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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.

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.

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.

The glutamine requirement for the in vitro proliferation of fish cells was investigated with cell lines from four different species and three tissues: goldfish skin (GFSk-S1), Chinook salmon embryo (CHSE-214), and rainbow trout liver (RTL-W1) and spleen (RTSp-W1). With a supplement of fetal bovine serum, the basal medium, Leibovitz's L-15, without glutamine supported the proliferation of all four cell lines as well, or nearly as well, as L-15 with 2 mM glutamine. This was true over short term assays of two to four weeks and for continuous propagation. CHSE-214 also grew as well with or without 2 mM glutamine in Minimum Essential Medium with fetal bovine serum. However, when the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in L-15 without glutamine. Therefore, glutamine was not required for growth in L-15, and in fact, was inhibitory in the absence of the dialyzable fraction of serum. By contrast, glutamine appeared to be important for growth in Minimum Essential Medium. When the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in Minimum Essential Medium with 2 mM glutamine. These results suggest that the glutamine requirement for the in vitro proliferation of fish cells is conditional and depends on the basal medium and serum supplement.

A histochemical, microdensitometric, and electron microscopic study of testes of the ratfish Hydrolagus colliei shows that an instance of the rare phenomenon of germ line chromatin diminution occurs in this vertebrate species. In primary spermatocytes at metaphase I a spherical mass of heterochromatin accumulates at one side of the metaphase plate. At anaphase I the heterochromatic mass is left in the equatorial cytoplasm and is passed into one of the two secondary spermatocytes formed during cytokinesis. As nuclear membranes are being restored, a double membrane envelope is also formed around the heterochromatic mass, which is then termed the 'chromatin diminution body' (CDB). At second meiotic division the CDB is included in the cytoplasm of one of the four spermatids and retained there, apparently unchanged, until mid-spermiogenesis. At that time the CDB becomes adherent to the spermatid plasma membrane and is pinched off from the spermatid by a process of apocrine exocytosis, taking a layer of spermatid plasma membrane along with it. Simultaneously this tri-membrane CDB is taken into the adjacent Sertoli cell by endocytosis, thereby acquiring a fourth membrane layer, a part of the Sertoli cell plasma membrane. The CDBs are subsequently phagocytized, possibly first fusing with dense, multilaminate bodies in the Sertoli cell cytoplasm. The CDB chromatin mass is strongly positive with the Feulgen method for DNA and the alkaline fast green method for histones. Microdensitometric analysis shows that the discarded chromatin amounts to about 10% of the diploid nuclear content and that it appears to be part of the normal diploid complement rather than DNA amplified during meiosis.

Nuclear shape and the organization of nuclei within the seminiferous follicles have been used to divide spermiogenesis in the longnose skate, Raja rhina, into eight stages and in the spiny dogfish, Squalus acanthias, into seven stages. Cytochemical techniques for basic proteins reveal that as spermatid nuclei begin to elongate they have the somatic histone complement, and as they complete elongation they contain a very arginine-rich, TCA-extractable complement, or the salmon sperm histones type. After the nuclei have developed their ultimate corkscrew shape the final transition takes place to a very arginine-rich, TCA-stable complement, or the mammalian sperm histone type. These nuclei were not rich in sulfhydryl groups, but they were extractable with sodium thioglycolate. In addition, a number of variables affecting TCA extraction and subsequent alkaline fast green staining are described.

Spermiogenesis in the ratfish (Hydrolagus colliei) is characterized by unusual changes in the basic proteins of the nucleus. Cytochemical analysis indicates that early spermatids in this cartilaginous fish contain the somatic type of histones. However, late spermatids contain the salmon type of sperm histone whereas spermatozoa display the mouse/grasshopper type designated by Bloch ('69). Such an unusual cytochemical shift from protamine in the spermatid to "stable protamine" in the sperm has not been documented heretofore.

For livers of lake trout from three Great Lakes sites, the 2,3,7,9-tetrachlorodibenzo-p-dioxin (TCDD) equivalent concentrations (TEQs) were determined in four ways. Bioassay-derived (BD) TEQs were measured with rat (H4IIE) and rainbow trout (RTL-W1) cell lines, and chemistry-derived (CD) TEQs were calculated with TCDD equivalency factors (TEFs) derived with H4IIE and RTL-W1, Generally, BD-TEQs and CD-TEQs for individual samples did not differ significantly for either H4IIE or RTL-W1, indicating that all of the AhR-active compounds in a sample were accounted for by the congener analysis and that these compounds acted in an additive fashion in both mammalian and piscine systems. However contributions of individual chemicals to overall CD-TEQs differed with H4IIE and RTL-W1 TEFs, and for some individual samples, TEQs did depend on the method used. Hepatic TEQs and etlioxyresorufin-O-deethylase (EROD) levels differed significantly between sites. For TEQs,the order was Glenora > Jackfish Bay > Black Bay; for EROD activity,the order was Jackfish Bay > Glenora = Black Bay. No correlation wasfound between hepatic TEQs and EROD activity, which suggests that the two measurements are evaluating different but related consequences of contaminant exposure. [ABSTRACT FROM AUTHOR]