Rainbow Trout Gill Research Articles

CYP1A expression in liver and gills of rainbow trout ( Oncorhynchus mykiss) after short-term exposure to dibenzothiophene (DBT)

Dibenzothiophene (DBT), a common component of crude oil, is a widespread environmental pollutant of known adverse effects to aquatic vertebrates. However, the molecular mechanism by which DBT exerts its effects still remains unknown. Our goal for this study was to examine DBT effects on CYP1A expression in liver and gills of rainbow trout after short-term exposure. Juvenile trout individuals were injected intraperitoneally with two doses of DBT (10 or 50 mg kg −1) and were kept in tanks for 8 and 24 h ( T = 14 °C), then their gene expression levels were evaluated by Real-Time qPCR and Western-blot analysis. Treatment with DBT at either dose decreased CYP1A mRNA levels through the exposure period, which resulted in the final decrease of CYP1A protein levels in liver and gills on the end of experiment (24 h). Thus, our results showing significant depletion of CYP1A molecules in metabolic tissues upon DBT treatment correlate with those previous reports that indicate a role of DBT in reducing CYP1A activity in fish.

Effect of seawater transfer on CYP1A gene expression in rainbow trout gills

During the transfer of rainbow trout from freshwater to seawater, the gills have to switch from an ion-absorption epithelium to an ion-secretion epithelium in order to maintain equilibrium of their hydromineral balance. After a change to ambient salinity, several gill modifications have already been demonstrated, including ion transporters. In order to identify new branchial mechanisms implicated in seawater acclimation, we carried out an extensive analysis of gene expression in gills using microarray technology. This strategy allowed us to show that CYP1A gene expression was up-regulated in the gills after salinity transfer. This increase was confirmed by real-time reverse transcription PCR. Furthermore, measurements of CYP1A enzyme activity (EROD) showed a significant increase after transfer to seawater. Immunohistochemistry analysis in the gills revealed that cells with a higher expression of CYP1A protein were principally pillar cells and those in the primary lamellae not in contact with the external medium. The results of this study suggest for the first time that CYP1A may be implicated in the seawater acclimation of the gills of rainbow trout.

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout ( Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B–aniline–sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS–polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC 50 values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.

The characterisation of the behaviour and gill toxicity of CdS/CdTe quantum dots in rainbow trout (Oncorhynchus mykiss)

The increasing commercial application of nanomaterials is raising concerns about their potential release to aquatic environments. The purpose of this study was to examine the behaviour in freshwater of capped CdS/CdTe quantum dots and the sublethal effects on gills of rainbow trout. Rainbow trout (Oncorhynchus mykiss) were exposed to increasing concentrations of CdS/CdTe or dissolved cadmium (CdSO4) for 48 h at 15°C. The initial Cd and Te concentration in the aquarium water and size fractionation were determined. After the exposure period, the gills were analysed for labile Cd content, changes in lactate and pyruvate, total and redox status of metallothioneins (MT), lipid peroxidation, protein chaperones of the heat shock protein (Hsp) 72 family and ubiquitin conjugates. QDs were mostly between 6.8 and 25 nm suggesting aggregates. Significant increases in total and metal-binding MT, gill lactate and pyruvate levels and Hsp 72 proteins were observed while ubiquitin protein conjugates were significantly decreased by the QDs only with dissolved Cd, LPO and lactate/pyruvate ratio were not affected by the QDs. A discriminant function analysis of the biomarker responses revealed that colloidal and dissolved Cd differed significantly from each other, suggesting different modes of action.

Efficacy of Intraperitoneally and Orally Administered ProVale, a Yeast β‐(1,3)/(1,6)‐D‐glucan Product, in Inhibiting Xenoma Formation by the Microsporidian Loma salmonae on Rainbow Trout Gills

