Mycotoxins in aquatic feeds and their effects on fish are becoming more important in aquaculture, as fishmeal and fish oil in feeds are being replaced with more sustainable plant protein. Here, we investigated the potential of the mycotoxin, beauvericin (BEA), to impact the rainbow trout (RT) intestine by using cultures of the epithelial cell line, RTgutGC. BEA was dosed in different ways and exposed at temperatures ranging from 4 to 26 °C before being evaluated for cell viability by the metabolic reduction of Alamar Blue, by the accumulation of Neutral Red (lysosomal activity), cytotoxicity (CellTox Green), and for wound healing. BEA induces cell death in RTgutGC cells. The lysosomes are the main target (Neutral Red assay is the most sensitive) while cytotoxicity and plasma membrane rupture (CellTox Green) occur at considerably higher concentrations. BEA caused a dose-dependent decline in Neutral Red reading at all tested temperatures but Alamar Blue readings did not decline at 4 °C. Under these conditions, BEA appears to impair only lysosomal activity. Wound healing was reduced at 4, 10, and 26 °C compared to 18 °C. Also BEA treatment, at non-cytotoxic concentrations, reduced wound healing, but the temperature had little influence on this. Different carrier vehicles (methanol, DMSO) and exposure methods (passive or active dispersal) for BEA exposure were also studied. Here, methanol and passive dispersal gave comparable results to exposure with DMSO and active dispersal. In contrast, when DMSO was dosed with passive dispersal, immediate cytotoxicity in combination with BEA was induced.

How temperature influences fish physiological systems, such as the intestinal barrier, is important for understanding and alleviating the impact of global warming on fish and aquaculture. Monolayers of the rainbow trout cell line, RTgutGC, with or without linear 500 mu m wide gaps (wounds) were the in vitro models used to study the integrity and healing of intestinal epithelial sheets at different temperatures. Cultures at hypothermic (4 degrees C) or hyperthermic (> 26 degrees C) temperatures were compared to normothermic control cultures (18-22 degrees C). Monolayers remained intact for at least a week at temperatures from 4 to 28 degrees C, but had lost their integrity after 3 h at 32 degrees C as the cells pulled away from one another and from the plastic surface. F-actin appeared as prominent stress fibers in cells at 28 degrees C and as blobs in cells at 32 degrees C. At normothermia and at 26 degrees C, cells migrated as sheets into the gaps and closed (healed) the gaps within 5-6 days. By contrast, wounds took 14 days to heal at 4 degrees C. At 28 degrees C some cells migrated into the gap in the first few days but mainly as single cells rather than collectively and wounds never healed. When monolayers with wounds were challenged at 32 degrees C for 3 h and returned to 18-22 degrees C, cells lost their shape and actin organization and over the next 6 days detached and died. When monolayers were subjected to 26 degrees C for 24 h and challenged at 32 degrees C for 3 h prior to being placed at 18-22 degrees C, cell shape and actin cytoskeleton were maintained, and wounds were healed over 6 days. Thus, intestinal epithelial cells become thermostabilized for shape, cytoskeleton and migration by a prior heat exposure.

Monolayer cultures of the rainbow trout cell line, RTgutGC, were used to study actions on the intestinal epithelium of a potential feed additive, the flavanone naringenin. Although having little effect on viability or morphology, naringenin altered the F-actin staining pattern. Stress fibers disappeared and peripheral actin became more intense. Naringenin enhanced barrier function, as measured by transepithelial electrical resistance (TEER) and Lucifer yellow permeability. When cell-free gaps or wounds were created, cells in L15/FBS migrated into and covered the gap but more slowly with naringenin. By contrast in L15, cells only partially migrated into the gap and high naringenin concentrations blocked migration. These cells in naringenin had fewer protrusion and stress fibers but some had prominent peripheral F-actin staining on the side facing the gap. Therefore, naringenin might be enhancing barrier function and impeding cell migration by a common mechanism, modulating actin dynamics.

The heat-shock protein 70 (Hsp70) inhibitor, VER-155008 (VER), was explored as a potential antiviral agent for two RNA viruses important to fish aquaculture, viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Studies were done at a temperature of 14 degrees C, and with cell lines commonly used to propagate these viruses. These were respectively EPC from fathead minnow for VHSV and CHSE-214 from Chinook salmon embryo for IPNV. Additionally, both viruses were studied with the Atlantic salmon heart endothelial cell line ASHe. For both VHSV and IPNV, 25 mu M VER impeded replication. This was evidenced by delays in the development of cytopathic effect (CPE) and the expression of viral proteins, N for VHSV and VP2 for IPNV, and by less production of viral RNA and of viral titre. As VER inhibits the activity of Hsp70 family members, these results suggest that VHSV and IPNV utilize one or more Hsp70s in their life cycles. Yet neither virus induced Hsp70. Surprisingly VER alone induced Hsp70, but whether this induction modulated VER's antiviral effects is unknown. Exploring this apparent paradox in the future should improve the usefulness of VER as an antiviral agent.

