Establishment and characterization of a gill cell line from pearl gentian grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀) and its application in cadmium toxicology.

A novel gill cell line from pearl gentian grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀, PGGG cell line) was established, its application in cadmium (Cd) toxicology was demonstrated in this study. Primary cultures and PGGG subcultures were carried out at 25 °C in Dulbecco's Modified Eagle medium/F12 medium (1:1; pH 7.2) supplemented with 15% fetal bovine serum (FBS). Primary PGGG cells were spindle-shaped, proliferated into a confluent monolayer within two weeks and were continuously subcultured over passage 60. The growth of cells at passages 20, 40, and 60 was examined. Chromosome analysis revealed that the chromosomal number of normal PGGG cells was 48, but the number of cells with the normal chromosomes number decreased during the passaging process. Cadmium is one of the most toxic metals in aquatic systems and has been associated with multiple animal and human health problems. To interpret the cytotoxicity and related mechanisms of cadmium, PGGG cells were used as an in vitro model. After treatment with cadmium at concentrations ranging from 1 µM to 500 µM, PGGG cells demonstrated dose- and time-dependent cytotoxicity, manifested as morphological abnormalities and a viability decline. Further, it was found that the reactive oxygen species (ROS) and malondialdehyde (MDA) levels were elevated following cadmium exposure, and related genes involved in the antioxidant system, including those encoding catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and Kelch-like- ECH-associated protein 1 (Keap1), were regulated differently. In addition, PGGG cells treated with cadmium had the typical features associated with apoptosis, including phosphatidylserine (PS) externalization; upregulated expression of caspase-3, -8, and -9; and apoptotic body formation. In general, the PGGG cell line may serve as a useful tool for studying the toxic mechanisms of cadmium or other toxicants or for toxicity testing and environment monitoring.

Gill structural integrity changes in fish deficient or excessive in dietary isoleucine: Towards the modulation of tight junction protein, inflammation, apoptosis and antioxidant defense via NF-κB, TOR and Nrf2 signaling pathways.

This study firstly aimed to test the impact of dietary isoleucine (Ile) on tight junction protein, inflammation, apoptosis, antioxidant defense and related signaling molecule gene expression in the gill of fish. Young grass carp (Ctenopharyngodon idella) (weighing 256.8 ± 3.5 g) were fed six diets containing graded levels of Ile, namely, 3.8, 6.6, 9.3, 12.5, 15.2 and 18.5 g/kg diet for 8 weeks. The results firstly revealed that Ile deficiency down-regulated the mRNA expressions of claudin-3, claudin-b, claudin-c, occludin and zonula occludens-1 (ZO-1) and up-regulated the mRNA expression of claudin-12, which led to the intercellular structure damage of fish gill. These effects were partially ascribed to the up-regulation of pro-inflammatory cytokines [interleukin 1ß (IL-1ß), interleukin 8 (IL-8) and tumor necrosis factor-α (TNF-α)] mRNA expressions that referring to up-regulated nuclear factor κB P65 (NF-κB P65) mRNA expression and down-regulated inhibitor factor κBα (IκBα) mRNA expression, and the down-regulation of anti-inflammatory cytokines [interleukin 10 (IL-10) and transforming growth factor ß1 (TGF-ß1)] mRNA expressions that referring to the down-regulated TOR and S6K1 mRNA expression. Interestingly, no change in claudin 15 mRNA level was observed among every treatment. At the same time, the results firstly indicated that Ile deficiency also resulted in the cellular structure damage of fish gill: (1) DNA fragmentation partially due to the up-regulation of caspase-3, caspase-8 and caspase-9 mRNA expression; (2) increase in protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the impaired antioxidant defense [indicated by decreased glutathione (GSH) level and depressed anti-superoxide anion (ASA), anti-hydroxyl radical (a-HR), copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) activities] that referring to the down-regulation of corresponding antioxidant enzyme mRNA expressions and the related signaling molecules Nrf2 mRNA expression. Ile excess caused similar negative effects that observed in Ile-deficient group, whereas these negative effects were reversed with appropriate Ile supplementation. In conclusion, our results indicated that Ile deficiency or excess disrupted the structural integrity of fish gill, partially due to the trigger of apoptosis, the impairment of antioxidant defense, and the regulation of tight junction protein, inflammatory cytokines, apoptosis-related, antioxidant enzymes and related signaling molecules mRNA expressions in the fish gill.

