Isolation and characterisation of two epithelial-like cell lines from the gills of Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon) and their use in aquatic toxicology.

In vitro gill models are becoming increasingly important in aquatic toxicology, yet the fish gill invitrome is underrepresented, encompassing approximately 0.1% of extant species. Here, we describe the establishment and characterisation of two gill-derived, epithelial-like cell lines isolated from fish species of significant importance to New Zealand: Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon). Designated CAgill1PFR (Chrysophrys auratus, gill 1, Plant & Food Research) and OTgill1PFR (Oncorhynchus tshawytscha, gill 1, Plant & Food Research), these cell lines have each been passaged greater than each 70 times over several years and are considered spontaneously immortalised. Both cell lines required serum for growth and exhibited differential responses to basal media formulations. CAgill1PFR was sensitive to low temperatures (4 °C) but replicated at high temperatures (30 °C), whereas OTgill1PFR was sensitive to high temperatures but remained viable at low temperatures, mirroring the natural environment of their host species. Immunostaining revealed expression of epithelial cell markers cytokeratin and E-cadherin, alongside positivity for the mesenchymal cell marker, vimentin. CAgill1PFR was more sensitive to the environmental toxin 3,4 dichloroaniline than OTgill1PFR through measurements of metabolic activity, membrane integrity, and lysosomal function. Furthermore, CAgill1PFR produced less CYP1A activity, indicative of ongoing biotransformation processes, in response to beta-naphthoflavone than OTgill1PFR. These cell lines expand the toolbox of resources and emphasise the need for species-specific aquatic toxicology research.

Distinguishing between ante factum and post factum properties of animal cell lines and demonstrating their use in grouping ray-finned fish cell lines into invitromes.

In this review, animal cell lines are considered to have two classes of attributes: "before-the-fact" (ante factum) and "after-the-fact" (post factum) properties. Fish cell lines from Actinopterygii (ray-finned fishes) are used to illustrate this distinction and to demonstrate how these properties can be used in various ways to categorize cell lines into groups or invitromes. Before-the-fact properties are set at initiation and are properties of the sample and species from which the cell line arose and of the scientist(s) who developed the cell line. On the basis of the Actinopterygii sample, invitromes exist for embryos, larvae, juveniles, adults, and spawning fish, and for most solid organs but rarely for biological fluids. For species, invitromes exist for only a small fraction of the Actinopterygii total. As to their development, scientists from around the world have contributed to invitromes. By contrast, after-the-fact properties are limitless and become apparent during development, characterization, use, and storage of the cell line. For ray-finned invitromes, cell lines appear to acquire immortality during development, are characterized poorly for differentiation potential, have numerous uses, and are stored formally only sporadically. As an example of applying these principles to a specific organ, the skeletal muscle invitrome is used. For ante factum properties, the cell lines are mainly from trunk muscle of economically important fish from 11 orders, 15 families, 19 genera, and 21 species of ray-finned fishes. For post factum properties, fibroblast-like and myogenic cell lines have been described but epithelial-like FHM is most widely used and curated. Considering cell lines by their before- and after-the-fact properties should facilitate integration of new cell lines into the literature and help incorporate the discipline of cell biology into other research areas, particularly the natural history of fishes.

A continuous myofibroblast precursor cell line from the tail muscle of Australasian snapper (Chrysophrys auratus) that responds to transforming growth factor beta and fibroblast growth factor.

Chrysophrys auratus (Australasian snapper) is one of the largest and most valuable finfish from capture fisheries in New Zealand, yet no cell lines from this species are reported in the scientific literature. Here, we describe a muscle-derived cell line initiated from the tail of a juvenile snapper which has been designated CAtmus1PFR (Chrysophrys auratus, tail muscle, Plant & Food Research). The cell line has been passaged over 100 times in 3 years and is considered immortal. Cells are reliant on serum supplementation for proliferation and exhibit a broad thermal profile comparable to the eurythermic nature of C. auratus in vivo. The impact of exogenous growth factors, including insulin-like growth factors I and II (IGF-I and IGF-II), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGFß), on cell morphology and proliferation was investigated. Insulin-like growth factors acted as mitogens and had minimal effect on cell morphology. TGFß exposure resulted in CAtmus1PFR exhibiting a myofibroblast morphology becoming enlarged with actin bundling. This differentiation was confirmed through the expression of smooth muscle actin (sma), an increase in type 1 collagen (col1a) expression, and a loss of motility. Expression of col1a and sma was decreased when cells were exposed to bFGF, and no actin bundling was observed. These data indicate that CAtmus1PFR may be myofibroblastic precursor cells descending from mesenchymal progenitor cells present in the tail muscle myosepta.

