Epithelioma Papulosum Cyprini Cell Line Research Articles

Multi-Level System to Assess Toxicity in Water Distribution Plants.

The toxicity of water samples from water distribution plants needs to be investigated further. Indeed, studies on the pro-oxidant effects driven by tap water are very limited. In this study, the water quality, pro-oxidant effects, and potential health risks driven by exposure to groundwater samples from two water plants (sites A and B) located in Northwestern Italy were investigated in a multi-level system. Physicochemical parameters and the absence of pathogens, cyanotoxins, and endocrine active substances indicated a good water quality for both sites. The 25 metals analyzed were found under the limit of quantification or compliant with the maximum limits set by national legislation. Water samples were concentrated by the solid-phase extraction system in order to assess the aquatic toxicity on Epithelioma papulosum cyprini (EPC) cell line. Levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase were evaluated through the Integrated Biomarkers Response (IBRv2) index. EPC cell line was found a sensible model for assessing the antioxidant responses driven by both water concentrates. A similar antioxidant response was shown by plots and IBRv2 suggesting a muted risk for the two sampling sites.

Synergistic Effect of Biosynthesized Silver Nanoparticles and Natural Phenolic Compounds against Drug-Resistant Fish Pathogens and Their Cytotoxicity: An In Vitro Study.

Fish pathogens causing disease outbreaks represent a major threat to aquaculture industry and food security. The aim of the presented study is to develop safe and effective bioactive agents against two bacterial isolates: Aeromonas hydrophila and Pseudomonas fluorescens. We employed a broth microdilution method to investigate the antibacterial effect of biosynthesized silver nanoparticles (AgNPs); rutin, a natural flavonoid extracted from Ruta graveneoles; and heliomycin, a secondary metabolite produced by marine actinomycetes AB5, as monotherapeutic agents. Moreover, AgNPs in combination with rutin (AgNP + R) and heliomycin (AgNPs + H) were examined for their synergistic effect. The cytotoxic effect of individual bioactive compounds and in combination with AgNPs was investigated on epithelioma papulosum cyprini (EPC) fish cell lines. Individual treatment of AgNPs, rutin, and heliomycin exhibited a dose-dependent antimicrobial activity against A. hydrophila and P. fluorescens. Rutin minimum inhibitory concentration (MIC) showed the lowest cytotoxicity when tested on EPC cell lines, while heliomycin MIC was highly cytotoxic. Combined subtherapeutic doses of AgNPs + R and AgNPs + H displayed additive and synergistic effects against A. hydrophila and P. fluorescens, respectively, with improved results and relative safety profile. The study findings demonstrate that a combination of AgNPs and natural bioactive compounds may represent novel therapeutics fighting fish pathogens potentially affecting the fish farming industry.

Novel insights into the immune regulatory effects of ferritins from blunt snout bream, Megalobrama amblycephala

Ferritins play vital roles in maintenance of iron homeostasis as iron storage proteins. Recently, the immune function of ferritins have attracted increasing attention, especially their roles in defense against pathogenic infections. However, the immune regulatory mechanism of fish ferritins are not well known. In the present study, comparative digital gene expression (DGE) profiling was performed to explore the regulatory effects of the Megalobrama amblycephala ferritins (MamFers) using MamFers overexpressed and control L8824 cells (Ctenopharyngodon idella hepatic cell line). Clean reads were aligned to the C. idella genome and differential expression analysis was conducted with representative differentially expressed genes pointed out. On that basis, further studies were performed to verify two pivotal regulated pathways in L8824 and EPC (Epithelioma Papulosum Cyprini cell line) cells, respectively. The results showed that NLRC5 (NOD-like Receptor Family CARD Domain Containing 5) mediated the regulation of MamFers on expression of MHC I (Major Histocompatibility Complex Class I) and its chaperone β2M (Beta-2-Microglobulin) in L8824 cells. Then, β2M further mediated the regulation of MamFers on hepcidin expression, indicating that MamFers regulated the expression of hepcidin via NLRC5/MHC I/β2M axis. In addition, MamFers regulated the adhesion of Aeromonas hydrophila to EPC cells by regulating the expression of two extracellular matrix proteins Intgβ1 (integrin β1) and FN (fibronectin). In a word, the present study provided novel insights into the immune regulatory functions of fish ferritins.

