Search

Arctic charr (Salvelinus alpinus) are the northernmost distributed freshwater fish and can grow at water temperatures as low as 0.2 °C. Other teleost species have impaired immune function at temperatures that Arctic charr thrive in, and thus, charr may maintain immune function at these temperatures. In this study, a fibroblastic cell line, named ACBA, derived from the bulbus arteriosus (BA) of Arctic charr was developed for use in immune studies at various temperatures. ACBA has undergone more than forty passages at 18 °C over 3 years, while showing no signs of senescence-associated β-galactosidase activity and producing nitric oxide. Remarkably, ACBA cells survived and maintained some mitotic activity even at 1 °C for over 3 months. At these low temperatures, ACBA also continued to produce MH class I proteins. After challenge with poly I:C, only antiviral Mx proteins were induced while MH proteins remained constant. When exposed to live viruses, ACBA was shown to permit viral infection and replication of IPNV, VHSV IVa and CSV at 14 °C. Yet at the preferred temperature of 4 °C, only VHSV IVa was shown to replicate within ACBA. This study provides evidence that Arctic charr cells can maintain immune function while also resisting infection with intracellular pathogens at low temperatures., (© 2017 John Wiley & Sons Ltd.)

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD + poly I:C, lipopolysaccharide (LPS), LPS + poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11 days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P < 0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression., (© 2016 John Wiley & Sons Ltd.)

A cell line, WE-cfin11e, with an epithelial-like morphology was developed from a caudal fin of walleye, Sander vitreus (Mitchill), characterized as distinct from the established walleye caudal fin fibroblast-like cell line, WE-cfin11f, and compared with WE-cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscopy were used to localize the intermediate filament protein, vimentin, the tight junction protein, zonula occludens-1 (ZO-1), the extracellular matrix protein, collagen I, and the viral protein, G. Although both cell lines contained vimentin, only WE-cfin11e stained for ZO-1 and only WE-cfin11f stained for collagen I. Ascorbic acid increased the accumulation of collagen I and caused the appearance of collagen fibres only in WE-cfin11f cultures. At 14 °C, both cell lines produced VHSV IVb, but the infection developed more rapidly in WE-cfin11f. At 4 °C, both cell lines became infected with VHSV IVb as judged by the expression of viral proteins, N and G, but only WE-cfin11f produced virus. The results suggest that cold temperatures can modulate viral tropism., (© 2015 John Wiley & Sons Ltd.)

Compared to fathead minnow, walleye demonstrate low susceptibility to experimental infection with VHSV IVb, regardless of route of exposure or water temperature at time of infection. In triplicate and duplicate groups, walleye were intraperitoneally (i.p.) injected (10 2 -10 8  pfu/fish) or waterborne-exposed (w; 1.4 × 10 7  pfu mL -1 ) with VHSV IVb. High cumulative mortality (64-100%) and severe gross lesions associated with VHSV IVb infection were evident only in fish i.p. injected with 10 8  pfu at 12 °C. These fish had multifocal necrosis of several tissues including the gill and heart. There was no difference in mortality between walleye infected (w or i.p.) at 12 °C (spring stocking) compared with a declining temperature profile from 18 to 12 °C (fall stocking). There were significant differences (P < 0.05) in mortality between four extant walleye strains following i.p. infection, indicating that the choice of walleye strain for stocking might be an important consideration. Viral antigen was found in both i.p. and w-exposed walleye using immunohistochemistry, mostly within the gill and skin of w-exposed fish and most prominently in dermal fibrocytes. VHSV IVb was detected in multiple tissues from 6 to 21 days post-infection using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)., (© 2014 John Wiley & Sons Ltd.)

The capacity of rainbow trout, Oncorhynchus mykiss, to be a host for frog virus 3 (FV3) was evaluated at the cellular level. Cell cultures from this species were tested for their ability to express FV3 major capsid protein (MCP) gene, to develop cytopathic effect (CPE), and to produce FV3. After FV3 addition, MCP transcripts were detected in six of six cell lines and in primary macrophage cultures. CPE developed in all cell culture systems, except primary lymphocytes. For the macrophage cell line, RTS11, and primary macrophages, cell death was by apoptosis because DNA laddering and Annexin staining were detected. By contrast, markers of apoptosis did not accompany CPE in three epithelial cell lines from the gill (RTgill-W1), intestine (RTgut-GC), and liver (RTL-W1) and in two fibroblast cell lines from gonads (RTG-2) and skin (RTHDF). Therefore, FV3 was able to enter and begin replicating in several cell types. Yet, FV3 was produced in only two cell lines, RTG-2 and RTL-W1, and only modestly. Overall, these results suggest that if tissue accessibility were possible, FV3 would have the capacity to induce injury, but the ability to replicate would be limited, likely making rainbow trout a poor host for FV3.

A cell line, WE-cfin11f, with a fibroblast-like morphology was developed from a walleye caudal fin and used to study the intersection of thermobiology of walleye, Sander vitreus (Mitchill), with the thermal requirements for replication of viral haemorrhagic septicaemia virus (VHSV) IVb. WE-cfin11f proliferated from 10 to 32 °C and endured as a monolayer for at least a week at 1-34 °C. WE-cfin11f adopted an epithelial shape and did not proliferate at 4 °C. Adding VHSV IVb to cultures at 4 and 14 °C but not 26 °C led to cytopathic effects (CPE) and virus production. At 4 °C, virus production developed more slowly, but Western blotting showed more N protein accumulation. Infecting monolayer cultures at 4 °C for 7 days and then shifting them to 26 °C resulted in the monolayers being broken in small areas by CPE, but with time at 26 °C, the monolayers were restored. These results suggest that at 26 °C, the VHSV IVb life cycle stages responsible for CPE can be completed, but the production of virus and the initiation of infections cannot be accomplished., (© 2013 John Wiley & Sons Ltd.)

The effects of Corexit 9500, a dispersant used to clean up oil spills, on invertebrates, lower vertebrates, birds, and human health have been examined, but there is a significant lack of study of the effect of this dispersant on aquatic viruses. In this study, the effects of Corexit 9500 on four aquatic viruses of differing structural composition were examined. Corexit 9500 reduced the titer of the enveloped viral hemorrhagic septicemia virus (VHSV) at all concentrations (10% to 0.001%) examined. The titer of frog virus 3 (FV3), a virus with both enveloped and nonenveloped virions, was reduced only at the high Corexit 9500 concentrations (10% to 0.1%). Corexit 9500 was unable to reduce the titer of nonenveloped infectious pancreatic necrosis virus (IPNV) but enhanced the titer of chum salmon reovirus (CSV) by 2 to 4 logs. With the ability to inactivate enveloped viruses and possibly enhance some nonenveloped viruses, Corexit 9500 has the potential to alter the aquatic virosphere.