AbstractThe objectives of the research were to compare the efficacy of ProVale yeast beta‐glucan (β‐glucan) with that of a previously tested research‐grade yeast β‐glucan preparation when administered as an intraperitoneal (IP) injection and to also pilot test the effectiveness of ProVale yeast β‐glucan as a feed additive for reducing Loma salmonae xenoma formation on the gills of rainbow trout Oncorhynchus mykiss. Rainbow trout received IP injections of ProVale (4, 10, or 20 mg of ProVale/kg of fish) or Sigma β‐glucan (4 mg/kg). Oral challenge with L. salmonae occurred 1 week after IP injection with the β‐glucan products. For the second objective, 400 rainbow trout were separated into tanks and duplicate groups were treated with 0, 50, 100, and 200 g of ProVale/1,000 kg of feed. Starting at 3 weeks prior to challenge and continuing 2 weeks after challenge, rainbow trout were fed the various ProVale doses daily at a feeding rate of 1% of the fish biomass. Commencing at 4 weeks postchallenge, the fish in each trial were evaluated for the presence of xenomas on the first left gill arch. The most protective IP dose of commercial ProVale was 10 mg/kg when compared with the laboratory‐grade IP dose of Sigma β‐glucan (4 mg/kg). Both of these intraperitoneally administered β‐glucan products were effective in reducing the mean xenoma count. ProVale used as a feed coating (200 g/1,000 kg) was able to reduce the mean xenoma count by 50%.

Flow cytometric analysis of BDE 47 mediated injury to rainbow trout gill epithelial cells

The polybrominated diphenyl ethers (PBDEs) are ubiquitous environmental contaminants whose residues are increasing in fish, wildlife and human tissues. However, relatively little is known regarding the mechanisms of cell injury caused by PBDE congeners in fish. In the present study, we employed flow cytometry-based analyses to understand the onset and mechanisms of cell injury in rainbow trout gill cells (RTgill-W1 cells) exposed to 2,2′,4,4′-tetrabromodiphenyl ether (BDE 47). Substantial optimization and validation for flow cytometry protocols were required during assay development for the trout gill cell line. Exposure to micromolar concentrations of BDE 47 elicited a significant loss in RTgill-W1 cell viability that was accompanied by a decrease in NAD(P)H autofluorescence, a marker associated with disruption of cellular redox status. This loss in NAD(P)H content was accompanied by a decrease in nonyl acridine orange fluorescence, indicating mitochondrial membrane lipid peroxidation. Furthermore, low doses of BDE 47 altered cellular forward angle light scatter (FS, a measure of cell diameter or size) and side light scatter properties (SS, a measure of cellular internal complexity), consistent with the early stages of apoptosis. These changes were more pronounced at higher BDE 47 concentrations, which led to an increase in the percentage of cells undergoing frank apoptosis as evidenced by sub-G1 DNA content. Apoptosis was also observed at a relatively low dose (3.2 μM) of BDE 47 if cells were exposed for an extended period of time (24 h). Collectively, the results of these studies indicate that exposure of rainbow trout gill cells to BDE47 is associated with the induction of apoptosis likely originating from disruption of cellular redox status and mitochondrial oxidative injury. The current report extends observations in other species demonstrating that oxidative stress is an important mechanism of BDE 47 mediated cellular toxicity, and supports the use of oxidative stress-associated biomarkers in assessing the sublethal effects of PBDEs and their replacements in fish. The application of flow cytometry endpoints using fish cell lines should facilitate study of the mechanisms of chemical injury in aquatic species.

Changes in selected immunological parameters and antioxidant status of rainbow trout exposed to malachite green ( Oncorhynchus mykiss, Walbaum, 1792)

In this study, the effects of malachite green on selected immunological parameters, oxidative stress and antioxidant status biomarkers in blood, liver, kidney, spleen and gill of rainbow trout, Oncorhynchus mykiss, was examined. During 5 days the malachite green was applied at concentrations of 1/15,000 and 1/150,000 for 30 s and 60 min, respectively. Immunological parameters (nitroblue tetrazolium (NBT) activity, total plasma protein (TP), total immunoglobulin (TI)) and biochemical parameters (lipid peroxidation (MDA), catalase (CAT) activity, reduced glutathione (GSH) levels) were evaluated after exposed to malachite green. It has been observed that NBT activity ( p < 0.05, p < 0.001), total protein ( p < 0.01, p < 0.001) and total immunoglobulin ( p < 0.05, p < 0.001) levels were decreased compared with control group. In the rainbow tout exposed to malachite green duration 5 days significantly increased lipid peroxidation, which might be associated to decreased levels of reduced glutathione and catalase activity in the whole tissues of O. mykiss ( p < 0.05, p < 0.01, p < 0.001 for each cases).

