Integrity and wound healing of rainbow trout intestinal epithelial cell sheets at hypo-, normo-, and hyper-thermic temperatures.

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 µm 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 °C) or hyperthermic (≥ 26 °C) temperatures were compared to normothermic control cultures (18-22 °C). Monolayers remained intact for at least a week at temperatures from 4 to 28 °C, but had lost their integrity after 3 h at 32 °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 °C and as blobs in cells at 32 °C. At normothermia and at 26 °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 °C. At 28 °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 °C for 3 h and returned to 18-22 °C, cells lost their shape and actin organization and over the next 6 days detached and died. When monolayers were subjected to 26 °C for 24 h and challenged at 32 °C for 3 h prior to being placed at 18-22 °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.

Responses of rainbow trout intestinal epithelial cells to different kinds of nutritional deprivation.

In order to develop an in vitro system to study the cell biology of starvation in the fish intestine, rainbow trout intestinal epithelial cells were subjected to three kinds of nutrient deprivation and evaluated for 7 days. The RTgutGC cell line was grown into monolayers in Leibovitz's basal medium supplemented with fetal bovine serum (L15/FBS) and then subjected to deprivation of serum (L15); of serum, amino acids, and vitamin (L15/ex); and of all nutrients (L15/salts). After 7 days of nutrient deprivation, the cells remained attached to the plastic surface as monolayers but changes were seen in shape, with the cells becoming more polygonal, actin and α-tubulin cytoskeleton organization, and in tight junction protein-1 (ZO-1) localization. Two barrier functions, transepithelial electrical resistance (TEER) and Lucifer Yellow (LY) retention, were impaired by nutrient deprivation. In L15/FBS, cells rapidly healed a gap or wound in the monolayer. In L15 and L15/ex, some cells moved into the gap, but after 7 days, the wound remained unhealed, whereas in L15/salts, cells did not even migrate into the gap. Upon nutrient replenishment (L15/FBS) after 7 days in L15, L15/ex, or L15/salts, cells proliferated again and healed a wound. After 7 days of nutrient deprivation, monolayers were successfully passaged with trypsin and cells in L15/FBS grew to again form monolayers. Therefore, rainbow trout intestinal epithelial cells survived starvation, but barrier and wound healing functions were impaired.

Invitromatics, invitrome, and invitroomics: introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout.

The literature on cell lines that have been developed from rainbow trout (RT) (Oncorhynchus mykiss) is reviewed to illustrate three new terms: invitromatics, invitrome, and invitroomics. Invitromatics is defined as the history, development, characterization, engineering, storage, and sharing of cell lines. RT invitromatics differs from invitromatics for humans and other mammals in several ways. Nearly all the RT cell lines have developed through spontaneous immortalization. No RT cell line undergoes senescence and can be described as being finite, whereas many human cell lines undergo senescence and are finite. RT cell lines are routinely grown at 18-22°C in free gas exchange with air in basal media developed for mammalian cells together with a supplement of fetal bovine serum. An invitrome is defined as the grouping of cell lines around a theme or category. The broad theme in this article is all the cell lines that have ever been created from O. mykiss, or in other words, the RT invitrome. The RT invitrome consists of approximately 55 cell lines. These cell lines can also be categorized on the basis of their storage and availability. A curated invitrome constitutes all the cell lines in a repository and for RT consists of 11 cell lines. These consist of epithelial cell lines, such as RTgill-W1, and fibroblast cell lines, such as RTG-2. RTG-2 can be purchased from a scientific company and constitutes the commercial RT invitrome. Cell lines that are exchanged between researchers are termed the informally shared invitrome and for RT consists of over 35 cell lines. Among these is the monocyte/macrophage cell line, RTS11. Cell lines whose existence is in doubt are termed the zombie invitrome, and for RT, approximately 12 cell lines are zombies. Invitroomics is the application of cell lines to a scientific problem or discipline. This is illustrated with the use of the RT invitrome in virology. Of the RT invitrome, RTG-2 was the most commonly used cell line to isolate viruses. Fifteen families of viruses were studied with RT invitrome. RT cell lines were best able to support replication of viruses from the Herpesviridae, Iridoviridae, Birnaviridae, Togaviridae, and Rhabdoviridae families.

The use of fish-derived cell lines for investigation of environmental contaminants: an update following OECD's fish toxicity testing framework No. 171.