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.

Screening of Fish Cell Lines for Piscine Orthoreovirus-1 (PRV-1) Amplification: Identification of the Non-Supportive PRV-1 Invitrome.

Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells.

VHSV IVb infection and autophagy modulation in the rainbow trout gill epithelial cell line RTgill-W1.

Autophagy modulation influences the success of intracellular pathogens, and an understanding of the mechanisms involved might offer practical options to reduce the impact of infectious disease. Viral haemorrhagic septicaemia virus (VHSV) can cause high mortality and economic loss in some commercial fish species. VHSV IVb was used to infect a rainbow trout gill cell line, RTgill-W1, followed by the treatment of the cells with different autophagy-modulating reagents. LC3II protein using Western blot was significantly (p < .05) decreased for two days following VHSV infection, and immunofluorescence confirmed that LC3II-positive intracytoplasmic puncta were also decreased. Infection with VHSV resulted in significantly decreased expression of the autophagy-related (Atg) genes atg4, at12, atg13 and becn1 after one day using quantitative PCR. Both viral gene copy number and VHSV N protein were significantly decreased by treating the cells with autophagy-blocking (chloroquine) and autophagy-inhibiting reagents (deoxynivalenol and 3-methyladenine) after three days, while autophagy induction (restricted nutrition and rapamycin) had limited effect. Only treatment of RTgill-W1 with deoxynivalenol resulted in a significant increase in expression of type I interferon. Therefore, the suppression of autophagy initially occurs after VHSV IVb infection, but the modulation of autophagy can also inhibit VHSV IVb infection in RTgill-W1 after three days.

Xela DS2 and Xela VS2: Two novel skin epithelial-like cell lines from adult African clawed frog (Xenopus laevis) and their response to an extracellular viral dsRNA analogue.

The skin epithelial layer acts as an important immunological barrier against pathogens and is capable of recognizing and responding to pathogen-associated molecular patterns (PAMPs) in human and mouse models. Although presumed, it is unknown whether amphibian skin epithelial cells exhibit the ability to respond to PAMPs such as viral double-stranded RNA (dsRNA). To address this, two cell lines from the dorsal skin (Xela DS2) and ventral skin (Xela VS2) of the African clawed frog (Xenopus laevis) were established. Xela DS2 and Xela VS2 cells have an epithelial-like morphology, express genes associated with epithelial cells, and lack senescence-associated beta-galactosidase activity. Cells grow optimally in 70% Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 26 °C. Upon treatment with poly(I:C), a synthetic analogue of viral dsRNA and known type I interferon inducer, Xela DS2 and Xela VS2 exhibit marked upregulation of key antiviral and pro-inflammatory transcripts suggesting frog epithelial cells participate in the recognition of extracellular viral dsRNA and production of local inflammatory signals; similar to human and mouse models. Currently, these are the only known Xenopus laevis skin epithelial-like cell lines and will be important for future research in amphibian epithelial cell biology, initial host-pathogen interactions, and rapid screening of the effects of environmental stressors, including contaminants, on frog skin epithelial cells.

Understanding the pathogenesis of Flavobacterium psychrophilum using the rainbow trout monocyte/macrophage-like cell line, RTS11, as an infection model.

The life cycle of Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), appears to involve interactions with spleen and head kidney macrophages. To develop an in vitro model for studying this, F. psychrophilum was incubated with a rainbow trout splenic monocyte/macrophage-like cell line (RTS11) and fundamental macrophage functions evaluated. The animal cell basal medium, L15, supplemented with bovine serum (FBS) supports RTS11 maintenance, and surprisingly, L15 with 2% FBS (L15/FBS) also supported F. psychrophilum growth. L15/FBS in which the bacteria had been grown is referred to as F. psychrophilum conditioned medium (FpCM). Adding FpCM to RTS11 cultures caused a small, yet significant, percentage of cells to die, many cells to become more diffuse, and phagocytosis to be temporarily reduced. FpCM also significantly stimulated transcript expression for pro-inflammatory cytokines (IL-1ß, TNFα and IL-6) and the anti-inflammatory cytokine (IL-10) after one day of exposure but this upregulation rapidly declined over time. Adding live F. psychrophilum to RTS11 cultures also altered the cellular morphology and stimulated cytokine expression more profoundly than FpCM. Additionally, the phagocytic activity of RTS11 was also significantly impaired by live F. psychrophilum, but not to the same extent as when exposed to FpCM. Adding heat-killed bacteria to RTS11 cultures elicited few changes. These bacteria/RTS11 co-cultures should be useful for gaining a deeper understanding of the pathogenesis of F. psychrophilum and may aid in the development of effective measures to prevent infection and spread of this troublesome disease.