Retail Baitfish in Michigan Harbor Serious Fish Viral Pathogens.

Indigenous small cyprinid fish species play an important role in Great Lakes ecosystems and also comprise the backbone of a multimillion‐dollar baitfish industry. Due to their widespread use in sport fisheries of the Laurentian Great Lakes, there are increasing concerns that baitfish may introduce or disseminate fish pathogens. In this study, we evaluated whether baitfish purchased from 78 randomly selected retail bait dealers in Michigan harbored fish viruses. Between September 2015 and June 2016, 5,400 baitfish divided into 90 lots of 60 fish were purchased. Fish were tested for the presence of viral hemorrhagic septicemia virus (VHSV), spring viremia of carp virus (SVCV), golden shiner reovirus (GSRV), fathead minnow nidovirus (FHMNV), fathead minnow picornavirus (FHMPV), and white sucker bunyavirus (WSBV). Using the epithelioma papulosum cyprini cell line and molecular confirmation, we demonstrated the presence of viruses in 18 of the 90 fish lots (20.0%) analyzed. The most prevalent virus was FHMNV, being detected in 6 of 30 lots of Fathead Minnow Pimephales promelas and 3 of 42 lots of Emerald Shiners Notropis atherinoides. We also confirmed GSRV in two fish species: the Golden Shiner Notemigonus crysoleucas (5 of 11 lots) and Fathead Minnow (3 of 30 lots). Two VHSV (genotype IVb) isolates were recovered from a single lot of Emerald Shiners. No SVCV, FHMPV, or WSBV was detected in any of the fish examined. Some of the infected fish exhibited clinical signs and histopathological alterations. This study demonstrates that live baitfish are a potential vector for the spread of viral pathogens and underscores the importance of fish health certifications for the Great Lakes baitfish industry.

Effects of Fetal Bovine Serum Concentration on the Propagation of Korean Viral Haemorrhagic Septicaemia Virus in an Epithelioma Papulosum Cyprini Cell Line

Effects of Fetal Bovine Serum Concentration on the Propagation of Korean Viral Haemorrhagic Septicaemia Virus in an Epithelioma Papulosum Cyprini Cell Line

Isolation, characterization and molecular identification of a novel aquareovirus that infects endangered fountain darter Etheostoma fonticola.

The fountain darter Etheostoma fonticola (FOD) is a federally endangered fish listed under the US Endangered Species Act. Here, we identified and characterized a novel aquareovirus isolated from wild fountain darters inhabiting the San Marcos River. This virus was propagated in Chinook salmon embryo (CHSE)-214, rainbow trout gonad-2 and fathead minnow cells at 15°C. The epithelioma papulosum cyprini cell line was refractory at all temperatures evaluated. High throughput sequencing technologies facilitated the complete genome sequencing of this virus utilizing ribosomal RNA-depleted RNA extracted from infected CHSE-214 cells. Conventional PCR primer sets were developed for the detection and confirmation of this virus to assist diagnostic screening methods. Phylogenetic analysis suggests this virus belongs to the Aquareovirus A genus. This research provides requisite initial data critical to support hatchery and refugia biosecurity measures for this endangered species.

Isolation, Characterization, Antioxidant, Antimicrobial and Cytotoxic Effect of Marine Actinomycete, Streptomyces Carpaticus MK-01, against Fish Pathogens

ABSTRACT Present study aim to evaluate the antimicrobial, antioxidant and cytotoxic potential of crude extract of Marine Streptomyces carpaticus MK-01 isolated from seawater collected from Daejeong-cost of Jeju Island. About 24 actinomycetes strains were isolated and subjected to morphological and molecular analysis that confirmed the isolate as S. carpaticus MK-01. Crude ethyl acetate extract of MK-01 strain showed extensive antibacterial activity against Gram-positive fish pathogenic bacteria namely Streptococcus iniae and S. parauberis with a maximum zone of inhibition (0.92±0.03mm) was recorded against S. parauberis at the minimum extract concentration (3.12µg/ml). The MK-01 ethyl acetate extract shows dose dependant significant increase in antioxidant activity. The 50% cytotoxic concentration (CC50) of MK-01 ethyl acetate extract was attained at 53.71 μg/ml and the effective concentration 50 (EC50) against virus-infected Epithelioma papulosum cyprini cell lines was 8.72 μg/ml of S. carpaticus MK-01 crude ethyl acetate extract.