Using ECIS (electric cell-substrate impedance sensing) to monitor the impedance of vertebrate cell monolayers provides a sensitive measure of toxicity for a wide range of chemical toxicants. One major limitation to using a cell-based sensor for chemical toxicant detection in the field is the difficulty in maintaining cell viability over extended periods of time prior to use. This research was performed to identify cell lines suitable for ECIS-based toxicity sensing under field conditions. A variety of invertebrate and vertebrate cell lines were screened for their abilities to be stored for extended periods of time on an enclosed fluidic biochip with minimal maintenance. Three of the ten cell lines screened exhibited favorable portability characteristics on the biochips. Interestingly, all three cell lines were derived from ectothermic vertebrates, and the storage temperature that allowed long-term cell survival on the enclosed fluidic biochips was also at the lower end of reported body temperature for the organism, suggesting that reduced cellular metabolism may be essential for longterm survival on the biochip. Future work with the ectothermic vertebrate cells will characterize their sensitivity to a wide range of chemical toxicants to determine if they are good candidates for use in a field portable toxicity sensor., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The ingestion and digestion of Escherichia coli by the ciliated protozoan, Tetrahymena thermophila, was investigated after an initial exposure to either water-soluble single-walled carbon nanotubes (SWNT) or to carbon black (CB). Both SWNT and CB were internalised and visible in food vacuoles of ciliates. When presented with E. coli expressing green-fluorescent protein (GFP), these ciliates internalised bacteria as well. However, ciliates that had first internalised SWNT but not CB subsequently externalised or egested vesicle-like structures with fluorescent bacteria inside. These egested bacteria were viable and less susceptible than planktonic E. coli to killing either by the antibiotic, chloramphenicol or the disinfectant, glutaraldehyde. These results suggest that SWNT can alter the intracellular trafficking of vesicles within ciliates, leading to bacterial prey being packaged externally and protected for a time from environmental killing, which could have implications for sewage treatment and for public health.

The two most prominent genotypes of viral hemorrhagic septicemia virus (VHSV) are -I in the Northeastern Atlantic region and -IV in North America, but much more is known about the cellular pathogenesis of genotype -I than -IV. VHSV genotype -IV is divided into -IVa from the Northeast Pacific Ocean and -IVb from the Great Lakes and both of which are less virulent to rainbow trout than genotype -I. In this work, infections of VHSV-IVa and -IVb have been studied in two rainbow trout cell lines, RTgill-W1 from the gill epithelium, and RTS11 from spleen macrophages. RTgill-W1 produced infectious progeny of both VHSV-IVa and -IVb. However, VHSV-IVa was more infectious than -IVb toward RTgill-W1: -IVa caused cytopathic effect (CPE) at a lower viral titre, elicited CPE earlier, and yielded higher titres. By contrast, no CPE and no increase in viral titre were observed in RTS11 cultures infected with either genotype. Yet in RTS11 all six VHSV genes were expressed and antiviral genes, Mx2 and Mx3, were up regulated by VHSV-IVb and -IVa. However, replication appeared to terminate at the translational stage as viral N protein, presumably the most abundant of the VSHV proteins, was not detected in either infected RTS11 cultures. In RTgill-W1, Mx2 and Mx3 were up regulated to similar levels by both viral genotypes, while VHSV-IVa induced higher levels of IFN1, IFN2 and LGP2A than VHSV-IVb., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A survey of immune-relevant genes that might be up-regulated in response to viral hemorrhagic septicaemia virus (VHSV) in the rainbow trout monocyte-macrophage cell line, RTS11, unexpectedly revealed an increased expression of perforin (PRF) and granzyme (GRZ) genes, which represent components of the major cytotoxic pathway. The natural killer-enhancing factor (NKEF), also known to modulate cytotoxic activity, was up-regulated at the gene but strikingly down-regulated at protein level. The expression of these genes was not affected in head kidney leukocytes (HKLs) infected with VHSV, leading us to evaluate the potential cytotoxic activity of RTS11 and HKLs. For the first time, the cytotoxic activity of RTS11 against xenogeneic targets has been demonstrated, although this was modest relative to HKLs. Yet the activity in RTS11 was significantly increased by VHSV, as in HKLs. This cytotoxic activity elicited by viral infection appeared to require viral gene expression because inactivated VHSV failed to increase RTS11 cytotoxic activity. As for other immune functions, RTS11 cells provide a model for further studying cytotoxic activities of fish monocyte-macrophages., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A zebrafish spleen cell line, ZSSJ, was developed and its growth arrest by gamma radiation determined and its capacity to stimulate the proliferation of the zebrafish blastula cell line, ZEB2J, measured. ZSSJ was initiated by explant outgrowth, grew adherent with mainly an epithelial-like morphology, and stained strongly for alkaline phosphatase. ZSSJ was not only grown in L-15 with 15% fetal bovine serum at 26 degrees C to 28 degrees degrees C but also grew at room temperature. Cultures of ZSSJ have undergone approximately 40 population doublings, had few cells staining for b-galactosidase activity, which is commonly present in senescent cultures, and many cells with an aneuploid karyotype, which is frequently associated with immortalization. ZSSJ growth was arrested by 30 to 50 Gy of g-irradiation, whereas after 20 Gy, some slight growth was observed. By contrast, growth of the rainbow trout spleen stromal cell line, RTS34st, which has been used as a feeder for zebrafish ES cell cultures, was arrested completely by 20 Gy. In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures.

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 have an epithelioid morphology and form tight monolayer sheets that can be used for testing epithelial resistance. The cells can be grown in regular tissue culture surfaces or in transwell membranes in direct contact with water on their apical surfaces. The ability of RTgill-W1 to withstand hypo- and hyper-osmotic conditions and their optimal growth capacity at room temperature, make these cells ideal sentinel models for in vitro aquatic toxicology as well as model systems to study fish gill function and gill diseases. RTgill-W1 support growth of paramyxoviruses and orthomyxoviruses like salmon anemia virus. RTgill-W1 also support growth of Neoparamoeba pemaquidensis, the causative agent of amoebic gill disease. The cells have been used to understand mechanisms of toxicity, ranking the potencies of toxicants, and evaluating the toxicity of environmental samples. These cells are also valuable for high throughput toxicogenomic and toxicoproteomic studies which are easier to achieve with cell lines than with whole organisms. RTgill-W1 cell line could become a valuable complement to whole animal studies and in some cases as gill replacements in aquatic toxicology.