Responses of antioxidant status and Na +–K +-ATPase activity in gill of rainbow trout, Oncorhynchus mykiss, chronically treated with carbamazepine

In recent years, chemical pollution by the residual pharmaceuticals has been increasingly important issue due to its widely present in the aquatic environment. However, the toxicological effects of residual pharmaceuticals on fish have not been adequately researched. The aim of this work is to investigate the toxic effect of CBZ, an anticonvulsant drug commonly present in aquatic environment, on antioxidant status and Na +–K +-ATPase in gill of rainbow trout exposed to sublethal CBZ (1.0 μg L −1, 0.2 mg L −1 and 2.0 mg L −1) for 7, 21 and 42 d. After prolonged exposure of CBZ at higher test concentration (0.2 or 2.0 mg L −1), oxidative stress was apparent as reflected by the significant higher LPO and CP levels in fish gill, as well as the significant inhibition of antioxidant enzymes activities including SOD, CAT, GR and GPx. Besides, reduced glutathione level and Na +–K +-ATPase activity were significantly lower than those of the control after 42 d of exposure to CBZ at higher test concentration (0.2 or 2.0 mg L −1). The results of this study indicate that chronic exposure of CBZ has altered multiple physiological indices in fish gill; however, before those parameters are used as special biomarkers for monitoring residual pharmaceuticals in aquatic environment, more detailed experiments in laboratory need to be performed in the future.

Phosphorylated intermediate of the ouabain-insensitive, Na +-stimulated ATPase in rat kidney cortex and rainbow trout gills

Several tissues from different animals, including the rat kidney and the freshwater rainbow trout gills, show an ouabain-insensitive, furosemide-sensitive, Na +-stimulated ATPase activity, which has been associated with the active control of the cell volume. This Na-ATPase is Mg 2+ dependent and it is inhibited by vanadate, which can be taken as an indication that this enzyme is a P-type ATPase. The P-type ATPases are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue at the enzyme's substrate site. In the current study, we partially characterized the phosphorylated intermediate of the ouabain-insensitive Na-ATPase of rat kidney cortex homogenates and that of gill microsomes from freshwater rainbow trout. While the kidney cortex homogenates, under our assay conditions, show both Na- and Na,K-ATPase activities, the gill microsomes, when assayed at pH 5.2, only show Na-ATPase activity. Both preparations showed a Mg 2+-dependent, Na +-stimulated phosphorylated intermediate, which is enhanced by furosemide. Incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH 2OH) showed that it is acid-stable and sensitive to hydroxylamine, either when phosphorylated in the presence or absence of furosemide. Addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation. Na + seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. In a E1–E2 reaction cycle of the Na-ATPase, furosemide seems to be blocking the transition step from Na·E1∼P to Na·E2-P.

Evaluating the cytotoxicity of palladium/magnetite nano-catalysts intended for wastewater treatment

Palladium/magnetite nanoparticulate catalysts were developed for efficient elimination of halogenated organic pollutants from contaminated wastewater. Particle recovery from treated water can be ensured via magnetic separation. However, in worst-case scenarios, this catalyst removal step might fail, leading to particle release into the environment. Therefore, a toxicological study was conducted to investigate the impact of both pure magnetite and palladium/magnetite nanoparticle exposure upon human skin (HaCaT) and human colon (CaCo-2) cell lines and a cell line from rainbow trout gills (RTgill-W1). To quantify cell viability after particle exposure, three endpoints were examined for all tested cell lines. Additionally, the formation of reactive oxygen species was studied for the human cells. The results showed only minor effects of the particles on the tested cell systems and support the assumption that palladium/magnetite nano-catalysts can be implemented for a new wastewater treatment technology in which advantageous catalyst properties outweigh the risks.

Measurement of total Zn and Zn isotope ratios by quadrupole ICP-MS for evaluation of Zn uptake in gills of brown trout ( Salmo trutta) and rainbow trout ( Oncorhynchus mykiss)

This study evaluates the potential use of stable zinc isotopes in toxicity studies measuring zinc uptake by the gills of brown trout ( Salmo trutta) and rainbow trout ( Oncorhynchus mykiss). The use of stable isotopes in such studies has several advantages over the use of radioisotopes, including cost, ease of handling, elimination of permit requirements, and waste disposal. A pilot study using brown trout was performed to evaluate sample preparation methods and the ability of a quadrupole inductively coupled plasma mass spectrometer (ICP-MS) system to successfully measure changes in the 67Zn/ 66Zn ratios for planned exposure levels and duration. After completion of the pilot study, a full-scale zinc exposure study using rainbow trout was performed. The results of these studies indicate that there are several factors that affect the precision of the measured 67Zn/ 66Zn ratios in the sample digests, including variations in sample size, endogenous zinc levels, and zinc uptake rates by individual fish. However, since these factors were incorporated in the calculation of the total zinc accumulated by the gills during the exposures, the data obtained were adequate for their intended use in calculating zinc binding and evaluating the influences of differences in water quality parameters.

