A comparison of cadmium-induced metallothionein gene expression and Me2+ distribution in the tissues of cadmiumsensitive (rainbow trout; Salmo gairdneri) and tolerant (stone loach; Noemacheilus barbatulus) species of freshwater fish

1. The accumulation of cadmium in the liver, kidney and gills of rainbow trout and stone loach was measured during exposure of the fish to the metal at 3 smg/l in their aquarium water. The pattern of accumulation of the toxic metal in the individual organs was different between the two species.2. The tissue concentrations of metallothionein-specific mRNA and metallothionein protein were also determined in these organs from the same fish. In rainbow trout, the induction of metallothionein gene expression resulted in a gradual increase in metallothionein concentration in gill over the course of the experiment whereas increases in metallothionein in the liver and kidney were detected only at the later time points of analysis (beyond 19 weeks). By contrast, in the same tissues from stone loach, relatively minor changes were quantified in specific mRNA and metallothionein concentrations.3. Throughout the experimental period, tissue concentrations of zinc and copper were determined in the liver, kidney and gills of the rainbow trout and stone loach. Subtle decreases were observed in the zinc concentration of gills in rainbow trout and substantial increases were observed in the hepatic copper concentrations in both species at the later time points of analysis.4. The ability of cadmium to induce metallothionein gene expression and its subsequent ability to compete for the sequestration sites on the newly-synthesized protein is discussed with regard to the relative levels of cadmium, zinc and copper in the organs studied and differing regimes of cadmium administration.     

A comparison of the accumulation and protein binding of environmental cadmium in the gills, kidney and liver of rainbow trout (Salmo gairdneri Richardson)

Rainbow trout were exposed to cadmium in their aquarium water for different lengths of time and at different concentrations. More than 99% of the total body loads accumulated under all such conditions of exposure was found in the liver, kidney and gills. Comparison of the metal-binding proteins in these organs indicated that two low mol. wt proteins sequestered the cadmium while zinc was bound to metallothionein. Cadmium was not found in association with metallothionein unless artificially high concentrations were introduced either by intraperitoneal injection of the fish or by dialysis of tissue extracts in vitro.     

A comparison of the sequestration of cadmium and zinc in the tissues of rainbow trout (Salmo gairdneri) following exposure to the metals singly or in combination

Rainbow trout were exposed to either cadmium (9 micrograms/l) or zinc (100 micrograms/l) in their aquarium water. They were then transferred to water containing concentrations of cadmium (54 micrograms/l) that would have otherwise proved fatal to the majority of the fish without the pretreatment. Most of the fish survived under both sets of conditions. However, two different mechanisms seem to be involved in the protection of the animals against the toxic manifestations of cadmium. In both cases, more than 99% of the total body load of cadmium was found in the liver, kidney and gills of the animals. Analysis of the metal-binding proteins in these organs was carried out. In the fish exposed to the two concentrations of cadmium, the toxic metal was found only in association with two low mol. wt specific binding proteins despite the presence of zinc- (and copper)-containing isometallothioneins in all three organs. On the other hand, cadmium was distributed between these binding-proteins and metallothioneins in the liver, kidney and gill of the trout pretreated with zinc before their exposure to cadmium.     

Sequestration of environmental cadmium by metallothionein in the roach (Rutilus rutilus) and the stone loach (Noemacheilus barbatulus)

Two species of coarse fish that are relatively resistant to cadmium poisoning were exposed to sub-lethal concentrations of the metal in their aquarium water. Thus, roach were exposed to cadmium concentrations between 30 and 500 micrograms/l for periods of 14-70 days whereas stone loach were exposed to 1250 micrograms Cd/l for 21-77 days. Under all conditions of exposure, it was found upon analysis of the major organs of accumulation of cadmium in the two species that the toxic metal was sequestered by a single isoform of metallothionein. The amino acid compositions of roach and stone loach metallothionein were determined and found to be similar to those reported for other piscine metallothioneins. The two proteins were found to contain Cd:Zn:Cu in approximate ratios of 4:1:2 per mole of protein. The sequestration of Cd by metallothionein in the two resistant species of fish is contrasted with the situation observed previously in a cadmium-sensitive species, the rainbow trout.     

The expression of a synthetic rainbow trout metallothionein gene in E. coli

A synthetic gene for rainbow trout metallothionein was constructed and inserted into a dual origin plasmid where expression was induced by a temperature shift in a proteinase-deficient strain of Escherichia coli. The recombinant protein was purified to homogeneity, and a partial amino acid sequence was determined to confirm its identity. Its immunochemical characteristics were similar to those of native metallothionein from rainbow trout. The amounts of recombinant metallothionein produced were quantified in soluble cell extracts by ELISA. Low concentrations were detected when growth was performed either in L-broth or defined (GMM-II) medium. Supplementation of the medium with zinc or copper had no effect on the amount of metallothionein produced. By contrast, when cadmium was included in either L-broth or GMM-II medium, much higher concentrations of the protein within the cells (approx. 13 micrograms/mg soluble cell protein) were detected. This stabilisation of the protein by metal reconstitution in vivo is considered in relation to the selective uptake/exclusion of metals by the cells and its significance for the scavenging of certain precious or toxic heavy metals is discussed.     