Changes in integrity of the gill during histidine deficiency or excess due to depression of cellular anti-oxidative ability, induction of apoptosis, inflammation and impair of cell-cell tight junctions related to Nrf2, TOR and NF-κB signaling in fish.

This study firstly explored the possible effects of dietary histidine on structural integrity and the related signaling factor gene expression in the gills of fish. Young grass carp (Ctenopharyngodon idella) were fed with six diets containing gradual levels of histidine for 8 weeks. The results firstly demonstrated that histidine deficiency caused increases in reactive oxygen species (ROS) contents, and severe oxidative damage (lipid peroxidation and protein oxidation) in the gills of fish, which was partially due to the decreased glutathione (GSH) content and antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR)]. Further investigations indicated that histidine deficiency caused depressions of those antioxidant enzyme activities are related to the down-regulation of corresponding antioxidant enzyme genes and the related signaling factor Nrf2 mRNA levels. Meanwhile, histidine deficiency induced DNA fragmentation via up-regulation of caspase-3, caspase-8 and caspase-9 expressions that referring to the down-regulation of TOR and S6K mRNA levels. Furthermore, His deficiency down-regulated claudin-b, claudin-c, claudin-3, claudin-12, claudin-15, occludin and ZO-1 transcription in fish gills. These effects were partially related to the up-regulation of pro-inflammatory cytokines, interleukin 1ß (IL-1ß), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α) and related signaling factor nuclear factor κB P65 (NF-κB P65) mRNA levels, and the down-regulation of anti-inflammatory cytokines, interleukin 10 (IL-10), transforming growth factor ß1 (TGF-ß1) and related signaling factor IκBα mRNA levels. Excessive histidine exhibited negative effects that were similar to histidine deficiency, whereas the optimal histidine levels reversed those negative effects. Taken together, our results showed that histidine deficiency or excess impaired the structural integrity of fish gill by disrupted fish antioxidant defenses and regulating the expression of tight junction protein, cytokines, apoptosis, antioxidant enzymes, NF-κB p65, IκBα, TOR, Nrf2, Keap1 and apoptosis-related genes in the fish gills.

Comparative study of the toxic effects of Chrysaora quinquecirrha (Cnidaria: Scyphozoa) and Chironex fleckeri (Cnidaria: Cubozoa) venoms using cell-based assays.

The venoms of jellyfish cause toxic effects in diverse biological systems that can trigger local and systemic reactions. In this study, the cytotoxic and cytolytic effects of Chrysaora quinquecirrha and Chironex fleckeri venoms were assessed and compared using three in vitro assays. Venoms from both species were cytotoxic to fish gill cells and rat cardiomyocytes, and cytolytic in sheep erythrocytes. Both venoms decreased cell viability in a concentration-dependent manner; however, the greatest difference in venom potencies was observed in the fish gill cell line, wherein C. fleckeri was 12.2- (P = 0.0005) and 35.7-fold (P < 0.0001) more potently cytotoxic than C. quinquecirrha venom with 30 min and 120 min cell exposure periods, respectively. Gill cells and rat cardiomyocytes exposed to venoms showed morphological changes characterised by cell shrinkage, clumping and detachment. The cytotoxic effects of venoms may be caused by a group of toxic proteins that have been previously identified in C. fleckeri and other cubozoan jellyfish species. In this study, proteins homologous to CfTX-1 and CfTX-2 toxins from C. fleckeri and CqTX-A toxin from Chironex yamaguchii were identified in C. quinquecirrha venom using tandem mass spectrometry. The presence and relative abundance of these proteins may explain the differences in venom potency between cubozoan and scyphozoan jellyfish and may reflect their importance in the action of venoms.

[A comparison of methods for identifying mast cells of Cyprinus carpio].