The heat-shock protein 70 (Hsp70) inhibitor, VER-155008 (VER), was explored as a potential antiviral agent for two RNA viruses important to fish aquaculture, viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Studies were done at a temperature of 14 °C, and with cell lines commonly used to propagate these viruses. These were respectively EPC from fathead minnow for VHSV and CHSE-214 from Chinook salmon embryo for IPNV. Additionally, both viruses were studied with the Atlantic salmon heart endothelial cell line ASHe. For both VHSV and IPNV, 25 μM VER impeded replication. This was evidenced by delays in the development of cytopathic effect (CPE) and the expression of viral proteins, N for VHSV and VP2 for IPNV, and by less production of viral RNA and of viral titre. As VER inhibits the activity of Hsp70 family members, these results suggest that VHSV and IPNV utilize one or more Hsp70s in their life cycles. Yet neither virus induced Hsp70. Surprisingly VER alone induced Hsp70, but whether this induction modulated VER's antiviral effects is unknown. Exploring this apparent paradox in the future should improve the usefulness of VER as an antiviral agent.

Rainbow trout cell cultures were exposed to three genotoxicants and examined for effects on γH2AX and p53 levels by western blotting and on cell viability using the indicator dyes Alamar Blue (AB) for energy metabolism and 5'-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for plasma membrane integrity. Bleomycin induced γH2AX and p53 in a dose- and time-dependent manner and had little cytotoxic effect. However, induction was first seen at 0.3 μM for γH2AX but not until 16.5 μM for p53. Methyl methanesulfonate (MMS) increased H2AX phosphorylation but diminished p53 levels as the dose was increased from 908 μM up to 2724 μM. Over this dose range cell viability was progressively lost. 4-nitroquinoline N-oxide (NQO) induced both γH2AX and p53, beginning at 62.5 nM, which was also the concentration at which cell viability began to decline. As the NQO concentration increased further, elevated γH2AX was detected at up to 2.0 μM, while p53 was elevated up to 1.0 μM. Therefore, H2AX phosphorylation was superior to p53 levels as a marker of DNA damage caused by genotoxicants that act by introducing double-stranded DNA breaks (bleomycin), alkyl groups (MMS), and quinoline adducts (NQO).

As global warming and environmental pollution modify aquatic environments, the thermal biology of fish could be affected by interactions between temperature and pollutants, such as selenium (Se). Therefore, selenomethionine (SeMet) was studied for effects on cell viability and on heat shock protein 70 (HSP70) levels in the rainbow trout intestinal epithelial cell, RTgutGC, at hypothermic (4 °C), normothermic (14 and 18 °C) and hyperthermic (26 °C) temperatures. RTgutGC cultures remained viable for at least a week at all temperatures, although energy metabolism as measured with Alamar Blue (resazurin) was appreciably diminished at 4 °C. Over a 7-day incubation, HSP 70 levels in cultures remained steady at 4 °C, declined at 18 °C, and increased slightly at 26 °C. When 125 μM SeMet was present, cultures remained viable and HSP70 levels were neither increased nor decreased relative to control cultures, regardless of the temperature. With 500 and 1000 μM SeMet, cell viability was profoundly impaired after 7 days in cultures at 14, 18 and 26 °C but was unchanged at 4 °C. Overall the results suggest that only hypothermia modulated the response of rainbow trout cells to SeMet.

Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC epsilon protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC epsilon activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras.c-Raf-1.PKC epsilon complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.

The p21-activated kinase, Pak, has recently been shown to phosphorylate Raf-1 on serine 338 (S338), a critical regulatory residue. The specificity requirements for Pak-mediated phosphorylation of S338 were examined by substitution analysis of Raf-1 peptides and conserved region 3 (CR3) proteins. Phosphorylation was found to be very sensitive to alterations in amino acid side chains proximal to S338. Loss of N-terminal arginines resulted in decreased peptide phosphorylation while loss of these residues, as well as C-terminal glutamates and bulky C-terminal hydrophobic residues, decreased phosphorylation of the CR3 protein. Phosphorylation of Raf-1 on tyrosine 341 is significant in epidermal growth factor- and Src-mediated signaling, suggesting that cooperativity may exist between Pak and Src phosphorylation of Raf-1. Purified Pak and Src were found not to be cooperative in phosphorylating peptides or purified CR3 protein. However, the phosphorylation of Raf-1 S338 by Pak was increased in the presence of Src. The complexity of this signaling module could thus account for the different levels of Raf-1 activation required for fulfillment of different biological roles within the cell.

C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serum-free medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A. (1998)Oncogene 16, 1417–1428), suggesting that Ha-Ras-mediated regulation of the MAPK cascade is dependent upon the action of an autocrine factor. Serum-free medium conditioned by V12H10 cells contains an activity that stimulates MAPK activity in quiescent fibroblasts. This MAPK stimulatory activity could be specifically blocked by the epidermal growth factor receptor (EGFR) inhibitors, PD153035 and PD158780. These inhibitors also blocked the serum-independent proliferation of V12H10 cells. Immunodepletion of conditioned medium with antibodies to transforming growth factor α and EGF significantly inhibited its ability to stimulate MAPK activity. Stable transfection of EGFR-negative NR6 and EGFR-positive Swiss3T3 cells with oncogenic (G12V)Ha-Ras demonstrated that only the Ha-Ras-transfected Swiss 3T3 cells possessed constitutive MAPK activity, and this activity was sensitive to PD153035. These data suggest that autocrine activation of the EGFR is required for the regulation of the MAPK cascade in cells minimally expressing oncogenic Ha-Ras.