Characterization of a bovine intestinal myofibroblast cell line and stimulation using phytoglycogen-based nanoparticles bound to inosine monophosphate.

The goal of the present study was to characterize a novel bovine intestinal myofibroblast (BT-IMF) cell line isolated from a fetal bovine intestine. This cell type is of importance as intestinal myofibroblasts play a key role in controlling intestinal epithelial cell proliferation, intestinal regulation, wound healing, epithelial cell turnover, and structural support. The present work demonstrates that BT-IMF cells could be successfully cryopreserved and thawed and cultured past 25 passages. Immunocytochemical staining of the BT-IMF cell line was positive for vimentin and smooth muscle actin (α-SMA) and negative for pancytokeratin, suggesting that the cells are myofibroblastic in type. Growth kinetic experiments demonstrate that hydrocortisone negatively impacts BT-IMF growth and non-essential amino acids enhance its proliferation. Inosine monophosphate (IMP) is a dietary nucleotide and is essential for supporting animal health. Stimulation with IMP bound to a novel phytoglycogen-based nanocarrier (IMP-NP) showed enhanced cell proliferation. BT-IMF provides a new tool for studying bovine cells in vitro and may be of particular interest for cultured meat manufacturing in the future.

Development of a walleye cell line and use to study the effects of temperature on infection by viral haemorrhagic septicaemia virus group IVb.

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.

Applications and potential uses of fish gill cell lines: examples with RTgill-W1.

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.

Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cells.

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.

Comparative oxygen radical formation and toxicity of BDE 47 in rainbow trout cell lines.

The polybrominated diphenyl ethers (PBDEs) constitute a class of flame retardants whose residues have markedly increased in fish and human tissues during the last decade. In particular, the levels of certain PBDE congeners in salmon have raised concern regarding potential risks associated with dietary PBDE exposures. However, little is known regarding PBDE-mediated cell injury in relevant in vitro cell models. We conducted a comparative study of oxyradical production and cell injury in rainbow trout gill (RTgill-W1) and trout liver cells (RTL-W1) exposed to 2,2',4,4'-tetrabromodiphenyl ether (BDE 47), a predominant BDE residue found in fish tissues such as salmonids. Exposure to low micromolar concentrations of BDE 47 elicited a significant loss in RTgill-W1 and RTL-W1 cell viability as measured by alamarBlue assay. The dose-response of BDE toxicity differed among the two cell lines, with the RTL-W1 liver cells showing greater resistance to toxicity at lower BDE 47 doses, but a more dramatic loss of viability relative to gill cells when challenged with higher (50 microM) doses. The sensitivity of the trout liver cells at higher BDE 47 exposures was reflected by a higher basal production of oxygen radical production by 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence that was markedly enhanced in the presence of BDE 47, suggesting an overwhelming of trout liver cell antioxidant defense pathways. Collectively, our data indicate that RTgill-W1 and RTL-W1 liver cells are sensitive to BDE 47-mediated cell injury through a mechanism that may involve oxidative stress. Our data also provide an in vitro basis for potential tissue differences in BDE 47-mediated cell injury.

Development of a continuous cell line, PBLE, from an American eel peripheral blood leukocyte preparation.

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.

High yield and rapid growth of Neoparamoeba pemaquidensis in co-culture with a rainbow trout gill-derived cell line RTgill-W1.