OBJECTIVE: To compare different identifying methods of the mast cells of red crucian carp (Cyprinus carpio). METHODS: The gill filaments were taken from Cyprinus carpio infected with Edwardsiella tarda and fixed in Bouin's solution. The hematoxylin-eosin (HE), toluidine blue (TB), Alcian blue (AB), neutral red, Masson trichrome, May-Grunwald Giemsa (MGG) stainings and streptavidin-biotin complex (SABC) immunohistochemical method were used to observe the mast cells. The smears of head kidney stained by Wright-Giemsa were used as a supplementary method. RESULTS: AB and TB were desirable staining solutions for clearly displaying mast cells, and MGG staining came second. Immunohistochemical method showed the small number of tryptase-positive mast cells and a weak positive reaction. The cells in smears were dispersed and showed different developmental stages. CONCLUSION: The immunohistochemistry and the histochemical staining combined with Wright-Giemsa-stained smears are two good ways to identify the mast cells of fish.

Origin and differentiation of ionocytes in gill epithelium of teleost fish.

This paper focuses on the environmental cues that transform the gills of euryhaline teleost fish from an oxygen exchange structure into a bifunctional organ that can control both gaseous movement and water/ion transport. The cellular development that allows this structure to accomplish these tasks begins shortly after fertilization of the egg. It involves alterations of structure and function of embryonic cells [ionoblasts (IB)] that are shed from the pharyngeal anlage area of the embryo. These IB contain unique protein-receptor domains in the plasma membrane. These receptors respond specifically to the environmental cues effecting a calcium-binding protein receptor [calcium-sensing receptor (CaSR)]. The CaSR containing IB act as stem cells and are acted upon by isotocin, a heteroprotein regulator which induces them to form progenitor ionocytes (pIC). The pIC form two types of cells. The first type becomes an aquaphilic ionocyte which regulates uptake of ions and through aquaporin molecules transports water out of the cell and controls body fluids of the fish. This mechanism is essential for freshwater living. The second type becomes a halophilic ionocyte and transports ions out of the cell and controls cell shrinkage by uptake of water via aquaporin molecules. This mechanism is essential for seawater living. These differentiating events in the pIC are controlled by the cross talking of genomic mechanisms found in the precursor IB. To unravel the cross talking events it is necessary to uncover how these genetic pathways are regulated by transcriptional and translational events coming from complementary DNA. Various gene families are involved such as those found in apoptosis mechanisms, regulatory volume regulators and ionic transport systems (cystic fibrosis transmembrane conductance regulator).

Effect of chloride on nitrite-induced methaemoglobinemia in Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus (Mitchill).

We evaluated the effects of chloride concentration on the clinical pathology in juvenile Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus (Mitchill), following semi-static exposures to 1 mg L(-1) nitrite for 96 h. In spring water naturally low in chloride (5 mg L(-1)), plasma nitrite concentrated to more than 40× environmental levels resulting in a severe methaemoglobinemia characterized by torpid behaviour, 30-fold increase in methaemoglobin fraction, anaemia, leucopenia and hyperkalaemia. Loss of intracellular water and potassium to extracellular space may have resulted in hyperkalaemia and haemodilution. Fish survived nitrite exposure, but 60% of torpid fish died following capture and tissue sampling. Fish acclimated to 10-fold higher chloride content (55 mg L(-1)) did not concentrate nitrite in the plasma above environmental levels or develop methaemoglobinemia, but did exhibit similar haematology and plasma chemistry changes. Plasma nitrite returned to preexposure levels by 14 days following nitrite exposures, but severity of clinical pathology changes persisted or increased, suggesting that Atlantic sturgeon have reduced capacity to recover from methaemoglobinemia. Fish that survive methaemoglobinemia may be susceptible to mortality from the cumulative effects of intoxication, handling and other stresses for two or more weeks following nitrite remediation. Chloride buffering in aquaculture systems reduces the toxic effects of nitrite accumulation.

Monitoring follow up of two areas affected by the Prestige oil four years after the spillage.