Regulation of endogenous antigen presentation in response to suboptimal temperatures in a walleye skin fibroblast cell line.

A skin fibroblast cell line WE-skin11f from walleye (Sander vitreus) was used to study the impact of temperature (26 °C, 20 °C, 14 °C, or 4 °C) on the transcript levels of genes involved in the endogenous antigen processing and presentation pathway (EAPP), which is an important antiviral pathway of vertebrates. Partial coding sequences were found for 4 previously unidentified walleye EAPP members, calreticulin, calnexin, erp57, and tapasin, and the constitutive transcript levels of these genes in WE-skin11f was unchanged by culture incubation temperature. The viral mimic poly (I:C) and viral haemorrhagic septicaemia virus (VHSV) IVb were used to study possible induction of EAPP transcripts (b2m, mhIa, and tapasin). The walleye cells were exquisitely sensitive to poly (I:C), losing adherence and viability at concentrations greater than 100 ng/mL, particularly at suboptimal temperatures. VHSV IVb viral particles were produced from infected WE-skin11f cells at 20 °C, 14 °C, and 4 °C but with much lower production at 4 °C. Under conditions where their impact on the viability of WE-skin11f cultures was slight, poly (I:C) and VHSV IVb were shown to induce b2m, mhIa, and tapasin transcript°s at 26 °C and 20 °C respectively. However, at 4 °C, the up-regulation of EAPP transcript levels was either delayed or completely impaired when compared to the 26 °C and 20 °C control temperatures of the respective experiments. These in vitro results suggest that suboptimal temperatures may be capable of modulating the regulation of the EAPP in walleye cells during viral infection.

Evolution of IFN subgroups in bony fish - 1:Group I-III IFN exist in early ray-finned fish, with group II IFN subgroups present in the Holostean spotted gar, Lepisosteus oculatus.

The present study helps clarify when the fish type I IFN groups/subgroups evolved, by examination of the IFN genes present in the Holostean spotted gar, Lepisosteus oculatus, in relation to the IFN genes present in the Chondrostea (sturgeon). It confirms that all three IFN groups (I-III), and group II subgroups, existed prior to the appearance of teleost fish. Preliminary expression analysis in a gar cell line (GARL) suggests these IFN genes will have a role in antiviral defence in Holostean fish, in that they are induced by poly(I:C). A refined model of IFN evolution within the actinopterygian fish is proposed.

Characterization of the continuous skin fibroblastoid cell line, WE-skin11f, from walleye (Sander vitreus).

A walleye dermal fibroblastoid cell line, WE-skin11f, was established and characterized. WE-skin11f was immunocytochemically positive for two known dermal fibroblast protein markers: vimentin and collagen I. At passage 26, WE-skin11f cultures contained both diploid and aneuploid populations. Ascorbic acid was required to produce extracellular collagen I fibres. Both of the skin fibroblastoid cell lines, WE-skin11f and rainbow trout-derived RTHDF, were not as good as the walleye caudal fin fibroblastoid cell line, WE-cfin11f, at forming abundant dense extracellular collagen matrices. The thermobiology of WE-skin11f was similar to that of other walleye cell lines with 26°C showing best temperature for growth and 4°C showing no growth but 100% viability. The transcript levels of b2m and mhIa genes of the major histocompatibility class I receptor in WE-skin11f were largely similar at all temperatures examined (4, 14, 20 and 26°C). Cortisol had a variety of effects on WE-skin11f cells: growth inhibition, morphological change from fibroblastoid to epithelioid, and enhancement of barrier function. Treatment of WE-skin11f cells with the physiologically relevant concentration of 100 ng/ml cortisol inhibited collagen I synthesis and matrix formation. Thus, WE-skin11f cell line could be useful in fish dermatology, endocrinology, and immunology research.

Improving a fish intestinal barrier model by combining two rainbow trout cell lines: epithelial RTgutGC and fibroblastic RTgutF.