The rainbow trout monocyte/macrophage-like cell line, RTS11, has been used to study homotypic aggregation (HA), which is a well-studied feature of leucocytes in mammals but less understood in fish. HA is the aggregation of cells of the same cell type. RTS11 underwent HA in response to polyinosinic:cytidylic acid (poly IC), polyadenylic acid (poly A), lipopolysaccharide (LPS), zymosan, and phorbol 12-myristate 13-acetate (PMA). Poly IC was the best inducer of HA and did so in a dose- and time-dependent manner. The induction of RTS11 aggregation by poly IC required divalent cations but was not blocked by either an inhibitor of lymphocyte function-associated molecule-1 (LFA-1) or the tripeptide integrin adhesion recognition sequence, RGD. Poly IC-induced HA was inhibited by colchicine and latrunculin B, which act on microtubules and microfilaments, respectively, implying the necessity for an intact cytoskeleton. HA induction by poly IC did not occur at 4 degrees C and was blocked by the transcriptional and translational inhibitors, actinomycin D and cycloheximide, respectively, suggesting the requirement for de novo protein synthesis. Poly IC-induced RTS11 aggregation was blocked by two inhibitors of dsRNA-dependent protein kinase (PKR). This is the first indication that PKR could have a role in the HA of leucocytes.

A continuous cell line, PBLE, was developed from the adherent cells in a culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. The cells were grown in Leibovitz's L-15 basal medium supplemented with 20% fetal bovine serum (FBS). Under normal culture conditions at 18 degrees C, the morphology of PBLE was fibroblast-like. The cultures have been subcultured over 80 times and have been cryopreserved successfully. These cells have a diploid karyotype of 38 chromosomes, survived temperatures from 5 to 36 degrees C, and proliferated at temperatures from 5 degrees C to at least 30 degrees C. PBLE underwent apoptosis in response to gliotoxin, but did not show a respiratory burst. Results suggest that PBLE may have arisen from a circulating mesenchymal stem cell. PBLE was susceptible to Chum salmon reovirus (CSV) and supported CSV replication. Therefore this cell line should be useful in studying eel specific virus-host interactions.

In experiments investigating the adhesive properties of the rainbow trout splenic monocyte-like cell line RTS11 it was found that the cells bound with low affinity to plates coated with bovine serum albumin (BSA) but that phorbol ester-induced activation/differentiation greatly increased adhesion to BSA. Similarly, pre-exposure to 500 microM MnCl(2) at time of plating, increased RTS11 adhesion to BSA coated plates, in agreement with the reported ability of divalent cations such as Mn(2+) to activate integrins. Integrins are a diverse family of heterodimeric cell surface glycoproteins that have been shown to mediate cell-cell and cell-extracellular matrix adhesion. Transcripts of the beta(2)-integrin CD18 were detected by PCR in RTS11 but not in RTG-2 cells, a fibroblastic lineage derived from rainbow trout gonads. These results suggest that differentiated RTS11 express molecules related to members of the beta(2)-integrin subfamily such as the macrophage lineage marker Mac-1 (CD11b/CD18) and/or p150,95 (CD11c/CD18) and possibly as well alpha(4)beta(1) of the beta(1)-integrin subfamily.

IFN-gamma is one of the key cytokines in defining Th1 immune responses. In this study, an IFN-gamma homologue has been identified in rainbow trout Oncorhynchus mykiss, and its biological activities have been characterized. The trout IFN-gamma cDNA is 1034 bp in length and translates into a 180-aa protein. The first intron of the trout IFN-gamma gene contains highly polymorphic GACA minisatellites and 44-bp DNA repeats, giving rise to at least six alleles. IFN-gamma is structurally conserved among vertebrates, and a signature motif has been identified. A nuclear localization sequence known to be crucial for IFN-gamma biological activities is also present in the C-terminal region of the trout IFN-gamma. The IFN-gamma expression was induced in head kidney leukocytes by stimulation with PHA or poly(I:C) and in kidney and spleen of fish injected with poly(I:C). rIFN-gamma produced in Escherichia coli significantly stimulated gene expression of IFN-gamma-inducible protein 10 (gammaIP-10), MHC class II beta-chain, and STAT1, and enhanced respiratory burst activity in macrophages. Deletion of 29-aa residues from the C terminus containing the nuclear localization sequence motif resulted in loss of activity with respect to induction of gammaIP-10 in RTS-11 cells. Moreover, IFN-gamma-induced gammaIP-10 expression was completely abolished by the protein kinase C inhibitor staurosporine, and partially reduced by U0126, a specific inhibitor for ERKs. Taken together, the present study has demonstrated for the first time a functional IFN-gamma homologue in a fish species, strongly suggesting a conserved Th1 immune response is most likely present in lower vertebrates.

Epithelial (CHSE-214), fibroblast (RTG-2) and macrophage (RTS11) cell lines from Chinook salmon and rainbow trout were tested for their sensitivity to gliotoxin, a fungal metabolite. Gliotoxin treatment for 6 or 24 h caused cell viability to decrease in a dose-dependent manner, with effective concentrations (EC50s) being similar for the three cell lines but varying with exposure time. Under some exposure conditions, hallmarks of apoptosis were detected. Apoptosis was evaluated by the appearance of fragmented nuclei upon H33258 staining and of genomic DNA laddering into 180 bp oligomers. Gliotoxin induced cell detachment in RTG-2 and CHSE-214 cultures, under some conditions. These were the only cultures of these two cell lines in which apoptosis was detected, and apoptotic cells appeared more frequent in the detached population. At the highest concentration, 15 microM, the cells died by an alternative mode, likely necrosis. By contrast, in RTS11 cultures cell detachment was not observed, and apoptosis occurred over a wider concentration range, even 15 microM, reaching levels of over 90%. The preferential death by necrosis for epithelial cells (CHSE-214) and by apoptosis for macrophages (RTS11) could be a beneficial host response to gliotoxin-producing fungi, leading respectively to the development and then resolution of inflammation.

The rainbow trout macrophage cell line RTS11 was found to be considerably more sensitive than rainbow trout fibroblast (RTG-2) and Chinook salmon epithelial (CHSE-214) cell lines to killing by macromolecular synthesis inhibitors, actinomycin D (AMD) and cycloheximide (CHX), a synthetic double stranded RNA (dsRNA), polyinosinic:polycytidylic acid (poly IC), and combinations of poly IC with AMD or CHX. Exposures of 24-30 h to AMD or CHX alone killed RTS11, but not CHSE-214 and RTG-2, in basal medium, L-15, with or without fetal bovine serum (FBS) supplementation. A two-week exposure to poly IC killed RTS11 in L-15, whereas RTG-2 and CHSE-214 remained viable. At concentrations that caused very little or no cell death, CHX or AMD pretreatments or co-treatments sensitized RTS11 to poly IC, causing death within 30 h. In all cases death was by apoptosis as judged by two criteria. H33258 staining revealed a fragmented nuclear morphology, and genomic degradation into oligonucleosomal fragments was seen with agarose gel electrophoresis. With AMD- or CHX-induced death, killing seemed caspase-independent as the pan caspase inhibitor, z-VAD-fmk, failed to block killing. By contrast, z-VAD-fmk almost completely abrogated killing by co-treatments of poly IC and low concentrations of AMD or CHX, suggesting caspase dependence. Killing by both types of treatments was blocked by 2 aminopurine (2-AP), which suggests the involvement of dsRNA-dependent protein kinase (PKR). The sensitizing of RTS11 to poly IC killing by AMD or CHX could be explained by a decrease in the level of a short-lived anti-apoptotic protein(s) and/or by the triggering of a ribotoxic stress.