The sinking of the oil tanker Prestige in November 2002 resulted in the spill of more than 63,000 tonnes of crude oil, and polluted more than 1,000 km of coastline, especially affecting Galicia (northwestern Spain). Four years after the accident, a new biological monitoring study was undertaken of two Galician areas intensely affected by the spill, Lira and Ancoradoiro, previously evaluated in the months following the accident ( Laffon et al. 2006 ). The mussel Mytilus galloprovincialis was employed as bioindicator organism to determine both polycyclic aromatic hydrocarbons (PAH) levels and genotoxic effects. PAH were determined chromatographically in seawater samples and mussel tissues collected from November 2006 to January 2008. The results obtained showed that PAH pollution was still present in these areas, but bioaccumulation of these compounds in mussels was low, compared to reference mussels, and lower than in our previous study. DNA damage assessment was also performed in gills and hemolymph cells by means of the alkaline comet assay. DNA damage levels were higher in mussels from the exposed areas than in reference mussels. DNA damage decreased after a 7-d recovery period in the laboratory, but prolonging the recovery period up to 14 d did not contribute to less DNA damage in gill cells. Hemolymph cells were more sensitive than gill cells to the induction of DNA damage.

An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish.

AIMS: To develop an in vitro screening method to be used for identifying potential effective chemotherapeutants to control Aeromonas hydrophila infections. METHODS AND RESULTS: Using catfish gill cells G1B and four chemicals (hydrogen peroxide, sodium chloride, potassium permanganate and D-mannose), the feasibility of using an in vitro screening method to identify potential effective chemotherapeutants was evaluated in this study. In vitro screening results revealed that, at concentration of 100 mg l⁻¹, H2O2 was the only chemical tested that was able to completely abolish the attachment and invasion of Aer. hydrophila to catfish gill cells. In vivo virulence studies using live channel catfish through bath immersion confirmed that H2O2 was the only chemical tested that was able to significantly (P < 0·001) reduce the mortality (from 90 or 100% to 0 or 20%) caused by Aer. hydrophila infections. CONCLUSIONS: The in vitro screening method using catfish gill cells G1B could be used to initially identify potential effective chemotherapeutants to control Aer. hydrophila. SIGNIFICANCE AND IMPACT OF THE STUDY: An in vitro screening method using catfish gill cells to identify potential effective chemotherapeutants described here will cut cost in research compared with the method of using live fish to screen lead compounds for fish disease control.

Specific expression and regulation of glucose transporters in zebrafish ionocytes.

Glucose, a carbohydrate metabolite, plays a major role in the energy supply for fish iono- and osmoregulation, and the way that glucose is transported in ionocytes is a critical process related to the functional operations of ionocytes. Eighteen members of glucose transporters (GLUTs, SLC2A) were cloned and identified from zebrafish. Previously, Na(+),K(+)-ATPase-rich (NaR), Na(+)-Cl(-) cotransporter-expressing (NCC), H(+)-ATPase-rich (HR), and glycogen-rich (GR) cells have been identified to be responsible for Ca(2+) uptake, Cl(-) uptake, Na(+) uptake, and the energy deposition, respectively, in zebrafish skin/gills. The purpose of the present study was to test the hypothesis of whether GLUT isoforms are specifically expressed and function in ionocytes to supply energy for ion regulatory mechanisms. On the basis of translational knockdown of foxi3a/3b (2 transcriptional factors related to the ionocytes' differentiation) and triple in situ hybridization/immunocytochemistry, 3 GLUT isoforms, zglut1a, -6, and -13.1, were specifically localized in NaR/NCC cells, GR cells, and HR cells, respectively. mRNA expression of zglut1a in embryos and adult gills were stimulated by the low Ca(2+) or low Cl(-) freshwater, which has been previously reported to upregulate the functions (monitored by epithelial Ca(2+) channel, NCC mRNA) of NaR/NCC cells, respectively while that of zglut13.1 was stimulated only by low Na(+), a situation to upregulate the function (monitored by carbonic anhydrase 15a mRNA) of HR cells. On the other hand, ambient ion compositions did not affect the zglut6 mRNA expression. Taken together, zGLUT1a, -6, and 13.1, the specific transporters in NaR/NCC cells, GR cells, and HR cells, may absorb glucose into the respective cells to fulfill different physiological demands.

65Zn2+ transport by isolated gill epithelial cells of the American lobster, Homarus americanus.

Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in 65Zn2+ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 muM verapamil, nifedipine, and the calcium ionophore A23187. 65Zn2+ influxes were hyperbolic functions of zinc concentration (1-1,000 microM) and followed Michaelis-Menten kinetics. Calcium reduced both apparent zinc binding affinity (K (m)) and maximal transport velocity (J (max)) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. 65Zn2+ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented.