Autophagy induced by infectious hematopoietic necrosis virus inhibits intracellular viral replication and extracellular viral yields in epithelioma papulosum cyprini cell line

Infectious hematopoietic necrosis virus (IHNV) is a common pathogen that causes severe disease in the salmonid aquaculture industry. Recent work demonstrated that autophagy plays an important role in pathogen invasion by activating innate and adaptive immunity. This study investigated the relationship between IHNV and autophagy in epithelioma papulosum cyprini cells. The electron microscopy results show that IHNV infection can induce typical autophagosomes which are representative structures of autophagy activation. The punctate accumulation of green fluorescence-tagged microtubule-associate protein 1 light chain 3 (LC3) and the protein conversion from LC3-I to LC3-II were respectively confirmed by confocal fluorescence microscopy and western blotting. Furthermore, the effects of autophagy on IHNV replication were also clarified by altering the autophagy pathway. The results showed that rapamycin induced autophagy can inhibit both intracellular viral replication and extracellular viral yields, while autophagy inhibitor produced the opposite results. These findings demonstrated that autophagy plays an antiviral role during IHNV infection.

Fathead minnow nidovirus infects spotfin shiner Cyprinella spiloptera and golden shiner Notemigonus crysoleucas.

Since the initial isolation of the fathead minnow nidovirus (FHMNV), concerns have been raised regarding the risks it may pose to other fish species. In this study, 7 fish species resident to the Laurentian Great Lakes were challenged intraperitoneally with 2 doses of FHMNV: 102.8 and 104.8 median tissue culture infective dose (TCID(50)) ml(-1). Infected spotfin shiner Cyprinella spiloptera and golden shiner Notemigonus crysoleucas suffered morbidity and mortality during the 40 d observation period, while other species, including creek chub Semotilus atromaculatus, rainbow trout Oncorhynchus mykiss, largemouth bass Micropterus salmoides and walleye Sander vitreus, showed no clinical signs or mortality. FHMNV was re-isolated on the epithelioma papulosum cyprini cell line from the tissues of infected spotfin shiner and golden shiner, which harbored high numbers of viral RNA copies as measured by quantitative loop-mediated isothermal amplification. Infected spotfin shiner and golden shiner exhibited external petechiae, exophthalmia, oedematous kidneys, and liver pallor. Histopathological analysis revealed multifocal areas of necrosis in the kidney, spleen and liver of infected fish. Spotfin shiner and golden shiner were then infected with 2 doses of FHMNV (10(3.5) and 10(3.9) TCID(50) ml(-1)) by immersion to mimic more natural modes of infection. Spotfin shiner experienced 60% mortality at both doses, while golden shiner did not experience mortality nor develop any clinical signs following a 40 d observation period. Overall, piscivorous fish tested in this study do not seem to be at risk for infection, while cyprinids appear to vary in their susceptibility to the strain of FHMNV used in this study.

Isolation and characterization of a novel reovirus from white bream Blicca bjoerkna.

During a fish health inspection in the Viennese waterway 'Old Danube', a virus was isolated exclusively from white bream Blicca bjoerkna (L.) (formerly Abramis bjoerkna L.), one of the most abundant cyprinids present and not known as a host species for this virus. The virus preferentially replicated in cultures of the epithelioma papulosum cyprini cell line where focal plaques of infection developed slowly. Examination of infected cell cultures by electron microscopy revealed non-enveloped 60 to 70 nm icosahedral virions that had characteristic multiple segregated protrusions of their outer capsid. A partial RNA-dependent RNA polymerase gene sequence was obtained and a BLAST search indicated 76% identity to golden shiner reovirus and grass carp reovirus. These results suggested that the virus belonged to the genus Aquareovirus (Family Reoviridae). Phylogenetic analysis placed the isolated virus within a clade of the species Aquareovirus C species. Accordingly, the virus was tentatively designated as white bream reovirus (WBRV) strain A-127/06 within the species Aquareovirus C.

