 hydrophobic and volatile chemicals from the fragrance palette, we showed that also the RTgutGC cell line can be used to predict fish acute toxicity of chemicals and yields intra-laboratory variability in line with other bioassays. By comparing the RTgutGC toxicity to a study employing the RTgill-W1 assay on the same group of chemicals, a fragrance specific relationship was established which reflects an almost perfect 1:1 relationship between in vitro and in vivo toxicity results. Thus, both cell lines can be used to predict fish acute toxicity, either by using the obtained in vivo-in vitro relationship or by taking the in vitro results at face value. We moreover demonstrate the derivation of non-toxic concentrations for downstream applications which rely on a healthy cell state, such as the assessment of biotransformation or chemical transfer.

Effect of ageing of bare and coated nanoparticles of zinc oxide applied to soil on the Zn behaviour and toxicity to fish cells due to transfer from soil to water bodies.

This study evaluated the influence of ageing of ZnO nanoparticles (NPs) applied to soil on the potential availability and chemical speciation of Zn, and also of their toxicity to aquatic organisms due to transfer of contaminants from soil to water. To this end, soil samples were spiked with two types of bare nanoparticles: b1ZnO NPs (rod- and elongated-shaped) and b2ZnO NPs (near-spherical shaped) and ZnO NPs coated with (3-aminopropyl)triethoxysilane (cZnO NPs) within the 0-800 mg Zn kg-1 soil dose range, and were left to age for 0, 30, 60 and 90 days. The available concentration and speciation of Zn in soil were determined by the DGT (diffusive gradients in thin films) technique and sequential extraction procedures, respectively. The toxicity of the aqueous extracts from the ZnO NP-treated soils was assessed in vitro in established fish cell lines (RTG-2). The highest distribution percentages of the applied Zn occurred in the organically complexed (OC), followed by the exchangeable (EXC) fraction, for all NP types, applied doses and incubation times. The toxicity of NPs depended on their intrinsic properties: b1ZnO NPs affected the membrane function, reductase enzyme activity and, to a lesser extent, reactive oxygen species (ROS) levels of fish cells, whereas b2ZnO NPs and cZnO NPs affected mainly ROS generation. Ageing increased Zn soil availability, but toxicity to fish cells showed no trend over time. The particle dissolution of ZnO NPs did not explain the observed toxicity, hence a nanoparticles-specific effect should be assumed. The findings of this study seem to indicate that the transfer of ZnO NP from contaminated soils to aquatic ecosystems should be addressed.

The application of the Comet assay in fish cell lines.

The use of fish models has been proven to be an effective and sensitive tool for the evaluation of genotoxicity of pure compounds and complex mixtures of chemicals in the context of environmental screening of pollutants and hazard assessment in aquatic toxicology. In particular, fish cell lines have been successfully introduced for detection of genotoxic effects and can serve as an alternative to animal testing in preliminary eco-/genotoxicological studies. For this purpose comet assay has been extensively used in fish cell lines for the evaluation of genotoxic potential of chemicals and complex environmental matrices. The most often used fish cell lines in the comet assay are RTG-2, RTgill-W1 and RTL-W1 derived from rainbow trout (Onchorhynchus mykiss) gonads, gills and liver, respectively, and ZFL and ZF4 cells established from zebrafish (Danio rerio) liver and embryos, respectively. The present review gives an overview of the most often-used permanent fish cell lines in genotoxicology and discusses their application in the comet assay.

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.