Histopathological changes in rainbow trout ( Oncorhynchus mykiss) after exposure to sublethal composite nitrogen fertilizers

Subchronic toxicity of composite inorganic fertilizers such as ammonium sulfate [fertilizer A; (NH 4) 2SO 4; 21% NH 4-N)], composite fertilizer 15-15-15 (fertilizer B; commercial formulation: 15% NH 4-N, 15% phosphorus, and 15% potassium oxide), and composite fertilizer 25-5-10 (fertilizer C; commercial formulation: 25% NH 4-N, 5% phosphorus, and 10% potassium oxide) on the skin, liver, kidney, pancreas, and gills of the juvenile rainbow trout ( Oncorhynchus mykiss) was studied in two-week toxicity tests under static-renewal test conditions. Fish exposed to sublethal concentrations of fertilizers did not show any behavioral abnormality compared to control groups. Histological lesions were observed in skin, gills, liver, pancreas, and trunk kidney of the fish. In the epidermis, degenerated/vacuolated epithelial cells, microcystic dilatations, and intracellular edema of mucus cell were observed. Liver had swollen and degenerated hepatocytes without losing adenoid structure. Hematopoietic tissues had necrosis and vacuolar degeneration on proximal tubules of the kidney. In order, the most affected organs were skin, liver, and kidney.

Agglomeration of tungsten carbide nanoparticles in exposure medium does not prevent uptake and toxicity toward a rainbow trout gill cell line

Agglomeration of tungsten carbide nanoparticles in exposure medium does not prevent uptake and toxicity toward a rainbow trout gill cell line

Agglomeration of tungsten carbide nanoparticles in exposure medium does not prevent uptake and toxicity toward a rainbow trout gill cell line

Due to their increased production and use, engineered nanoparticles are expected to be released into the aquatic environment where particles may agglomerate. The aim of this study was to explore the role of agglomeration of nanoparticles in the uptake and expression of toxicity in the rainbow trout ( Oncorhynchus mykiss) gill cell line, RTgill-W1. This cell line was chosen as model because it is known to be amenable to culture in complete as well as greatly simplified exposure media. Nano-sized tungsten carbide (WC) with or without cobalt doping (WC-Co), two materials relevant in the heavy metal industry, were applied as model particles. These particles were suspended in culture media with decreasing complexity from L15 with 10% fetal bovine serum (FBS) to L15 to L15/ex, containing only salts, galactose and pyruvate of the complete medium L15. Whereas the serum supplement in L15 retained primary nanoparticle suspensions, agglomerates were formed quickly in L15 and L15/ex. Nevertheless, scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) elemental analysis revealed an uptake of both WC and WC-Co nanoparticles into RTgill-W1 cells irrespective of the state of agglomeration of nanoparticles. The localisation seemed to be restricted to the cytoplasm, as no particles were observed in the nucleus of cells. Moreover, reduction in cell viability between 10 and 50% compared to controls were observed upon particle exposure in all media although the pattern of impact varied depending on the medium and exposure time. Short-term exposure of cells led to significant cytotoxicity at the highest nominal particle concentrations, irrespective of the particle type or exposure medium. In contrast, long-term exposures led to preferential toxicity in the simplest medium, L15/ex, and an enhanced toxicity by the cobalt-containing WC nanoparticles in all exposure media. The composition of the exposure media also influenced the toxicity of the cobalt ions, which may dissolve from the WC-Co nanoparticles, with cells reacting much more sensitively toward cobalt ions in the absence of FBS. However, the toxicity observed by ionic cobalt alone did not explain the toxicity of the WC-Co nanoparticles, suggesting that the combination of metallic Co and WC is the cause of the increased particle toxicity of WC-Co. Taken together, our findings indicate that minimal exposure media can lead to rapid agglomeration of nanoparticles but that agglomeration does not prevent uptake into cells and the expression of toxicity.