MicroRNA profile analysis of Epithelioma papulosum cyprini cell line before and after SVCV infection

MicroRNAs (miRNAs) play significant roles in regulating almost all of the biological processes in eukaryotes. An accumulating body of evidence shows that miRNAs are associated with cellular changes following viral infection. Spring viremia of carp virus (SVCV) is the pathogen of Spring viremia of carp (SVC), which results in heavy losses in the cultured common carp (Cyprinus carpio) industry in many countries. To study the involvement of miRNAs during SVCV infection, we adopted the Solexa sequencing technology to sequence small RNA libraries from the Epithelioma papulosum cyprini (EPC) cell line before and after infection with SVCV. In this study, a total of 161 conserved and 26 novel miRNAs were identified. Subsequently, the expression patterns of these miRNAs were compared between the uninfected (control library, M) and SVCV-infected (infection library, E) libraries. In addition, to verify the Solexa sequencing results, the expression patterns of 14 randomly selected miRNAs were validated by qRT-PCR. The targets of the significantly differentially expressed miRNAs were then predicted, and the miRNAs that could directly target the SVCV genome were also predicted. No miRNA encoded by SVCV itself was detected. To the best of our knowledge, this study presents the first miRNA profiling assessment in association with fish rhabdovirus infection, and the data presented lay a foundation for further investigations to determine the roles of miRNAs in regulating the molecular mechanism during SVCV infection.

Isolation and Infectious Temperature Optimization of Genetically Similar VHSV Isolates in Farmed Olive Flounder, Paralichthys olivaceus

Viral hemorrhagic septicemia virus (VHSV) was isolated from farmed olive flounder (Paralichthys olivaceus). The viral N-gene was amplified by reverse transcriptase PCR, cloned, and sequenced for phylogenetic analysis to identify the genotype (I-IV). Virus isolates were cultured on Epithelioma papulosum cyprini cell line and, after completion of the cytopathic effects, the supernatant was collected and used to challenge virus-free flounder. The infected founder were reared in 16°C, 21°C, or 25°C, and compared to an unchallenged control. Virus titration was measured in the head kidneys, spleens, livers, brains, muscles, and gills of challenged fish using real-time quantification of the VHSV G-gene. Phylogenetic analysis confirmed that the isolates were VHSV Genogroup IV. The VHSV-challenged fish in the 16°C group showed 100% mortality with significantly increased expression of viral G-gene mRNA in the spleen, compared to fish reared in other temperatures and the control fish, suggesting that fish reared in 16°C are more susceptible to VHSV infection.

Experimental susceptibility of Caspian white fish, Rutilus frisii kutum to Spring viraemia of carp virus

Caspian white fish (Rutilus frisii kutum) is a fish of the family Cyprinidae, which is commercially harvested from the Caspian Sea. Experimental infection with Spring viraemia of carp virus (SVCV) was conducted in order to examine susceptibility of caspian White Fish and clinical impacts of infection. Fingerling fish were injected intra-peritoneally or waterborne-exposed with SVCV and were monitored daily for 7weeks. Dead fish and those survived at the end of experimental period were collected for virus isolation and reverse transcriptase polymerase chain reaction analysis. Epithelioma papulosum cyprini cell line was used to re-isolate the virus and indirect fluorescent antibody test was conducted to identify the isolated virus. Infection trials showed that SVCV was highly pathogenic for the Caspian White Fish with mortality rate ranging from 75 to 85%, depending on the viral challenge model. SVCV genome was detected from dead and apparently healthy fish tissues of both virus exposure models, which showed Caspian White Fish not only can be regarded as a susceptible host, but also serve as a vector of the virus.

Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout–derived cell line, RTG‐2

Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout-derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