An in vitro model of the fish intestine is of interest for research and application in diverse fields such as fish physiology, aquaculture and chemical risk assessment. The recently developed epithelial barrier model of the fish intestine relies on the RTgutGC cell line from rainbow trout (Oncorhynchus mykiss), cultured in inserts on permeable membranes. Our aim was to extend the current system by introducing intestinal fibroblasts as supportive layer in order to reconstruct the epithelial-mesenchymal interface as found in vivo. We therefore initiated and characterized the first fibroblast cell line from the intestine of rainbow trout, which has been termed RTgutF. Co-culture studies of RTgutGC and RTgutF were performed on commercially available electric cell substrate for impedance sensing (ECIS) and on newly developed ultrathin, highly porous alumina membranes to imitate the cellular interaction with the basement membrane. Cellular events were examined with non-invasive impedance spectroscopy to distinguish between barrier tightness and cell density in the ECIS system and to determine transepithelial electrical resistance for cells cultured on the alumina membranes. We highlight the relevance of the piscine intestinal fibroblasts for an advanced intestinal barrier model, particularly on ultrathin alumina membranes. These membranes enable rapid crosstalk of cells cultured on opposite sides, which led to increased barrier tightening in the fish cell line-based epithelial-mesenchymal model.

Effect of suboptimal temperature on the regulation of endogenous antigen presentation in a rainbow trout hypodermal fibroblast cell line.

Rainbow trout (Oncorhynchus mykiss) face low environmental temperatures over winter months and during extreme low temperature events. Suboptimal temperatures are known to negatively impact the teleost immune system, although there is mixed evidence in rainbow trout as to the effect on the endogenous antigen processing and presentation pathway (EAPP). The EAPP is an important pathway for antiviral defense that involves the presentation of endogenous peptides on the cell surface for recognition by cytotoxic T cells. Using a rainbow trout hypodermal fibroblast (RTHDF) cell line as an in vitro model, we determined that constitutive EAPP transcript levels are not impaired at low temperature, but induction of up-regulation of these transcripts is delayed at the suboptimal temperature following exposure to poly(I:C) or viral haemorrhagic septicaemia virus IVb, which was still able to enter and replicate in the cell line at 4 °C, albeit with reduced efficiency. The delay in the induction of EAPP mRNA level up-regulation following poly(I:C) stimulation coincided with a delay in ifn1 transcript levels and secretion, which is important since interferon-stimulated response elements were identified in the promoter regions of the EAPP-specific members of the pathway, implying that IFN1 is involved in the regulation of these genes. Our results suggest that the ability of rainbow trout to mount an effective immune response to viral pathogens may be lessened at suboptimal temperatures.

VER-155008 induced Hsp70 proteins expression in fish cell cultures while impeding replication of two RNA viruses.

The heat-shock protein 70 (Hsp70) inhibitor, VER-155008 (VER), was explored as a potential antiviral agent for two RNA viruses important to fish aquaculture, viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Studies were done at a temperature of 14 °C, and with cell lines commonly used to propagate these viruses. These were respectively EPC from fathead minnow for VHSV and CHSE-214 from Chinook salmon embryo for IPNV. Additionally, both viruses were studied with the Atlantic salmon heart endothelial cell line ASHe. For both VHSV and IPNV, 25 µM VER impeded replication. This was evidenced by delays in the development of cytopathic effect (CPE) and the expression of viral proteins, N for VHSV and VP2 for IPNV, and by less production of viral RNA and of viral titre. As VER inhibits the activity of Hsp70 family members, these results suggest that VHSV and IPNV utilize one or more Hsp70s in their life cycles. Yet neither virus induced Hsp70. Surprisingly VER alone induced Hsp70, but whether this induction modulated VER's antiviral effects is unknown. Exploring this apparent paradox in the future should improve the usefulness of VER as an antiviral agent.

Effect of selenomethionine on cell viability and heat shock protein 70 levels in rainbow trout intestinal epithelial cells at hypo-, normo-, and hyper-thermic temperatures.

As global warming and environmental pollution modify aquatic environments, the thermal biology of fish could be affected by interactions between temperature and pollutants, such as selenium (Se). Therefore, selenomethionine (SeMet) was studied for effects on cell viability and on heat shock protein 70 (HSP70) levels in the rainbow trout intestinal epithelial cell, RTgutGC, at hypothermic (4 °C), normothermic (14 and 18 °C) and hyperthermic (26 °C) temperatures. RTgutGC cultures remained viable for at least a week at all temperatures, although energy metabolism as measured with Alamar Blue (resazurin) was appreciably diminished at 4 °C. Over a 7-day incubation, HSP 70 levels in cultures remained steady at 4 °C, declined at 18 °C, and increased slightly at 26 °C. When 125 µM SeMet was present, cultures remained viable and HSP70 levels were neither increased nor decreased relative to control cultures, regardless of the temperature. With 500 and 1000 µM SeMet, cell viability was profoundly impaired after 7 days in cultures at 14, 18 and 26 °C but was unchanged at 4 °C. Overall the results suggest that only hypothermia modulated the response of rainbow trout cells to SeMet.