Introduction: Hematology

Interleukin-6 (IL6) is a pleiotropic cytokine with important immunoregulatory functions. Its expression is inducible in immune cells and tissues of several fish species. We also found that IL6 mRNA abundance was significantly increased in spleen, liver, and gill of rainbow trout after experimental infection with Aeromonas salmonicida. Genomic DNA sequences of IL6 orthologs from three salmonid species revealed a conserved exon/intron structure and a high overall nucleotide identity of >88%. To uncover key mechanisms regulating IL6 expression in salmonid fish, we amplified a fragment of the proximal IL6 promoter from rainbow trout and identified in-silico conserved binding sites for NF-κB and CEBP. The activity of this IL6 promoter fragment was analyzed in the established human embryonic kidney line HEK-293. Luciferase- and GFP-based reporter systems revealed that the proximal IL6 promoter is activated by Escherichia coli. Essentially, both reporter systems proved that NF-κB p50, but not NF-κB p65 or CEBP, activates the IL6 promoter fragment. Truncation of this fragment caused a significant decrease in IL6 promoter activation. This characterization of the proximal promoter of the IL6-encoding gene provides basic knowledge about the IL6 gene expression in rainbow trout.

Gene Expression and Enzyme Activity of Mitochondrial Proteins in Irradiated Rainbow Trout (Oncorhynchus Mykiss, Walbaum) TissuesIn Vitro

In recent years ethical, legislative and economic pressures have created a renewed interest in the development of alternatives to in vivo animal experiments. In vitro studies, particularly those using cell cultures, have been used increasingly as tools to assess the degree of toxicity associated with or present in particular environments. While cell cultures are useful to give relative toxicity values, genotypic and phenotypic integrity may be compromised in the continuous artificial environment they experience. In addition, cell cultures lack the complexity of functional organs and thus do not truly represent the effects that toxins exert on organ and organism functionality. In this study, ex vivo tissue cultures of rainbow trout gill, skin and spleen samples were analyzed for variation of expression in genes associated with oxidative phosphorylation after exposure to ionizing radiation. Significant radiation-induced changes in gene expression and enzyme activity associated with the mitochondrial oxidative phosphorylation process were identified. The tissues examined in this study demonstrated an exposure threshold at which radiation dose stimulates an alteration in the regulatory activity of mitochondrial-associated genes. Spleen tissues exposed to low levels of radiation (0.1 Gy) appeared most sensitive whereas skin tissues proved least sensitive, reacting only to higher doses (>1 Gy). We propose this investigative approach as an innovative alternative to in vivo studies because it identifies toxic exposure in vitro and could significantly reduce the number of live-animal toxicity tests required.

Cellular mechanisms of Cl−transport in trout gill mitochondrion-rich cells

We have studied Cl(-) transport mechanisms in freshwater rainbow trout gill mitochondrion-rich (MR) cells using intracellular pH (pH(i)) imaging. Scanning electron microscopy demonstrated maintenance of cellular polarity in isolated MR cells. MR cell subtypes were identified by Na(+) introduction to the bath, and Cl(-) transport mechanisms were subsequently examined. Cl(-)-free exposure resulted in an alkalinization of pH(i) in both MR cell subtypes, which was dependent on HCO(3)(-) in the bath and inhibited by 1 mM DIDS. Recovery of pH(i) from an acidified state in Na(+)-free conditions was also DIDS sensitive. These results are the first functional evidence for Cl(-)/HCO(3)(-) exchangers in fish gill MR cells. A direct switch from NaCl to Cl(-)-free conditions caused a pH(i) acidification in a subset of MR cells, which was enhanced in the absence of HCO(3)(-). The acidification was replaced by an alkalinization when Cl(-) removal was performed in the presence of NPPB (500 microM) or EIPA (500 microM). Finally, we found that the Na(+)-induced alkalinization of pH(i) found in a previous study is inhibited by EIPA. This inhibitor profile's results suggest the presence of a Cl(-)-dependent Na(+)/H(+) exchange mechanism.

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.

