Niels C. Bols

ECOTOXICOLOGY AND INNATE IMMUNITY IN FISH

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.

Identification and Bioactivities of IFN-γ in Rainbow Trout Oncorhynchus mykiss: The First Th1-Type Cytokine Characterized Functionally in Fish

IFN-γ is one of the key cytokines in defining Th1 immune responses. In this study, an IFN-γ homologue has been identified in rainbow trout Oncorhynchus mykiss, and its biological activities have been characterized. The trout IFN-γ cDNA is 1034 bp in length and translates into a 180-aa protein. The first intron of the trout IFN-γ gene contains highly polymorphic GACA minisatellites and 44-bp DNA repeats, giving rise to at least six alleles. IFN-γ is structurally conserved among vertebrates, and a signature motif has been identified. A nuclear localization sequence known to be crucial for IFN-γ biological activities is also present in the C-terminal region of the trout IFN-γ. The IFN-γ 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-γ produced in Escherichia coli significantly stimulated gene expression of IFN-γ-inducible protein 10 (γIP-10), MHC class II β-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 γIP-10 in RTS-11 cells. Moreover, IFN-γ-induced γIP-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-γ homologue in a fish species, strongly suggesting a conserved Th1 immune response is most likely present in lower vertebrates.

Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity.

A cell line RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. RTL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants.

Ability of 16 priority PAHs to be directly cytotoxic to a cell line from the rainbow trout gill

Sixteen polycyclic aromatic hydrocarbons (PAHs) were screened for their ability to be directly cytotoxic to a cell line from the rainbow trout gill, RTgill-W1. Exposure times of 2 h or less were sufficient for direct cytotoxicity to be detected, which appeared to be caused by a common mechanism, the general perturbation of membranes. This was judged by the similarity of results obtained for three fluorescent indicator dyes, alamar Blue, 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and neutral red. Among the 16 PAHs tested, just two- and three-ring PAHs were found to be directly cytotoxic. These were naphthalene approximately = acenaphthylene approximately = acenaphthene > fluorene approximately = phenanthrene. The results suggest that water solubility and lipophilicity are the critical properties determining the direct cytotoxicity of PAHs and that they do so by influencing PAH accumulation in membranes. Only naphthalene was effective at concentrations well below its water solubility limit. Therefore, direct cytotoxicity is likely to be most environmentally relevant only with naphthalene.

Methodology for demonstrating and measuring the photocytotoxicity of fluoranthene to fish cells in culture

Methodology was developed for quantifying the photocytotoxicity of fluoranthene to a gill cell line from rainbow trout for future use in screening polycyclic aromatic hydrocarbons for their relative photocytotoxicity to fish. Solubilization in a modified culture medium was achieved with and without foetal bovine serum (FBS) and with and without dimethyl sulfoxide (DMSO). FBS caused most of the fluoranthene to remain in solution and blocked photocytotoxicity if present during UV irradiation. DMSO had little effect on fluoranthene distribution in cell cultures but caused cells to be slightly more sensitive to the phototoxicity of fluoranthene. The indicator dyes alamar Blue() and 5-carboxyfluorescein diacetate acetoxymethyl ester were used to quantify cytotoxicity in two different ways-singly in two separate assays, and mixed together in a novel single assay, which saved time and material. With UV irradiation for 2 hr at a photon fluence rate of either 1.4 mumol UV-B/m(2)/sec (UV-A:UV-B, 1.5) or 1.1 mumol UV-B/m(2)/sec (UV-A:UV-B, 9.7), both dyes indicated increasing loss of viability with increasing doses of fluoranthene. EC(50) values ranged from 18 to 44 ng/ml (89-217 nM), with the alamar Blue assay being slightly more sensitive.

The production and bioactivity of rainbow trout (Oncorhynchus mykiss) recombinant IL-1β

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.

Production of zebrafish germ-line chimeras from embryo cell cultures

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.

Functional characterisation of the recombinant tumor necrosis factors in rainbow trout, Oncorhynchus mykiss.

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.

Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, environmental contaminants that pose a potential risk to fish populations. Both field and laboratory studies suggest that exposure of the early life stages of fish to PAH can mimic the embryotoxic effects of the planar halogenated hydrocarbons (PHHs), the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin. PHH toxicity is mediated by the aryl hydrocarbon receptor (AHR) and PHH potency is predicted by its AHR-binding affinity and CYP1A induction potency. However, the role of the AHR, if any, in mediating the developmental effects of PAH to fish remains unknown. In this study we looked at the AHR binding affinity of a test set of PAH that had been previously ranked for their potency for inducing teleost CYP1A. PAH that induced CYP1A inhibited [3H]TCDD binding to in vitro-expressed AHRs from rainbow trout and the AHR expressed in PLHC-1 fish hepatoma cells. Generally, the relative rank order for AHR binding affinity predicted the rank order of these same PAH for inducing CYP1A reported in other studies. There was a strong, positive relationship between binding to the PLHC-1 AHR (stimulus) and the EC50s for CYP1A induction (response) in whole juvenile trout and in RTL-W1 cells, but EC50s were much higher than expected for a 1:1 stimulus/response relationship. These data show that the ability of PAH to bind to teleost AHR predicts PAH potency for CYP1A induction. If PAH toxicity is receptor-mediated and predicted by induction potencies, we will have a powerful mechanistic-based tool for rapidly assessing the risk of toxicity to fish of PAH from any source.

Technology and uses of cell cultures from the tissues and organs of bony fish.

SummaryFor a wide range of purposes, primary cell cultures and/or cell lines have been prepared from most tissues and organs of a small fraction of the estimated 20,000 species of bony fish. These cell cultures usually have been maintained with mammmalian sera. For many applications their usefulness would be enhanced by a more piscine and defined environment. However, the piscine equivalents of mammalian polypeptide growth and differentiation factors are largely unknown and are unlikely ever to be available commercially. In the future they might be obtained from the medium in which fish cells have been grown. Therefore, by being a potential source of fish polypeptide growth and differentiation factors, a cell line from a fish organ might be utilized as a Rossetta stone to decipher thein vitro proliferation and differentiation of other cells from this or other organs from the same or different species.