Glycogen phosphorylase in glycogen-rich cells is involved in the energy supply for ion regulation in fish gill epithelia.

The molecular and cellular mechanisms behind glycogen metabolism and the energy metabolite translocation between mammal neurons and astrocytes have been well studied. A similar mechanism is proposed for rapid mobilization of local energy stores to support energy-dependent transepithelial ion transport in gills of the Mozambique tilapia (Oreochromis mossambicus). A novel gill glycogen phosphorylase isoform (tGPGG), which catalyzes the initial degradation of glycogen, was identified in branchial epithelial cells of O. mossambicus. Double in situ hybridization and immunocytochemistry demonstrated that tGPGG mRNA and glycogen were colocalized in glycogen-rich cells (GRCs), which surround ionocytes (labeled with a Na(+)-K(+)-ATPase antiserum) in gill epithelia. Concanavalin-A (a marker for the apical membrane) labeling indicated that GRCs and mitochondria-rich cells share the same apical opening. Quantitative real-time PCR analyses showed that tGPGG mRNA expression levels specifically responded to environmental salinity changes. Indeed, the glycogen content, glycogen phosphorylase (GP) protein level and total activity, and the density of tGPGG-expressing cells (i.e., GRCs) in fish acclimated to seawater (SW) were significantly higher than those in freshwater controls. Short-term acclimation to SW caused an evident depletion in the glycogen content of GRCs. Taken altogether, tGPGG expression in GRCs is stimulated by hyperosmotic challenge, and this may catalyze initial glycogen degradation to provide the adjacent ionocytes with energy to carry out iono- and osmoregulatory functions.

Activation and nuclear translocation of ERK in response to ligand-dependent and -independent stimuli in liver and gill cells from rainbow trout.

The mitogen-activated protein kinase ERK is an important signalling molecule involved in the control of cell proliferation, differentiation and cell death, targeting molecules at the cell membrane, in the cytosol, and in the nucleus. This study investigated the activation pattern and subcellular distribution of ERK in liver and gill cells of rainbow trout upon hypo-osmotic shock, addition of epidermal growth factor (EGF) and copper treatment. It further set out to characterize the hypothetical role of nuclear-export signal (NES)-dependent relocation of ERK after nuclear entry and the potential involvement of the ERK activator MEK. Although, in primary hepatocytes, ERK was activated in all conditions in a stimulus-specific manner, it did not accumulate in the nucleus, irrespective of the absence or presence of the inhibitor of NES-dependent export leptomycin B (LB). Similarly, in trout hepatoma cells, where pERK levels increased upon osmotic and mitotic stimulation, but not after toxic insult, no significant nuclear translocation was observed. In a gill cell line, levels of pERK increased after osmotic and mitotic stimulation and showed a decrease during incubation with a toxicant. Again, none of these conditions triggered nuclear accumulation of pERK in the gill cells in the absence of LB, but in contrast to the observation in liver cells, both osmotic and mitotic stimulation caused nuclear accumulation in the presence of the inhibitor. The ERK activator MEK, which possesses a NES-sequence, was apparently not involved in nuclear export, as it did not seem to enter the nucleus. Altogether, ERK is activated in trout cells in a stimulus- and cell type-specific manner, and our data suggest that it acutely acts primarily on cytoplasmic or membrane-situated targets in liver cells, whereas it presumably triggers rapid transcriptional activities in gill cells.

Induction of metallothionein-like proteins by mercury and distribution of mercury and selenium in the cells of hepatopancreas and gill tissues in mussel Mytilus galloprovincialis.