Applications and future directions in invertebrate and fish cell culture

This collection of papers results from the 12th International Conference on Invertebrate and Fish Cell Culture, which took place concurrently with the 2008 World Congress on In Vitro Biology in Tucson, Arizona from June 14 to 18, 2008. The World Congress drew a total of 251 abstracts, including 54 oral presentations as well as numerous poster presentations. Attendees (342 in total) included many undergraduate and graduate students, postdoctoral associates, and junior and senior scientists, encouraging interaction at all levels. As part of this stimulating atmosphere, the 12th International Conference on Invertebrate and Fish Cell Culture offered six oral presentations covering two topics: “Cell Interactions and Signal Mechanisms” and “Fish and Amphibian Cells in Ecotoxicological Research”. Additionally, a round-table discussion encouraging lively discussion on the state of our field was also convened. Lastly, a joint session with the SIVB Animal Section included three presentations on “Virus-Cell Interactions in Vertebrate and Invertebrate Systems”. In the first symposium, three excellent presentations were given on “Prostaglandin Actions in an Established Insect Cell Line” by Dr. David Stanley (USDA/ARS/ BCIRL, Columbia, MO), “Signaling Interactions Between Olfactory Receptor Axons and Glial Cells in the Axon Sorting Zone of the Developing Moth Olfactory Pathway” by Dr. Lynne A. Oland (University of Arizona, AZ, USA), and “Intercellular Signaling Regulates Heart Development in Drosophila” by Dr. Stuart J. Newfeld (Arizona State University, AZ). The second symposium began with a presentation on “Applications and Potential Uses of Rtgill-W1, a Cell Line Derived from Gills of Rainbow Trout” by Dr. Lucilia E. J. Lee (Wilfrid Laurier University, ON, Canada), followed by “Unique Cell Characteristics for the Development of a Portable Cell-based Toxicity Sensor for Drinking Water Protection” by Dr. Mark W. Widder (US Army Center for Environmental Health Research, Fort Detrick, MD), and ended with “Evaluation of EP-1, a Cell Line fromAnguilla japonica, to Study the Life Cycle of the Microsporidian Heterosporis anguillarum” by Mrs. S.R. Monaghan (University of Waterloo, ON, Canada). The objectives of the round-table discussion were to assess the current and prospective uses for invertebrate and fish cell and tissue cultures, as well as to discuss their limitations and strengths. The topics included physiological investigations, the proliferation and study of pathogens, detection of environmental toxins, and the elucidation of toxin mechanism(s) of action. Additionally, the joint symposium on virus–cell interactions included a presentation by Dr. Suzanne M. Thiem (Michigan State University) on “Baculovirus Genes Affecting Host Functions”, one by Dr. Brenda G. Hogue (Arizona State University) on “Coronavirus Assembly at Internal Cellular Membranes”, and one by Dr. William T. Gerthoffer (University of South Alabama) on “Viral Gene Transfer Vectors in Studies of Human Smooth Muscle Function”. During our conference, we also had a special opportunity to honor Dr. Arthur McIntosh for his nearly 40 yr of research in insect cell culture and pathology with the 2008 SIVB Fellow Award. As a pioneer in insect cell culture, his In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-009-9178-5

AN ENRICHED STABLE-ISOTOPE APPROACH TO DETERMINE THE GILL–ZINC BINDING PROPERTIES OF JUVENILE RAINBOW TROUT (ONCORHYNCHUS MYKISS) DURING ACUTE ZINC EXPOSURES IN HARD AND SOFT WATERS

The objective of the present study was to employ an enriched stable-isotope approach to characterize Zn uptake in the gills of rainbow trout (Oncorhynchus mykiss) during acute Zn exposures in hard water (approximately 140 mg/L as CaCO3) and soft water (approximately 30 mg/L as CaCO3). Juvenile rainbow trout were acclimated to the test hardnesses and then exposed for up to 72 h in static exposures to a range of Zn concentrations in hard water (0-1000 microg/L) and soft water (0-250 microg/L). To facilitate detection of new gill Zn from endogenous gill Zn, the exposure media was significantly enriched with 67Zn stable isotope (89.60% vs. 4.1% natural abundance). Additionally, acute Zn toxicity thresholds (96-h median lethal concentration [LC50]) were determined experimentally through traditional, flow-through toxicity tests in hard water (580 microg/L) and soft water (110 microg/L). Following short-term (< or =3 h) exposures, significant differences in gill accumulation of Zn between hard and soft water treatments were observed at the three common concentrations (75, 150, and 250 microg/L), with soft water gills accumulating more Zn than hard water gills. Short-term gill Zn accumulation at hard and soft water LCS0s (45-min median lethal accumulation) was similar (0.27 and 0.20 microg/g wet wt, respectively). Finally, comparison of experimental gill Zn accumulation, with accumulation predicted by the biotic ligand model, demonstrated that model output reflected short-term (<1 h) experimental gill Zn accumulation and predicted observed differences in accumulation between hard and soft water rainbow trout gills. Our results indicate that measurable differences exist in short-term gill Zn accumulation following acclimation and exposure in different water hardnesses and that short-term Zn accumulation appears to be predictive of Zn acute toxicity thresholds (96-h LC50s).