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.

Activation of an Aquareovirus, Chum Salmon Reovirus (CSV), by the Ciliates Tetrahymena thermophila and T. canadensis.

For the first time, ciliates have been found to activate rather than inactivate a virus, chum salmon reovirus (CSV). Activation was seen as an increase in viral titre upon incubation of CSV at 22 °C with Tetrahymena canadenesis and two strains of T. thermophila: wild type (B1975) and a temperature conditional mutant for phagocytosis (NP1). The titre increase was not likely due to replication because CSV had no visible effects on the ciliates and no vertebrate virus has ever been shown unequivocally to replicate in ciliates. When incubated with B1975 and NP1 at 30 °C, CSV was activated only by B1975. Therefore, activation required CSV internalization because at 30 °C only B1975 exhibited phagocytosis. CSV replicated in fish cells at 18 to 26 °C but not at 30 °C. Collectively, these observations point to CSV activation being distinct from replication. Activation is attributed to the CSV capsid being modified in the ciliate phagosomal-lysosomal system and released in a more infectious form. When allowed to swim in CSV-infected fish cell cultures, collected, washed, and transferred to uninfected cultures, T. canadensis caused a CSV infection. Overall the results suggest that ciliates could have roles in the environmental dissemination of some fish viral diseases.

CK-2 of rainbow trout (Oncorhynchus mykiss) has two differentially regulated alleles that encode a functional chemokine.

Rainbow trout chemokine 2 (CK-2) is currently the only known CC chemokine to have a mucin stalk. Further analysis of the mucin stalk region revealed a second, related CC chemokine sequence, denoted here as CK-2.1. This second sequence was determined to be an allele of CK-2 following genomic PCR analysis on several outbred individuals. Furthermore, in both in vivo and in vitro trials, CK-2 and CK-2.1 were both present, but appeared to have differential tissue expression in both control and PHA stimulated samples. Upon the development of a polyclonal antibody to rCK-2, CK-2 was only observed in the brain, liver and head kidney of PHA stimulated rainbow trout tissues. In comparison, when using the rainbow trout monocyte/macrophage-like cell line, RTS-11, CK-2 protein was observed in both control and PHA stimulated conditions. When studying the function of CK-2, a chemotaxis assay revealed that both peripheral blood leukocytes and RTS-11 cells migrated towards rCK-2 significantly at all concentrations studied when compared to truncated ß2m. Interestingly, this migration was lowest at both the highest concentration and the lowest concentrations of CK-2. Thus, teleostean chemokine receptors may become desensitized when overstimulated as has been observed in mammalian models. The observed chemotactic function was indeed due to rCK-2 as cell migration was inhibited through pre-treatment of both the cells and the polyclonal antibody with rCK-2. As has been observed thus far with all other chemokines, CK-2 does appear to function through binding to a G-coupled protein receptor as chemotaxis could be inhibited through pre-treatment with pertussis toxin. Overall, the results of this study indicate that CK-2 is a functional chemokine that is encoded by two differentially expressed alleles in rainbow trout, CK-2 and CK-2.1.

The characterization of γH2AX and p53 as biomarkers of genotoxic stress in a rainbow trout (Oncorhynchus mykiss) brain cell line.

Rainbow trout cell cultures were exposed to three genotoxicants and examined for effects on γH2AX and p53 levels by western blotting and on cell viability using the indicator dyes Alamar Blue (AB) for energy metabolism and 5'-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for plasma membrane integrity. Bleomycin induced γH2AX and p53 in a dose- and time-dependent manner and had little cytotoxic effect. However, induction was first seen at 0.3 µM for γH2AX but not until 16.5 µM for p53. Methyl methanesulfonate (MMS) increased H2AX phosphorylation but diminished p53 levels as the dose was increased from 908 µM up to 2724 µM. Over this dose range cell viability was progressively lost. 4-nitroquinoline N-oxide (NQO) induced both γH2AX and p53, beginning at 62.5 nM, which was also the concentration at which cell viability began to decline. As the NQO concentration increased further, elevated γH2AX was detected at up to 2.0 µM, while p53 was elevated up to 1.0 µM. Therefore, H2AX phosphorylation was superior to p53 levels as a marker of DNA damage caused by genotoxicants that act by introducing double-stranded DNA breaks (bleomycin), alkyl groups (MMS), and quinoline adducts (NQO).