The binding of mercury (Hg) to metallothioneins (MTs) and the relation between Hg and selenium in supernatants of hepatopancreas and gill tissues of the common mussel Mytilus galloprovincialis (Lamarck, 1819) was investigated. The mussels were exposed to different Hg concentrations in laboratory conditions: 2.5 microgHg/L, 4 d exposure (short term) and 60 microgHg/L, 33 d exposure (long term). In addition, the results were compared to those found for mussels from nature (polluted and unpolluted region). In control and short-term-exposed mussels, the level of Hg extraction (cytosol) from hepatopancreas and gill cells was very low with respect to the total Hg concentrations in the corresponding tissues, around 10% in control and around 20% after exposure. As expected, Hg exposure was followed by Se increase. For Se, the levels of extraction were higher, around 20% in control and up to 50% (heaptopacrease) of 70% (gills) after exposure. In order to study the distribution of Hg and Se in the cells of these organs, the total Hg and Se concentrations were analyzed in the subcellular fractions obtained after differential centrifugation. Although after exposure the concentrations of both element increased in all subcellular fractions, their percentages in particular fractions were lower or higher. In this study, the convincing binding of Hg to metallothionein-like proteins was perceived after long-term laboratory exposure (gills, heapatopancreas) and in wild mussels collected near industrial port (hepatopancreas). In latter case, we also detected the traces of Se bound to the MT fractions after size-exclusion chromatography.

Induction of micronuclei and nuclear abnormalities in Oreochromis niloticus following exposure to petroleum refinery and chromium processing plant effluents.

The genotoxic effects of effluents from a petroleum refinery and a chromium processing plant were evaluated in Oreochromis niloticus (Pisces: Perciformes) using the micronucleus test. Fish were exposed to different concentrations (5, 10 and 20%, v/v) of the effluents for 3, 6 and 9 days. Micronucleus analyses were carried out on gill epithelial cells and peripheral blood erythrocytes. Nuclear abnormalities other than micronuclei, considered as genetic damage indicators, were also evaluated on erythrocytes. Cyclophosphamide at a single dose of 4 mg/L was used as a positive control. The results of this study showed that both effluents had genotoxic potential. On the other hand, the level of genetic damage induced by petroleum refinery effluent was considerably higher than that of chromium processing plant effluent. Our results further indicate that nuclear abnormalities other than micronuclei, such as blebbed and lobed nuclei, may also be used as indicators of genotoxic damage.

Analyses of impact of metal ion contamination on carp (Cyprinus carpio L.) gill cell suspensions.

The decline in fish population because of water contamination is problem. As a result of direct exposure in water, it has been readily accepted that the gills are the main site of water contamination and toxicity (e.g., metal ions). In the present study, we investigated metal ion contamination on the functional capacity of carp gill cells with antioxidant interactions in an in vitro study. The extent of cellular membrane damage, lipid peroxidation (LPO) (as TBARS levels), and glutathione (GSH) content were investigated after the addition of two metal ion compounds (viz., CuSO(4) and HgCl(2)) in various concentrations (300, 500, 700, 1000, and 3000 microM) to gill cell preparation of the freshwater fish carp (Cyprinus carpio L.) with modulations by bovine serum albumin (BSA) (0.5% and 1.0%) and dimethyl sulfoxide (DMSO) (0.5%) as free-radical scavengers. The Comet assay technique was also performed for the highest concentrations of the two mentioned metal ions as an index of DNA breaks. The outcomes were as follows: (1) Copper and mercury increased the rate of LPO dose dependently (r=+0.995 and r=+0.993, respectively; p<0.001), but the GSH content was only marginally affected (r = -0.787 and r = -0.844, respectively; p <0.05). (2) Depletion of GSH molecules by copper had a wider range than mercury. (3) In the highest concentration of metal ions (3000 microM), both DMSO and 1.0% BSA showed a pro-oxidative potential to elevate the levels of TBARS (p<0.001), but for other concentrations when supplemented with three scavengers, a fall in the levels of the latter was found. (4) The addition of 1.0% BSA to medium containing 3000 microM of metal ions caused a significant decline in GSH content (p<0.01). (5) Copper and mercury could cause a high rate of DNA breaks (single stranded) in carp gill cell suspensions as a Comet appearance. These findings indicate that copper and mercury have a deleterious influence on membrane integrity and GSH content in a relatively dose-dependent manner. The complexes of metal ions and thiol (SH) residues of cell proteins could also act as a potential cell toxicant leading to disturbances in cell functions causing cell death. DNA fragmentation is frequent in metal ion contamination.

Effects of cortisol and prolactin on Na+ and Cl- transport in cultured branchial epithelia from FW rainbow trout.