Interleukin (IL)-11 is a multifunctional cytokine that stimulates hematopoietic progenitor cells and exerts a series of important immunomodulatory effects. It was believed to be restricted to mammals, but here we report the first nonmammalian IL-11 gene, in rainbow trout (Oncorhynchus mykiss). A trout IL-11 cDNA clone was isolated that contains a 5'-untranslated region (UTR) of 400 bp, an open reading frame of 612 bp and a large 3'-UTR of 1924 bp. Analysis of a genomic DNA clone from a trout lambda library revealed that the trout IL-11 gene has the same five exon/four intron gene organization, as well as the same intron phase, as mammalian IL-11 genes. The 204 amino acid trout IL-11 translation has a predicted signal peptide of 26 amino acids and mature peptide of 178 amino acids, with a calculated molecular mass of 20.5 kDa and a theoretical pI of 9.83. The mature peptide contains a cysteine residue and a potential N-linked glycosylation site that are not present in mammals. Phylogenetic analysis clearly grouped trout IL-11 with IL-11 molecules from other species and separated from other members of the IL-6 family. The IL-11 gene is highly expressed in intestine and gills in healthy fish and its expression can also be detected in spleen, head kidney, brain, skin and muscle. Bacterial infection of rainbow trout markedly up-regulates IL-11 expression in liver, head kidney and spleen. IL-11 expression is also up-regulated in RTS-11 cells (a trout macrophage cell line), which constitutively expressed the lowest level of IL-11 of the four trout cell lines examined, after stimulation with bacteria, lipopolysaccharide, poly(I:C) and recombinant trout IL-1beta. Only a single transcript of 3.2 kb could be detected in lipopolysaccharide or recombinant IL-1beta-stimulated RNA samples by northern blotting. The expression results, showing that IL-11 is widely distributed and modulated by infection and other cytokines, suggest that fish IL-11 is an active player in the cytokine network and the host immune response to infection.

Induction of cytochrome P450 (CYP1A), as measured by liver ethoxyresorufin-O-deethylase (EROD) activity in juvenile rainbow trout (Oncorhynchus mykiss), was used to derive relative potency factors (RPFs) for several polycyclic aromatic hydrocarbons (PAHs), chosen for their induction potency in a rainbow trout liver cell line (RTL-W1). Potency for causing induction was estimated as the median effective concentration (EC50) from exposure-response curves. With the exception of phenanthrene, all PAHs tested induced EROD activity in juvenile trout, ranked as: benzo[k]fluoranthene>benzo[b]fluoranthene>benzo[b]fluorene>beta-napthoflavone>retene (7-isopropyl-1-methylphenanthrene). When induction potency was expressed relative to benzo[k]fluoranthene, RPFs ranged from 0.02 to 1, and the rank order in vivo was identical to the rank order with RTL-W1-derived values. The additivity of PAHs in mixtures in RTL-W1 cells was compared to whole-fish results from a previous study. EROD induction showed additive interactions for PAHs with exposure-response curves of similar slopes. This study demonstrates that assays of CYP1A induction using rainbow trout liver cells in culture would be a convenient substitute for assays with whole fish as part of testing programs for risk assessment of PAHs.

A homologue of interleukin 18 has been identified from rainbow trout, Oncorhynchus mykiss. The trout IL-18 gene spans 3.7 kb and consists of six exons and five introns, sharing the same gene organization with its human counterpart. The putative translated protein is 199 amino acids in length with no predicted signal peptide. Analysis of the multiple sequence alignment reveals a conserved ICE cut site, resulting in a mature peptide of 162 amino acids. The trout IL-18 shares 41-45% similarity with known IL-18 molecules and contains an IL-1 family signature motif. It is constitutively expressed in a wide range of tissues including brain, gill, gut, heart, kidney, liver, muscle, skin and spleen. Transcription is not modulated by lipopolysaccharide, poly(I:C) or trout recombinant IL-1beta in primary head kidney leucocyte cultures and RTS-11 cells, a macrophage cell line. However, expression is downregulated by lipopolysaccharide and rIL-1beta in RTG-2 cells, a fibroblast-like cell line. An alternatively spliced form of IL-18 mRNA has also been found and translates into a 182 amino acid protein with a 17 amino acid deletion in the precursor region of the authentic form. This alternatively spliced form is also widely expressed although much lower than the authentic form. Interestingly, its expression is upregulated by lipopolysaccharide and poly(I:C), but is not affected by rIL-1beta in RTG-2 cells. The present study suggests that alternative splicing may play an important role in regulating IL-18 activities in rainbow trout.

A lambda clone containing a rainbow trout IL-1beta1 gene was isolated by a PCR screening strategy from a genomic library cloned in lambda GEM-11, and an EcoRI fragment from this clone was fully sequenced, and contained 1680 bp 5'-flanking sequence, the whole IL-1beta1 gene open reading frame, and the 3'-flanking region with two potential poly A signals and poly A sites. This clone encoded a protein that shared 99.8% identity to the previously published trout IL-1beta1 cDNA sequence, with only three base substitutions. The main difference was that this clone had an additional complete HpaI SINE insertion in the 3rd intron making intron III 211 bp larger (834 bp via 623 bp). Thus this sequence was designated as allele B (Big intron III) of IL-1beta1 and the previously reported sequence as allele S (Short intron III). Three lines of evidence (allele specific PCR, cloning and sequencing, and direct sequencing of PCR products) revealed that allele B was constitutively expressed and could respond to stimulation with lipopolysaccharide or trout recombinant IL-1beta. Searching of the GenBank database with the HpaI SINE sequence resulted in three additional HpaI loci being identified in rainbow trout. Another SINE retroposition was also identified in the same intron of both alleles of IL-1beta1 by comparison with the trout IL-1beta2 gene. This novel SINE sequence, sharing high homology with the HpaI SINE at the 3'-end region, is present in EST databases of several species including human, mouse and fish. The consensus of this novel SINE shares 57 to 61% identities to tRNA-Leu from different species. Another older retroposition event in the same intron of IL-1beta1 has also been hypothesised, recognised as six adenines, that may function as a RNA polIII terminator. A model for the IL-1beta1 allele formation is proposed. Following the earliest retroposition into one of the two IL-1beta genes that resulted from a genome duplication in salmonids, the proper environment for successive PV SINE retroposition was created. A recent retroposition of the HpaI SINE in IL-1beta1 resulted in the formation of the two alleles of IL-1beta1. Examination of the SINEs insertion and their host gene microenvironments revealed that the SINE retroposition does not appear random, both in the site selection and the direction of insertion. The mechanism governing this outcome is discussed.