Protocols for evaluating chemical toxicity at the cellular level using fish cell lines are described in this unit. Routine methodologies for growing salmonid cell lines, and using them in aquatic toxicology studies that support the mandate of the Organization for Economic Co-operation and Development (OECD) to reduce the use of whole animals in toxicity testing, are presented. Rapid, simple, cost-effective tests evaluating viability of cells with three indicator dyes per sample provides a broad overview of the sensitivity of cells to chemical contaminants. This fluorometric assay involves: (1) alamar blue for metabolic activity, (2) CFDA-AM for membrane integrity, and (3) neutral red for lysosomal function. These protocols are conveniently performed in semi-unison within the same multiwell plates and read at three different wavelengths. Detailed step-by-step descriptions of the assays, parameters to consider, troubleshooting, and guidelines for data interpretation are provided as essential tools for investigating environmental aquatic contaminants at the cellular level.

Ammonia-containing industrial effluents, lethal to rainbow trout, induce vacuolisation and Neutral Red uptake in the rainbow trout gill cell line, RTgill-W1.

Nine samples of whole effluent from the operation of an industrial plant over the course of one year, were tested on rainbow trout for lethality and on the rainbow trout gill cell line, RTgill-W1, for metabolic activity, plasma membrane integrity, and lysosomal activity, as measured by using the alamar Blue (AB), 5-carboxyfluorescein diacetate acetoxymethyl (CFDA-AM), and neutral red (NR) assays, respectively. None of the nine samples caused a loss of plasma membrane integrity, and only two caused a transitory decline in metabolism. Three samples caused massive vacuolisation in RTgill-W1 cells, which was accompanied by increased uptake of NR, and only these three samples were lethal to the rainbow trout. The addition of ammonia to RTgill-W1 cultures also induced vacuolisation and NR uptake, with little change in plasma membrane integrity or metabolism. Subsequently, the effluent source was identified as a nitrogen product producer, and variable levels of ammonia were found in the nine samples. Three of the four samples with the highest non-ionised ammonia levels were those which were toxic to rainbow trout and which caused vacuoles in RTgill-W1 cells. The close correlation between rainbow trout-killing and RTgill-W1 vacuolisation by the effluents, suggests that vacuolisation of RTgill-W1 cells could be used to indicate effluents which would be toxic to rainbow trout as a result of their ammonia content.

Use of Tetrahymena thermophila to study the role of protozoa in inactivation of viruses in water.

The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4 degrees C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality.

Cytotoxicity of metals common in mining effluent to rainbow trout cell lines and to the ciliated protozoan, Tetrahymena thermophila.

As fish cell lines and ciliates are potential alternatives to whole animals in testing environmental samples for toxicity, two cell lines from rainbow trout, RTgill-W1 and RTL-W1, and the ciliate, Tetrahymena thermophila, were compared for their sensitivity to the toxicity of five metals that are commonly found in mining effluents: copper, cadmium, zinc, nickel, and iron. Adding copper to growth medium for either cell system failed to elicit toxicity. Therefore, metal exposures were done in simple buffers, which allowed all metals to exert toxicity, except for nickel on fish cells. Cell viability was measured successfully with alamar Blue (AB) for metabolic activity and 5'-carboxyfluoroscein diacetate acetoxymethyl ester (CFDA-AM) for membrane integrity, whereas neutral red uptake, which was quantified fluorometrically, gave confounding results with copper. Cadmium was the most toxic metal to Tetrahymena and killed the ciliate at approximately a 10-fold lower concentration than needed to kill fish cells. Fish cells were more sensitive than Tetrahymena to the cytotoxic action of zinc. Copper and iron were cytotoxic to fish cells and Tetrahymena at similar concentrations. Therefore, the relative sensitivity of fish cells and Tetrahymena to the cytotoxicity of metals depended on the metal, being similar for some but very different for others.

Comparing a ciliate and a fish cell line for their sensitivity to several classes of toxicants by the novel application of multiwell filter plates to Tetrahymena.

Although ciliated protozoa such as Tetrahymena have many desirable properties as toxicological test organisms, their attributes would be better realized if multiple cultures could be simultaneously exposed to toxicants, quickly washed to terminate toxicant exposure, and conveniently evaluated for changes in cellular functions. Therefore, multiwell filter plates (MWFPs), manufactured primarily for biochemical applications, were used to expose Tetrahymena thermophila to copper, Triton X-100, and gliotoxin and compared to results of exposure in microcentrifuge tubes (MCTs). For MWFP, removal of toxicant solutions and retention of Tetrahymena in wells was done by placing plates on a manifold and applying pressure with a vacuum pump. Retained cells were resuspended in the same wells and their functions assessed with the fluorescent indicator dyes, Alamar blue to measure energy metabolism, and 5'-carboxyfluorescein diacetate acetoxymethyl ester to evaluate membrane integrity. For MCTs, exposures were terminated by centrifugation, and resuspended Tetrahymena were transferred to conventional multiwell plates for viability assessment with the same fluorescent dyes. Results were measured with a fluorescent multiwell plate reader and dose-response curves were obtained successfully with both procedures. However, MWFPs were much more convenient and rapid, potentially allowing 96 cultures to be processed at a time. Exposing Tetrahymena in MWFPs also allowed the ciliate and a rainbow trout gill cell line, RTgill-W1, to be compared for their sensitivity to toxicants under similar conditions of exposure and by common viability assays. Both cell systems showed toxic responses to Triton X-100 and copper at similar concentrations, but RTgill-W1 was more sensitive to gliotoxin.