The electrophysiological and ion-transporting properties of cultured gill epithelia from freshwater (FW) rainbow trout were examined in the presence of cortisol and prolactin as media supplements. Epithelia were of the double-seeded insert (DSI) type containing both pavement cells (PVCs) and mitochondria-rich cells (MRCs) and were grown in Leibovitz's L15 media on filters allowing exposure to different apical media conditions. Experiments were carried out in two series after 7-9 days symmetrical (L15 apical-L15 basolateral) culture. In both series, 100% L15 was maintained as the basolateral medium throughout and supplemented with physiologically relevant doses of either prolactin (50 ng/ml), cortisol (500 ng/ml), or cortisol + prolactin (500 + 50 ng/ml, respectively). In series 1, epithelia were exposed to progressively diluted apical media (100, 75, 50, 25, 12.5% L15, and FW) at 24-h intervals. The preparations retained integrity [high transepithelial resistance (TER); low ion efflux rates] during this prolonged dilution protocol. Cortisol, or cortisol + prolactin, resulted in a greater TER and reduced ion efflux rates during dilution, likely an effect on junctional permeability of PVCs, but did not promote active Na+ and Cl- uptake from apical FW. In series 2, epithelia were directly exposed to apical FW and ion fluxes measured over the first 6 h. Under these conditions, cortisol or cortisol + prolactin promoted active uptake of both Na+ and Cl- simultaneously from apical FW, probably attributable to actions on the MRCs. However, Na+-K+-ATPase activities were not significantly altered by any of the treatments in either series. Overall, prolactin alone did not appear to promote FW adaptation but exhibited synergism with cortisol. These results provide further support for the cultured DSI epithelium as an in vitro model for ion transport in FW fish.

Induction of glutathione-S-transferases in primary cultured digestive gland acini from the mollusk bivalve Pecten maximus (L.): application of a new cellular model in biomonitoring studies.

In the last three decades, marine invertebrates have been used to monitor environmental health conditions and potential pollution, e.g. in the Mussel Watch Program. The whole animal or specific organs are used to determine contamination levels and disturbances. In the present study, a new in vitro cell culture model was validated for pollution monitoring. A commercial species, the scallop Pecten maximus, was tested for the presence and induction of phase II glutathione-S-transferase (GST) enzymes. These activities were monitored for a year, and the results were found to be consistent with those in the literature. Tributyltin, ethylmethane sulfonate and the water-soluble fraction of crude oil were assayed in, in vitro induction studies. A rapid increase of GST activities was observed within 24 h with all compounds tested, and a time- as well as a dose-response was established. This in vitro cell culture model seems suitable for routine use to predict the effects of pollutants on whole organisms within an ecosystem and in fisheries.

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller).

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.

Cultured gill epithelia from freshwater tilapia (Oreochromis niloticus): effect of cortisol and homologous serum supplements from stressed and unstressed fish.

Procedures for the preparation and culture of branchial epithelia from dispersed gill cells of freshwater tilapia (Oreochromis niloticus) are described. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on both the apical and basolateral side with isotonic media containing 6% fetal bovine serum (FBS). When the apical medium was replaced with freshwater (pseudo in vivo asymmetrical culture conditions), transepithelial resistance (TER) increased markedly, transepithelial potential became negative, and paracellular permeability decreased. The physiological effects of cortisol and 10% homologous (tilapia) serum were investigated. Tilapia serum (TS) was prepared from unstressed and stressed fish and therefore allowed comparison between the effects of homologous serum derived from fish in differing physiological states. Under both symmetrical and asymmetrical culture conditions, cortisol significantly elevated TER across cultured tilapia gill epithelia, indicative of a significant increase in epithelial "tightness." Cortisol reduced transepithelial Na + and Cl? movement and paracellular permeability. The glucocorticoid agonist dexamethasone elicited a similar response, which was inhibited by the glucocorticoid antagonist (receptor blocker) RU486. Cortisol did not stimulate active ion transport across epithelia under either symmetrical or asymmetrical culture conditions. In epithelia supplemented with TS from stressed fish, physiological changes in cultured preparations were consistent with those observed in FBS + cortisol-supplemented epithelia. Differences between the physiological status of epithelia supplemented with TS from unstressed and stressed fish could be abolished with RU486. Using TS as a medium supplement did not stimulate active ion transport under asymmetrical culture conditions, although Na +-K +-ATPase activity increased in TS-supplemented epithelia relative to FBS-supplemented preparations.