Recently a number of cytokine homologs have been cloned in teleost fish, including several that resemble chemokines, but to date few have been confirmed using functional assays. Chemokines are a family of cytokines that are able to induce chemotaxis in leucocytes. In this study CK-1, a rainbow trout chemokine, was functionally characterised. Recombinant CK-1 is able to attract rainbow trout peripheral blood leucocytes (PBL) in a micro-chemotaxis chamber. A greater number of PBLs migrated in response to CK-1 than to negative controls, either media alone or equivalent concentrations of beta2M, while comparable numbers migrated to the positive control, recombinant human C5a. The tissue distribution of CK-1 mRNA was also assessed by Northern blotting of RT-PCR and showed that expression is constitutive in the liver and gut, and is inducible by intraperitoneal injection of phytohemagglutinin in PBL and the head-kidney. Continuous cell lines generated from the gut and pituitary gland of the rainbow trout also express CK-1 message, whilst Southern analysis shows that CK-1 is a single copy gene. Finally, CK-1 shows the greatest amino acid similarity CCL20/LARC/Mip-3alpha as well as similar gene structure and expression pattern.

Tumor necrosis factor (TNF) is a key mediator in regulating the inflammatory response. Previously two TNF genes have been cloned and sequenced from rainbow trout, Oncorhynchus mykiss. In this study, the mature peptides of the two TNF molecules were produced in bacteria, purified under native conditions and their bioactivities evaluated in vitro. Both trout rTNF1 and rTNF2 induced gene expression of a number of proinflammatory factors including IL1beta, TNF1, TNF2, IL8 and COX2 in freshly isolated head kidney leucocytes and the macrophage cell line RTS11. The stimulatory doses of both rTNFs were >or=10 ng/ml. Moreover, leucocyte migration and phagocytic activity were enhanced in vitro by the rTNFs in a dose dependent manner. Western blot analysis revealed the presence of multiple forms of rTNF structures including monomeric, dimeric and trimeric forms, suggesting that formation of a homotrimeric structure may be essential for the TNF bioactivities.

A full-length cDNA encoding CCAAT/enhancer-binding protein beta (C/EBPbeta) was cloned from rainbow trout by anchored PCR. The putative 291 amino acid protein has 53% and 32% identity to the zebrafish and Japanese flounder sequences, respectively, and 30-34% identity to tetrapod homologues. This clone contains most conserved C/EBPbeta domains except the second transactivation domain just like the zebrafish homologue. Also similar to zebrafish, rainbow trout produces only shorter C/EBPbeta isoforms (LAP and LIP) but not the longer isoform (LAP*). However, unlike the zebrafish and Japanese flounder homologues, trout C/EBPbeta has the short open reading frame (uORF) upstream of the start codon for LAP but in an alternate reading frame, a feature of tetrapod C/EBPbeta genes. In normal rainbow trout, C/EBPbeta mRNA was detected in peripheral blood leukocytes, head kidney, posterior kidney, liver, spleen, gills, intestine and muscle. RT-PCR revealed that transcript levels of trout C/EBPbeta are clearly higher in sodium alginate-induced peritoneal cells than in head kidney and peritoneal cells of saline-injected fish or head kidney cells of alginate-injected fish. Together with expression in immunologically important tissues, this indicates that C/EBPbeta is likely to be involved in the immune response just as in mammals. Southern hybridization suggested that C/EBPbeta is a single copy gene. There are no introns in this C/EBPbeta gene, just like the mammalian homologues. These data suggest that we have obtained the trout ortholog of C/EBPbeta.

In comparison to mammals, relatively few of the molecules involved in teleost immune responses have been isolated and characterized. A rapid method of isolating molecules important for immune function is subtractive hybridization. One such experiment using infectious hematopoietic necrosis virus-infected Atlantic salmon produced several cDNA clones with similarity to mammalian immune-specific genes, including granzyme M (accession no. AF434669) and CD9. After cloning the rainbow trout version of CD9, sequence analysis showed that both salmonid sequences contained many features of the tetraspanin receptor family to which CD9 belongs. Phylogenetic analysis revealed a close association of the trout and salmon sequences to known CD9 and CD81 receptors. Southern blotting demonstrated that the rainbow trout gene is single copy. Reverse transcriptase PCR showed strong expression of this clone in many tissues, but liver expression was very low - an observation consistent with the clone being a CD9, not a CD81, equivalent. The evidence suggests that the sequences reported here are bona fide teleost CD9 homologues and we are currently producing recombinant proteins and polyclonal antisera for use in functional studies.

A sequence encoding a CXC - type chemokine from rainbow trout was found to most resemble members of the CXCL9/CXCL10/CXCL11 sub-family. In mammals, all 3 chemokines are regulated by IFN-gamma and are chemotactic for activated T lymphocytes. The trout chemokine (gammaIP1), with a message of 787 nucleotides, contains 100 amino acids in a typical non-ELR CXC chemokine arrangement. A second sequence (gammaIP2), with 6 nucleotide differences in the coding region when compared to the first, was also identified although it is not known whether this is a second functional gene or a second allele. The gene is separated onto 4 exons, and the introns intervene in conserved positions according to the mammalian equivalents. The sequence encoded by the second exon shares the highest amino acid identity (37%) with CXCL10, with lower values of identity to other CXC chemokines (17-31%). Furthermore, phylogenetic analysis groups the trout chemokine with mammalian CXCL9, CXCL10 and CXCL11 peptides. Constitutive expression of gammaIP is seen in trout gill and low level expression in spleen, head kidney and liver. In RTS-11 cells, gammaIP expression can be induced with poly I:C, but not by LPS, suggesting virus-mediated regulation of gammaIP. Intraperitoneal injection of recombinant trout TNF-alpha caused elevation in gammaIP mRNA levels in trout head kidney.

Cortisol at 1,000 and 100 ng/ml, and less consistently at 10 ng/ml, inhibited increases in cell number and 3H-thymidine incorporation by cultures of the rainbow trout (Oncorhynchus mykiss) monocyte/macrophage cell line, RTS11. Cell viability was not altered by cortisol, although a small decline in the capacity of cultures to reduce the redox dye, Alamar Blue was observed. In cortisol-treated cultures, more round and fewer spread cells were evident. Similar results were observed with dexamethasone but not cortisone. The glucocorticoid receptor antagonist, RU-486, prevented the effects of cortisol on RTS11 proliferation, and shape. In co-culture with the spleen stroma cell line (RTS34st) or in medium conditioned by RTS34st, the proliferation of RTS11 was enhanced. Treating RTS11/RTS34st co-cultures or RTS11 cultures in RTS34st conditioned medium with cortisol did not inhibit RTS11 proliferation. Overall these experiments suggest that proliferation of rainbow trout macrophages is regulated by cortisol, but the effect is modulated by the cellular micro-environment, possibly through the release of cytokines.