Neoparamoeba pemaquidensis is an ubiquitous amphizoic marine protozoan and has been implicated as the causative agent for several diseases in marine organisms, most notably amoebic gill disease (AGD) in Atlantic salmon. Despite several reports on the pathology of AGD, relatively little is known about the protozoan and its relationship to host cells. In this study, an in vitro approach using monolayers of a rainbow trout gill cell line (RTgill-W1, ATCC CRL-2523) was used to rapidly grow large numbers of N. pemaquidensis (ATCC 50172) and investigate cell-pathogen interactions. Established cell lines derived from other tissues of rainbow trout and other fish species were also evaluated for amoeba growth support. The amoebae showed preference and highest yield when grown with RTgill-W1 over nine other tested fish cell lines. Amoeba yields could reach as high as 5 x 10(5) cells mL(-1) within 3 days of growth on the gill cell monolayers. The amoebae caused visible focal lesions in RTgill-W1 monolayers within 24 h of exposure and rapidly proliferated and spread with cytopathic effects destroying the neighbouring pavement-like cells within 48-72 h after initial exposure in media above 700 mOsm kg(-1). Disruption of the integrity of the gill cell monolayers could be noted within 30 min of exposure to the amoeba suspensions by changes in transepithelial resistance (TER) compared with control cell monolayers maintained in the exposure media. This was significantly different by 2 h (P < 0.05) compared with control cells and remained significantly different (P < 0.01) for the remaining 72 h that the TER was monitored. The RTgill-W1 cell line is thus a convenient model for growing N. pemaquidensis and for studying host-pathogen interactions in AGD.

Cell adhesion characteristics of a monocytic cell line derived from rainbow trout, Oncorhynchus mykiss.

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.

Gill and liver histopathological changes in yellow perch (Perca flavescens) and goldfish (Carassius auratus) exposed to oil sands process-affected water.

The extraction of bitumen from the Athabasca oil sands (Alberta, Canada) produces significant volumes of process-affected water containing elevated levels of naphthenic acids (NAs), ions, and polycyclic aromatic hydrocarbons (PAHs). The sublethal response of aquatic organisms exposed to oil sands constituents in experimental aquatic environments that represent possible reclamation options has been studied. In this study, the effects of process-affected waters on gill and liver tissues in yellow perch (Perca flavescens) and caged goldfish (Carassius auratus) held in several reclamation ponds at Syncrude's Mildred Lake site have been assessed. Following a 3-week exposure, significant gill (epithelial cell necrosis, mucous cell proliferation) and liver (hepatocellular degeneration, inflammatory cell infiltration) histopathological changes were noted in fish held in waters containing high levels of oil sands process-affected water. In addition, measurements of gill dimensions (gill morphometrical indices) proved sensitive and provided evidence of a physiological disturbance (gas exchange) with exposure to oil sands materials. Due to the complexity of oil sands process-affected water, the cause of the alterations could not be attributed to specific oil sands constituents. However, the histopathological parameters were strong indicators of exposure to oil sands process-affected water and morphometrical data were sensitive indicators of pathological response, which can be used to identify the interactive effects of ionic content, NAs, and PAHs in future laboratory studies.

The effects of salinity on naphthenic acid toxicity to yellow perch: gill and liver histopathology.

Naphthenic acids (NAs) are naturally occurring saturated linear and cyclic carboxylic acids found in petroleum, including the bitumen contained in the Athabasca Oil Sands deposit in Alberta, Canada. The processing of these oil sands leads to elevated concentrations of NAs, as well as increased salinity from produced waters as a result of ions leaching from the ores, the process aids, and the water associated with the deeper aquifers. These changes can result in waters that challenge reclamation of impacted waters associated with oil sands development. Laboratory tests examined the effects of salinity on NA toxicity using local young-of-the-year yellow perch exposed to a commercially available mixture of NAs (CNA) and an NA mixture that was extracted from oil sands process-affected water (ENA), with and without the addition of sodium sulfate (Na(2)SO(4)). Gill and liver histopathological changes were evaluated in the surviving fish after 3 weeks of exposure. At 6.8 mg/L ENA and 3.6 mg/L CNA, 100% mortality was observed, both with and without the addition of salt. Exposure of yellow perch to 25% of the NA required to give an LC100 (0.9 mg/L CNA; 1. 7 mg/L ENA) resulted in high levels of gill proliferative (epithelial, mucous, and chloride cell) changes, a response that was increased with the addition of 1g/L salt (Na2SO4) for the ENA. The significance of these changes was a reduced gill surface area, which likely caused a reduction in both the transport of NAs within the fish and the exchange of vital respiratory gases. While the gills were affected, no liver alterations were identified following NA or NA+salt exposures. Differences in the chemical composition of the NAs tested may explain the differences in the lethality and histopathology of yellow perch.