Applying whole water samples to cell bioassays for detecting dioxin-like compounds at contaminated sites.

Methodology was developed in order to rapidly and cost-efficiently screen whole water samples without extraction for the presence of dioxin-like compounds using a cell bioassay approach. Presence of dioxin-like compounds was indicated by the induction in the rainbow trout (Oncorhynchus mykiss) liver cell line, RTL-W1, of cytochrome CYP1A, which was measured as 7-ethoxyresorufin-O-deethylase (EROD) activity. Two simple culture media, L-15/ex and Earle's-G, prepared in tissue culture water and supplemented with 5% serum, proved suitable for supporting RTL-W1 cell viability and induction of EROD activity by the model inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (BaP). Preparation of the same simplified media using whole surface and ground water instead of tissue culture water again allowed EROD induction by spiked TCDD and BaP to be detected but higher concentrations of inducers were necessary. Despite this reduced sensitivity, RTL-W1 cells responded to 4 out of 40 ground water samples from a former oil and lignite processing site with significant EROD induction. In the future, the value of the bioassay is as an inexpensive means of quickly screening ground and surface water samples to identify high contaminant levels particularly at industrial sites, where detailed site-investigations and long-term monitoring programs are required.

Evaluating the toxicity of Triton X-100 to protozoan, fish, and mammalian cells using fluorescent dyes as indicators of cell viability.

Three viability assays using fluorescent dyes effectively detected a loss of viability in cultures of three mammalian cell lines (H4IIE, Caco2, and HepG-2), two fish cell lines (RTgill-W1 and RTL-W1), and a ciliated protozoan, Tetrahymena thermophila, after exposure to Triton X-100, used as a model toxicant. The dyes were Alamar Blue (AB), neutral red (NR), and propidium iodide, which respectively monitored energy metabolism, lysosomal activity, and membrane integrity. A fourth fluorescent dye, 5-carboxyfluorescein diacetate acetoxymethyl ester, was problematic. For 2-h Triton X-100 exposures, mammalian cell lines were as susceptible as piscine cell lines, whereas T. thermophila was approximately twofold less sensitive as detected with AB and NR. Despite being less sensitive, cytotoxicity tests on T. thermophila could be done in spring water, which means that unlike animal cells they could be directly exposed to most industrial effluents without osmolality adjustments. Therefore, T. thermophila could be a useful complement to animal cells as alternatives to fish in toxicity testing.

The use of fish-derived cell lines for investigation of environmental contaminants.

This unit describes protocols for growing salmonid cell lines and using them in in vitro toxicology studies. Cell viability of cultures is assessed with three indicator dyes: alamar blue for metabolic activity, CFDA-AM for membrane integrity, and neutral red for lysosomal activity. These protocols are essential tools for investigating environmental toxicity at the cellular level.

Applying whole-water samples directly to fish cell cultures in order to evaluate the toxicity of industrial effluent.

Methodology was developed for presenting to fish cells in culture whole-water samples without extraction and used to evaluate the toxicity to a rainbow trout gill cell line, RTgill-W1, of more than 30 whole-water samples collected from a paper mill over approximately a year of operation. Presentation to cells was achieved by adding to water samples the amounts of salts, galactose and sodium pyruvate, as solids, that were necessary to give concentrations and osmolality of the basal growth medium, Leibovitz's L-15. Cell viability was measured with three fluorescent indicator dyes: alamar Blue for metabolism, 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for membrane function, and neutral red for lysosomal activity. Eighteen samples were tested with the Daphnia lethality bioassay and 11 of these were toxic. None of these were judged cytotoxic to RTgill-W1. Sixteen samples were tested with the rainbow trout lethality bioassay and only one was toxic. This sample was also the only sample that was cytotoxic to RTgill-W1. Therefore, these methods for presenting water samples and measuring their cytotoxicity to RTgill-W1 are a promising substitute for toxicity tests of industrial effluent with rainbow trout but not with Daphnia.