An interleukin 8 (IL-8) homologue has been identified in the rainbow trout Oncorhynchus mykiss. The transcript contains an open reading frame of 294 nucleotides that translates into a 97 amino acid putative peptide, with 5' and 3' untranslated regions (UTR) of 171 and 453 nucleotides, respectively. As with previously sequenced lamprey and flounder genes, the trout amino acid sequence lacks the typical ELR motif upstream of the first pair of cysteines, where DLR is present. The trout IL-8 gene contains four exons divided by three short introns of 341, 247 and 292bp, and occupies 1824bp of genomic DNA. RT-PCR reveals a low level constitutive expression of the IL-8 homologue in many tissues, including spleen, heart, liver, head kidney and gill. Expression was not detectable in the brain. Whilst no apparent affect of lipopolysaccharide (LPS) on IL-8 expression was observed in vivo, stimulation of a trout macrophage cell line (RTS-11) with either LPS or poly I:C did result in clear up-regulation of IL-8 expression, detectable by RT-PCR and Northern blot analysis.

Ten microsatellite loci (Omy27DU,Omy325(A3)UoG, OmyFGT5TUF,OmyFGT14TUF, OmyFGT15TUF,OmyFGT23TUF, Omy77DU,Ssa20.19NUIG, Ots1BML, andOne18ASC) were amplified using the polymerase chain reaction to create genetic profiles for nine cell lines (RTG-2, RTH-149,RTL-W1,RTgill-W1, RTS-11, RTS-34st, RTP-2, RTP-91E and RTP-91F) from rainbow trout(Oncorhynchus mykiss) and one cell line (CHSE-214) from Chinook salmon (O. tschawytscha). A cell line (PHL) from anon-salmonid, the Pacific herring (Clupea harengus pallasi), was included as a control. The ten loci clearly revealed the uniqueness of each cell line, except for two cell lines (RTP-91E andRTP-91F) from the same fish. RTP-91E and RTP-91F were identical at all loci except Ssa20.19NUIG. The most useful locus for demonstrating uniqueness was Ots1BML. The information was used to demonstrate that an uncharacterized rainbow trout cell line (Clone 1A)was in fact CHSE-214, illustrating the usefulness of multiplexed microsatellites for the creation of genetic profiles for salmonid cell lines and for the testing of cell line cross-contamination.

This review summarizes the scattered literature on the effects of toxicants on the external and internal innate immunity of fish. Insecticides, heavy metals and surfactants have been the most frequently examined toxicants, whereas dioxins, furans and polychlorinated biphenyls have been tested less frequently. Studies to date have been conducted at the levels of cells in vitro, of fish in the laboratory and microcosms, and also of fish in the field. Among innate immune parameters, phagocyte respiratory burst appears especially sensitive to toxicants. Toxicant-induced alterations in external mucous production have also been observed repeatedly. Field studies have occasionally examined changes to melano-macrophage centers, but the meaning of such changes is not clear. Advances in basic knowledge of fish innate immunity should lead to improvements in monitoring fish health and predicting the impact of toxicants on fish populations, which is a fundamental ecotoxicological goal.

Polybrominated diphenyl ethers (PBDEs) have become widely distributed as environmental contaminants due to their use as flame retardants. Their structural similarity to other halogenated aromatic pollutants has led to speculation that they might share toxicological properties such as hepatic enzyme induction. In this work we synthesized a number of PBDE congeners, studied their affinity for rat hepatic Ah receptor through competitive binding assays, and determined their ability to induce hepatic cytochrome P-450 enzymes by means of EROD (ethoxyresorufin-O-deethylase) assays in human, rat, chick, and rainbow trout cells. Both pure PBDE congeners and commercial PBDE mixtures had Ah receptor binding affinities 10(-2)-10(-5) times that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast with polychlorinated biphenyls, Ah receptor binding affinities of PBDEs could not be related to the planarity of the molecule, possibly because the large size of the bromine atoms expands the Ah receptor's binding site. EROD activities of the PBDE congeners followed a similar rank order in all cells. Some congeners, notably PBDE 85, did not follow the usual trend in which strength of Ah receptor binding affinity paralleled P-450 induction potency. Use of the gel retardation assay with a synthetic oligonucleotide indicated that in these cases the liganded Ah receptor failed to bind to the DNA recognition sequence.

The predicted rainbow trout mature interleukin-1 beta (IL-1 beta) peptide has been produced as a recombinant protein in E. coli. The bioactivity of this molecule has been studied using trout head kidney cell preparations and a trout macrophage cell line (RTS11). Trout rIL-1 beta was shown to increase the expression level of IL-1 beta, cyclooxygenase (COX2) and MHC class II beta chain transcription, as determined by Northern blot analysis. Stimulatory doses of rIL-1 beta were typically > or =10 ng/ml. Induction of IL-1 beta expression occurred within 1h post-stimulation with trout rIL-1 beta and was maximal 3-6h post-stimulation. Trout rIL-1 beta was also able to increase murine D10.G4.1 cell proliferation and trout head kidney leukocyte phagocytic activity, in a dose-dependent manner. However, equivalent D10.G4.1 cell proliferation was induced with approximately 1000-fold lower doses of human rIL-1 beta. That LPS contamination did not contribute to the effects seen was confirmed by determining its concentration in the trout rIL-1 beta preparation, and demonstrating that the rIL-1 beta activity was inhibited by heating or pre-incubation with a polyclonal anti-trout rIL-1 beta antibody.

An outdoor microcosm study was conducted in order to evaluate the kinetics of immunomodulation in rainbow trout (Oncorhynchus mykiss) exposed to liquid creosote. Fish were sampled on d 7, 14, 21, and 28 from microcosms dosed initially with 0, 3, and 10 microl/L of creosote. Pronephros leukocytes were monitored for phagocytic activity, oxidative burst, and surface immunoglobulin-positive (Slg+) B-cell counts. Oxidative burst was inhibited by creosote exposure; however, by sampling d 28, the burst response returned to near control levels. Phagocytic activity, on the other hand, was stimulated, peaking on sampling d 7, then returning to near control levels by d 28. Although control Slg+ B-cell counts were quite variable across sampling days, Slg+ B-cell counts were also elevated in creosote-exposed fish after seven days of exposure. Slg+ B-cell numbers decreased significantly to near control levels during the remainder of the study. The overall results from this study confirm that creosote has the potential to alter certain immune parameters, and emphasize the importance of monitoring changes in the immune system during exposure. Polycyclic aromatic hydrocarbons (PAHs), a major constituent of liquid creosote, are the suspected immune-altering agents.