Butyltin species in benthic and pelagic organisms of the Saguenay Fjord (Canada) and Imposex occurrence in common whelk (Buccinum undatum).

The distribution and accumulation of butyltins in various tissues of 13 benthic and pelagic species living in the sub-Arctic Saguenay Fjord (Canada) were investigated. Butyltin contamination was ubiquitous in this ecosystem with tributyltin (TBT) biota to sediment accumulation factors (BSAF) ranging between 0.22 and 11, but without any important biomagnification between trophic levels. The large range of butyltin compounds accumulating within different tissues of the species collected from all trophic levels was from 7 to 1238 ng Sn g(-1) d.w. and indicates an exceptional contamination level only found in northern coastal areas exposed to an intensive traffic of commercial ships. Results show that bioaccumulation in organisms depends on three main factors: (1) the actual contamination level in their habitat, (2) their assimilation pathway by water, sediment or diet, and (3) their ability to metabolize TBT and excrete metabolites. By their lack of an efficient TBT degradation system, bivalves are subject to accumulate more butyltins (from 890 to 993 ng Sn g(-1) d.w. for TBT and from n.d to 138 ng Sn g(-1) d.w. for metabolites) whereas most burrow-dwelling organisms are able to degrade TBT and their butyltin levels ranged from 86 to 239 ng Sn g(-1) d.w. for TBT and from 7 to 106 ng Sn g(-1) d.w. for metabolites. Acadian redfish (Sebastes fasciatus) feeding preferentially on shrimps and small crustaceans rich in TBT showed a contamination level about three times higher than eelpout (Licodes vahlii). The latter species living in contact with the sediment and feeding on worms and other burrowing species had a lower proportion of TBT in their tissues. Finally, deleterious effects of butyltins in the Saguenay Fjord were assessed by the significant occurrence of Imposex in common whelk (Bucinum undatum) in two sites from Baie des Ha! Ha!. Results revealed that the effects of Imposex were accentuated close to the source of contamination, at Port-Alfred harbour, since the total of imposexed whelks collected at site A (the mouth of Baie des Ha! Ha!) was 12.5% and reached 52.6% at site B (Port Alfred). Although the incidence or frequency of imposex was low in site A compared to site B, the relative penile length index (RPL) values, a measure of the degree or severity of imposex, was similar at both sites indicating the presence of TBT with higher concentrations in site B.

Effect of corticosteroids on viability and proliferation of the rainbow trout monocyte/macrophage cell line, RTS11.

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.

Integrin-like substrate adhesion in RTG-2 cells, a fibroblastic cell line derived from rainbow trout.

Fluorometric cell attachment assays together with competitive inhibitors of adhesion were used to probe for the presence of integrins, a diverse family of heterodimeric cell-surface glycoproteins involved in cell-cell and cell-extracellular matrix adhesion, in the fibroblastic rainbow trout cell line, RTG-2. The adhesive properties of this cell line were evaluated. RTG-2 cells adhered poorly to TC plastic in the absence of serum but as little as 2.5% fetal bovine serum allowed over 75% of the cells to attach after 5 h. Surfaces coated with the extracellular matrix proteins collagen I, collagen IV, fibrin, fibrinogen, or fibronectin were able to support attachment of RTG-2 cells. Adhesion of RTG-2 cells to fibronectin varied linearly with fibronectin coating densities in the range 0 to 65 ng/mm(2). Oligopeptides containing the sequence Arg-Gly-Asp (RGD) caused dose-dependent inhibition of adhesion to microtiter plates coated with fibrin, fibrinogen, and fibronectin, whereas attachment to collagen I and collagen IV was less severely affected. In all cases, peptides containing Arg-Gly-Glu (RGE) or Asp-Gly-Arg (DGR) sequences caused no reduction of cell attachment. Since many integrins mediate adhesion by binding to RGD sequences in their target ligands, these results suggest the presence of integrin-like adhesion molecules on the surface of RTG-2 cells.