Propagation and isolation of ranaviruses in cell culture

The optimal in vitro propagation procedure for a panel of ranavirus isolates and the best method for isolation of Epizootic haematopoietic necrosis virus (EHNV) from organ material in cell-culture were investigated. The panel of ranavirus isolates included: Frog virus 3 (FV3), Bohle iridovirus (BIV), Pike-perch iridovirus (PPIV), European catfish virus (ECV), European sheatfish virus (ESV), EHNV, Doctor fish virus (DFV), Guppy virus 6 (GF6), short-finned eel virus (SERV) and Rana esculenta virus Italy 282/102 (REV 282/102). Each isolate was titrated in five cell lines: bluegill fry (BF-2), epithelioma papulosum cyprini (EPC), chinook salmon embryo (CHSE-214) rainbow trout gonad (RTG-2) and fathead minnow (FHM), and incubated at 10, 15, 20, 24 and 28 °C for two weeks. BF-2, EPC and CHSE-214 cells performed well and titers obtained in the three cell lines were similar, whereas FHM and RTG-2 cells consistently produced lower titers than the other cell lines at all temperatures. The optimal temperature for propagating the isolates collectively to high titers in vivo was 24 °C. Additionally, three established methods for re-isolation of virus from EHNV-infected organ material were compared. Challenged fish were sampled twice weekly and 7 organs were processed separately according to the three methods. Samples incubated on BF-2 cells at 22 °C for 2 weeks + 1 week sub-cultivation (method 1) provided more positive results than the other 2 methods and when using the EPC cell line. Virus was most frequently isolated from the kidney, followed by brain, muscle, heart, liver, gills and lastly spleen.

First detection and confirmation of spring viraemia of carp virus in common carp, Cyprinus carpio L., from Hamilton Harbour, Lake Ontario, Canada

In June 2006, 150 wild common carp were sampled from Hamilton Harbour, Lake Ontario, Canada. Tissue pools consisting of kidney, spleen and encephalon were screened for viruses as a condition facilitating the export of live carp to France. Cytopathic effect (CPE), indicative of a viral infection, became evident after 8 days of incubation at 15 degrees C. Eighteen of 30 tissue pools (five fish per pool) eventually demonstrated viral CPE. The viral pathogen was initially cultured and isolated on the epithelioma papulosum cyprini cell line and subsequently shown to produce CPE in the fathead minnow and bluegill fin cell lines. Electron microscopy demonstrated the virus to be a rhabdovirus. Reverse transcriptase-polymerase chain reaction assay and nucleotide sequence analysis identified the isolate as spring viraemia of carp virus (SVCV). Phylogenetic analysis of a 533 bp region of the glycoprotein gene grouped the Canadian isolate in SVCV genogroup Ia together with isolates from Asia and the USA. Sequence comparisons revealed the Hamilton Harbour, Lake Ontario isolate to be most similar to an isolate obtained from common carp in the Calumet Sag Channel in Illinois in 2003 (98.9% nucleotide identity). This is the first report of the detection of SVCV in Canada.

Effects of temperature on infectivity and of commercial freezing on survival of the North American strain of viral hemorrhagic septicemia virus (VHSV)

Temperature affected the growth of the North American strain of viral hemorrhagic septicemia virus (VHSV) in experimentally infected cell cultures and in Pacific sardine Sardinops sagax. In addition, commercial freezing significantly reduced the infectivity of VHSV in tissues of experimentally infected sardine. Isolates of VHSV representing the geographic range of North American VHSV replicated in the EPC (Epithelioma papulosum cyprini) cell line at 10, 15 and 20 degrees C, but the more northern isolates from British Columbia, Canada, demonstrated significantly reduced growth at 20 degrees C compared to VHSV from more southern locations (p <0.001). An injection challenge of Pacific sardine with VHSV from California resulted in 66.7% mortality at a seawater temperature of 13 degrees C compared to 6.7% at 20 degrees C. Commercial blast-freezing of sardine experimentally infected with VHSV reduced median concentrations of virus in the kidney and spleen from 5.25 x 10(6) to 5.5 x 10(3) pfu (plaque-forming units) g(-1). Decreased growth of the California isolate of VHSV at higher temperatures following experimental infection of the sardine and reduced virus survival following commercial freezing of infected sardine are factors that would lessen the risk of transmission of VHSV through frozen baitfishes.