The ability of the environmental contaminant phenanthrene (PH) and its photooxidized product phenanthrenequinone (PHQ) to disrupt progesterone secretion was examined in a model system of in vitro suspensions of luteal cells from the rat. Treatment with PHQ dramatically inhibited luteinizing hormone (LH) stimulated progesterone secretion. PHQ also generated a significant increase in reactive oxygen species (ROS). In the absence of LH, however, PHQ stimulated a small increase in basal progesterone secretion. The parent compound, PH, did not alter progesterone or ROS release. Since there is evidence that PHQ lowers the activity of nitric oxide synthase (NOS) and that nitric oxide (NO) affects progesterone production, we examined the response to the NOS inhibitors N-monomethyl-L-arginine, Zn protoporphyrin-9, and aminoguanidine in luteal cells. However, there was no effect of these agents on LH stimulated progesterone secretion. These results indicated that PHQ is a potent disrupter of progesterone secretion and should perhaps be considered in assessing the risk of PH to humans.

2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is a fluorogenic probe commonly used to detect cellular production of reactive oxygen species (ROS), for example in the respiratory burst of granulocytes and mononuclear phagocytes. This method depends on the de-acetylation of H2DCFDA by cellular esterases, to form the oxidant-sensitive compound, 2',7'-dichlorodihydrofluorescein (H2DCF). Importantly, however, not all cells possess sufficient esterase activity to produce the H2DCF needed for accurate measurement of ROS. In this study, we used chemically de-acetylated probe (H2DCF) to assess the phorbol-ester-triggered respiratory burst of rainbow trout macrophages, which, like some mammalian mononuclear phagocytes, appear to have low probe-esterase activity. We compared this approach to the use of intact H2DCFDA and the cytochrome c reduction assay. The H2DCF and cytochrome c reduction assays gave similar portrayals of the kinetics of the macrophage respiratory burst, while H2DCFDA did not. We therefore recommend the use of H2DCF over H2DCFDA for quantification of the production of reactive oxygen species. Additionally, we stress the need to test reaction buffers or culture media used with H2DCF(DA) for their ability to oxidize the probe directly or indirectly. As an example, we have observed that tyrosine combined with ubiquitous metal contaminants of physiological buffers can result in high levels of oxidation, which may be incorrectly interpreted as cellular activity.

The ability of fractionated petroleum refinery effluent to cause cellular responses in fish cell lines was evaluated. The cellular responses, which included direct and indirect cytotoxicity, photocytotoxicity and induction of 7-ethoxyresorufin-O-deethylase (EROD) activity, may potentially be linked to sublethal effects observed in effluent-exposed fish and fish larvae. In order to be able to quantify cellular responses rapidly, microtitre plates were used along with fluorescent probes. For the quantification of cyto- and photocytotoxicity, the fluorescent probes were alamar Blue and carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which monitor metabolic activity and cell membrane integrity, respectively. EROD activity was measured as the rate of conversion by EROD of the substrate 7-ethoxyresorufin to its fluorescent product, resorufin. Effluent from an Ontario refinery was fractionated into aqueous and particulate phase. As well, a solid phase extract (SPE) was used to prepare concentrated effluent for testing in the cell lines. The effluent was able to elicit all of the responses of interest although significant cyto- and photocytotoxicity required effluent equivalent concentrations above 100% effluent and could only be revealed upon exposure of cells to the SPE concentrated effluent. Based on their retention on C18, the cytotoxicants are likely to be non-polar to moderately polar chemicals. The presence of polar compounds affecting cellular metabolism was indicated by the responses of exposed cells to a 90% aqueous phase effluent. In contrast to cyto- and photocytotoxicity, EROD induction occurred at effluent equivalent concentrations well below 100% effluent and was elicited by the SPE and the particulate fraction thereby suggesting that most EROD-inducers were particle-bound. Among other applications, the described techniques could help to determine the source of causative agents of sublethal effects in the refining process.

A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino-acid putative peptide, with a 5' untranslated region (UTR) of 140 bp and a 3' UTR of 506 bp. Two potential N-linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT-PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up-regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)-1 beta. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL-1 beta, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF-alpha is discussed.

In order to investigate cell-specific differences in the response of in vitro models to environmental toxicants, we compared the capacity of nine polycyclic aromatic hydrocarbons (PAHs) to induce cytochrome P4501A (CYPIA) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes and a rainbow trout liver cell line, RTL-W1. Induction of CYPIA was estimated from the catalytic activity of 7-ethoxyresorufin-O-deethylase (EROD) and compared by median effective concentration (EC50) values, induction spans, and benzo[a]pyrene induction equivalency factors for inducing PAHs. The influence of culture conditions was investigated with respect to the presence or absence of serum and varying exposure times. Both in vitro systems lead to an identical classification of the PAHs in noninducing (anthracene, fluoranthene, phenanthrene, and pyrene) and inducing compounds with a similar ranking of inducing PAHs. Mean EC50 values in RTL-W1 cells were, respectively, 343 and 266 nM for benzo[a]anthracene, 57 and 92 nM for BaP, 134 and 283 nM for benzo[b]fluoranthene, 455 and 270 nM for chrysene, and 98 and 116 nM for 3-methylcholanthrene. Compared to primary hepatocytes, the RTL-W1 cell line was more sensitive in its EROD response to the presence or absence of serum and to the increase in exposure time, which led to higher EC50 values.

Although the zebrafish possesses many characteristics that make it a valuable model for genetic studies of vertebrate development, one deficiency of this model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem cell cultures are routinely used for gene transfer and provide the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. Transgenic animals possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of a chimeric embryo. Although zebrafish embryo cell cultures that exhibit characteristics of embryonic stem cells have been described, successful contribution of the cells to the germ-cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures maintained in the presence of cells from a rainbow trout spleen cell line (RTS34st) are able to produce germ-line chimeras when introduced into a host embryo. Messenger RNA encoding the primordial germ-cell marker, vasa, was present for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cell differentiation in the embryo cell cultures. These results suggest that the RTS34st splenic-stromal cell line will be a valuable tool in the development of a cell-based gene transfer approach to targeted gene inactivation in zebrafish.

The induction of 7-ethoxyresorufin-o-deethylase (EROD) activity was examined in three rainbow trout pituitary cell lines: RTP-91E, RTP-91F and RTP-2. RTP-91E and RTP-91F were developed from the pituitary of a male and have epithelial-like and fibroblast-like morphologies, respectively. RTP-2, which was described previously, was developed from the pituitary of a female and has an epithelial-like shape. In all cell lines EROD activity was induced by 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD). Immunoblotting with the polyclonal antibody, anti-trout CYP1A1(277-294)/KLH, confirmed induction of a 58-kDa polypeptide. Potential inhibitors of the aryl hydrocarbon receptor, geldanamycin and alpha-naphthoflavone, inhibited EROD induction by TCDD. Other compounds inducing EROD activity were 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126), and 3-methylcholanthrene (3MC). When judged by the concentration eliciting 50% of the maximal response (EC50), induction was similar in RTP-2 and RTP-91E, and less effective in RTP-91F. Regardless of the cell line, the rank order from most to least potent inducer on the basis of EC50 value was TCDD> or =PCDD>TCDF>PCB 126>>3MC. When induction potencies were expressed relative to TCDD, the values obtained with the pituitary cell lines were similar to previously published values derived with a rainbow trout liver cell line.

The lack of cell lines from mononuclear phagocytes of salmonid fish has impeded the study of immune function at a cellular level in these economically and ecologically important animals. Here, we report on the further characterization of RTS11, a previously described macrophage-like cell line from the rainbow trout spleen, with regard to its expression of a number of immunologically relevant genes. Analysis of gene expression by reverse transcription-polymerase chain reaction, using rainbow-trout-specific primers demonstrated that RTS11 cells express the beta chain of the class II major histocompatibility complex, the cytokines transforming growth factor-beta (TGF-beta) and interleukin-1beta (IL-1beta), and cyclo-oxygenase-2 (COX-2). Inducing the cells with lipopolysaccharide led to increased expression of IL-1beta and COX-2, as determined by Northern blotting. These results together suggest that RTS11 retains many of the characteristics expected of mature macrophages, and should be a valuable tool for further study of the expression and function of these immunomodulatory proteins in fish.

Cytochrome P4501A (CYP1A) metabolizes a wide array of lipophilic xenobiotics. In fish liver, CYP1A is constitutively expressed at low levels, but xenobiotics can strongly induce CYP1A expression via a receptor-mediated pathway. While induction of hepatic CYP1A in teleosts by xenobiotics is well investigated, very little is known on the regulation of constitutive CYP1A expression and its induction by factors other than xenobiotics. In the present study we show that in the rainbow trout liver cell line, RTL-W1, CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity can be induced by a change of the culture medium, in the absence of xenobiotics. The increase in cellular EROD levels is of transient nature. Experiments with cell incubation solutions supplemented with various medium components indicate that photooxidized tryptophan is the agent causing the increase of EROD activity after medium change.

Screening for environmental contaminants in milk is generally conducted by chemical analysis, yet such an approach may be time-consuming and expensive, and is not indicative of the physiological consequences of such exposure. The focus of this review is to summarize those constituents of milk that may be altered by maternal exposure to one of the most biologically active environmental pollutants, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and to develop the case for their use as biomarkers of response. Several chemical and/or cellular components of milk are potentially useful as biomarkers, and may be developed as convenient, biologically relevant indicators of maternal exposure to dioxin-like compounds.

Previously, exposure of fish to polycyclic aromatic hydrocarbons (PAHs) in both field and laboratory settings has been associated with eye damage, but this has only been expressed qualitatively. In this study, an automated scanning laser system has been employed to quantitatively evaluate changes in lens optical quality in rainbow trout (Oncorhynchus mykiss) following their in vivo exposure to creosote, which is a complex mixture with many PAHs. Rainbow trout were placed in 12,000-L outdoor microcosms dosed with 0, 3, or 10 microl/L liquid creosote for a 28-day period. Collected fish were examined for changes in focal length variability (FLV), lens size, and weight. These measurements were compared with induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity and hepatic and water concentrations of priority pollutant PAHs. The optical quality of rainbow trout lenses was significantly reduced following creosote exposure, as indicated by increased FLV. Lens damage was significantly related to hepatic EROD activity, and both effects rose with creosote dose. Analytical measurements of microcosm water indicated elevated concentrations of PAHs in creosote-dosed ponds, including compounds capable of inducing rainbow trout EROD activity in vitro. Hepatic concentrations of PAHs were low and not related to creosote dose, likely due to rapid metabolism and elimination. This study demonstrates for the first time employment of a highly sensitive and quantitative technique to measure lens damage in fish exposed to contaminants in situ. The relationship between this effect and hepatic CYP1A activity may suggest a mechanistic linkage, which could lead to the use of EROD activity as an indicator of toxic effect rather than just chemical exposure.

The optical properties of rainbow trout lenses were investigated after in vitro exposure to polycyclic aromatic hydrocarbons (PAHs) and ultraviolet (UV) irradiation, both because PAHs frequently contaminate aquatic environments and because UV exposure has generally increased with the decline of the ozone layer. Lenses were exposed to UV irradiation for 12 hr while immersed in culture medium. UV irradiation, with or without the presence of PAHs, was accomplished with one UVA and one UVB photoreactor lamp to yield a photon fluence rate of 9.27 micromol m(-2)s(-1)UVA (UVA:UVB 10.8, radiant exposure of 13.4 Jcm(-1)). Individual PAHs studied were fluorene, fluoranthene and benzo(a)pyrene. In addition, lenses were exposed to a solution of creosote, a wood preservative used in the aquatic environment that contains many PAHs. All PAH exposures, including creosote, were carried out either in the dark or concurrently with UV irradiation. A scanning laser monitor system was used to evaluate the optical properties of lenses for up to 236 hr after the UV/PAH treatments. Mean focal length variability (FLV) increased with time after concurrent exposure to UV irradiation and high concentrations of either fluoranthene (4900 n m), benzo(a)pyrene (265 n m) or creosote (70 microg ml(-1)), with FLV values ranging from, 0.21-0.41, 0.21-0.64 and 0.15-0.22 mm, respectively, 72 hr after termination of the UV/PAH treatment. UV irradiation alone or exposure to PAHs in the dark brought about no changes in the optical properties of lenses. Also, fluorene in the presence or absence of UV had no effect, even at concentrations as high as 128 microm. Lenses were also unchanged by 12 hr exposures in the dark to solutions of either fluorene, fluoranthene, benzo(a)pyrene or creosote that had been previously UV irradiated for 12 hr. This meant that photomodified products of the individual PAHs or creosote were not cataractogenic and emphasized that simultaneous exposure to UV and PAHs or creosote was necessary for the increased FLV. The results point for the first time to an interaction between UV irradiation and PAHs as a potential contributing factor to cataract formation in fish., (Copyright 2000 Academic Press.)

A technique for the clonal culture of rainbow trout leukocytes in a methylcellulose matrix can be used to identify growth factors and other substances affecting cell proliferation and development in fish. Methylcellulose supports colony formation by rainbow trout leukocytes isolated from the major hemopoietic organ, the pronephros. The addition of rainbow trout serum dramatically increased the number of colonies formed, scored by counting colonies. As an alternative measure of cell proliferation, 3H-thymidine incorporation by cells can be easily measured in methylcellulose cultures. This method requires only small amounts of test substances, is rapid, and is superior for assessing the growth-stimulating ability of some substances, such as bacterial lipopolysaccharide, which stimulated growth but not the formation of discreet colonies by rainbow trout cells.