Pathogenesis of Iridovirus: in vitro Influence on Macrophage Activity and Cytokine-Like Protein Production in Fish

Pathogenesis of Iridovirus: in vitro Influence on Macrophage Activity and Cytokine-Like Protein Production in Fish

A small RNA virus isolated from apparently healthy wild sandbar shiners,Notropisscepticus(Jordan &amp; Gilbert)

As part of the National Wild Fish Health Survey, the US Fish and Wildlife Service initiated a survey of pathogens present in wild fish populations. During that survey, a filterable agent was isolated from a pooled sample of sandbar shiners, Notropis scepticus (Jordan & Gilbert), at the Warm Springs Fisheries Center, Fish Health Laboratory, Warm Springs, Georgia. The shiners were collected from North Carolina’s Deep River at the SR 1456 bridge on October 22 1998. The liver, kidney, spleen and intestine of five apparently healthy sandbar shiners were pooled and homogenized in Hank’s balanced salt solution (HBSS, Sigma) at a dilution of 1:10 (w/v). The homogenate was further diluted in HBSS to 1:100 and centrifuged at 2500 g for 20 min at 4 °C. The resulting supernatant was stored overnight at 4 °C and inoculated onto epithelioma papulosum cyprini (EPC) and fathead minnow (FHM) cell lines cultured in minimum essential medium (Gibco) supplemented with 10% foetal bovine serum (FBS) (Fryer & Lannan 1994). Cell cultures were incubated at 20 °C; when a cytopathic effect (CPE) was observed, the supernatant was removed, passed through a 0.45-mm syringe filter, and transferred to fresh EPC and FHM cells. A CPE of focal cell rounding occurred in the FHM cell line 72-h post-inoculation. Focal aggregates of refractile, rounded cells became numerous and at 13 days post-inoculation the monolayer was destroyed. The EPC cell line was also susceptible to the viral agent; however, the CPE was more diffuse and exhibited cellular hypotrophy in addition to cell rounding. Monolayers of this cell line were completely destroyed following a 13-day incubation at 25 °C. Inoculation of the rainbow trout gonad cell line (RTG-2) with the isolate resulted in a focal CPE similar to that observed in the FHM cells (Fig. 1). Determination of particle size and morphology was achieved using electron microscopy of negatively stained particles. Briefly, the monolayers of EPC cells were inoculated with the virus suspension and incubated at 25 °C until the CPE was extensive. Monolayers were scraped from the flask surface and suspended in the culture supernatant. Cell-associated virions were released from the cells with two freeze–thaw cycles and the supernatant was clarified using centrifugation (Marathon 6K, Fisher Scientific, Pittsburgh, PA, USA) at 6000 g for 30 min. The supernatant was removed and centrifuged at 106000 g for 60 min in SSC buffer (0.15 M sodium chloride, 0.15 M sodium citrate, pH 7.4). The resulting pellet was suspended in 20 L SSC buffer. Negatively stained preparations examined by electron microscopy revealed spherical virions measuring c. 20–25 nm in diameter (Fig. 2). The thymidine analogue, 5-iodo-2-deoxyuridine (IDU), was used to determine the nucleic acid composition of the viral genome. Epithelioma papulosum cyprini cells were seeded at c. 90% confluency on a 48-well plate and incubated overnight in a 50-mg mL IDU solution at 25 °C (Rovozzo & Burke 1973). Media was removed from the cell monolayers; the cells were then inoculated with tenfold dilutions of the virus suspension in IDU solution and incubated at room temperature for 1 h. Fresh IDU solution supplemented with 10% FBS was then added to the Correspondence A E Goodwin, Aquaculture/Fisheries Center, University of Arkansas at Pine Bluff, 1200 N. University Drive, P.O. Box 4912, Pine Bluff, AR 71611, USA (e-mail: agoodwin@uaex.edu)

Development of a fish in vitro cell culture model to investigate oxidative stress and its modulation by dietary vitamin E

When cultured in Dulbecco's minimal essential medium the established epithelioma papulosum cyprini cell line from carp was found to be vitamin E-deficient due to the very low level of vitamin E in the medium and the foetal calf serum used as supplement. The toxicity of oxidative stressors to this cell line was evaluated by means of the neutral red cytotoxicity assay and it was found that an organic hydroperoxide, t-butylhydroperoxide was extremely cytotoxic and that the redox-cycling agents diquat and menadione were less toxic. When grown under vitamin E supplementation (25 μM), the toxicity of these chemicals was reduced by at least an order of magnitude in concentration demonstrating the protective effect of vitamin E. These data show the importance of vitamin E status for interpretation of in vitro and in vivo data and that this in vitro system is useful for mechanistic studies.