Correlation between metallothionein and energy metabolism in sea bass,Dicentrarchus labrax,exposed to cadmium

Specimens of sea bass (Dicentrarchus labrax) were exposed to two different cadmium concentrations (0.5 and 5 μg Cd²⁺/ml seawater) for a period of 7 days. Cadmium accumulated in the tissues of D. labrax in the following order: kidney > liver > gills at both concentrations. Accumulation patterns in fish exposed to 0.5 μg Cd²⁺/ml seawater were different with respect to 5.0 μg Cd²⁺/ml seawater. At both Cd concentrations a similar stress situation occurred during the first 4 hr as noted by the depletion of glycogen stores and the increase in free glucose in the muscle; metallothionein was induced in the liver, but failed to bind all the cytosolic Cd, which was in part bound to high-molecular-weight ligands. Fish recovered from this initial stress situation within 24 hr as indicated by the increase in glycogen and the decrease of glucose. Long-term effects were clearly dependent upon metal concentration: at lower Cd exposure, metallothionein induction increased linearly with time and counteracted the toxic effect of the metal; on the other hand, when fish were exposed to 5.0 μg Cd²⁺/ml seawater a clear stress occurred at the end of the exposure, as indicated by the notable decrease of glycogen stores, the increase of free glucose, the decrease of AEC in the muscle and the increase of Cd bound to high-molecular-weight ligands in the liver.     

Endogenous and heavy-metal-ion-induced metallothionein gene expression in salmonid tissues and cell lines

Endogenous levels of metallothionein (MT) mRNA were detected by RNA probes in several somatic and germ-line tissues of rainbow trout, such as eggs, ovaries and immature testis. These levels may be related to metal-ion homeostasis in the observed tissues. The induction kinetics of trout MT isoform B (MT-B) mRNA were studied after single intraperitoneal injections of CdCl2, CuCl2 and ZnCl2. MT-B mRNA was induced within 12 h in liver, kidney, spleen and gills. However, over the 48-h experimental period, the kinetics of MT-B mRNA accumulation differed in response to the three metal salts, possibly due to differential handling of the salts by these tissues. Multiple metal-salt injections induced high levels of MT-B mRNA in the four tissues studied. In the rainbow trout hepatoma cell line, ZnCl2 was a better inducer of the MT-B gene, as compared to CdCl2 and CuCl2. The expression of the exogenous trout MT-B promoter in Chinook salmon embryonic cell line indicates the presence of MT regulatory factors. In contrast, the endogenous MT genes in these cells are quiescent, possibly due to the methylation of their promoter region.     

The effect of digested sludge on the common carp, Cyprinus carpio L.

The effects of sewage sludge on the survival of the common carp, Cyprinus carpio, were investigated. It was noted that, when the concentrations of the sludge used for rearing carp exceeded 0.6%, the fish died within 42 days, whereas those reared in 0.2% sludge remained alive and healthy during the trial period. The body and the gills of the dead fish were covered by a veil-like film which looked like coagulated mucus and was formed by the heavy metal ions from the sludge reacting with some constituents of the mucus secreted by the gill. The histopathological symptoms of the various organs further demonstrated that internal injury in liver and kidney was also an important feature of the intoxicants as well as external damage, especially the particles observed around the gills. It was concluded that the optimum concentration of sludge to any species of fish should be determined by testing a series of concentrations before using it as a supplementary fish feed or fish pond fertilizer.     

Selenium-induced autometallographic demonstration of endogenous zinc in organs of the rainbow trout, Salmo gairdneri

Autometallographic (AMG) silver enhancement of endogenous zinc was studied in seven organs of the rainbow trout Salmo gairdneri. Groups of trout were injected intraperitoneally with sodium selenite in doses ranging from 0.08 to 25 ppm, administered 1 h before being killed. The concentration of selenium obtained by each organ was determined by gamma-spectrometry, and compared with the autometallographic deposition of silver grains. The relative accumulation of selenium in the organs was: liver greater than spleen greater than kidney greater than intestine greater than gills greater than brain greater than muscle. In the fish labelled with 10 and 25 ppm Se, AMG-deposits were found (1) within lysosomes of liver cells, (2) within the granules and on the nuclear membrane of melanophores in the spleen, (3) on the microvilli and in the apical cytoplasm of renal proximal tubular cells, (4) within the granules and along the plasma membrane of intestinal eosinophilic granule cells, and in the apical portion of the intestinal epithelium, and (5) in the gills, within granule cells and on the surface of the ionocytes. In the trouts injected with 5 ppm Se, silver grains were still observed in the liver, the intestine, and the gills, whereas, no such grains were found in preparations from fish having received 1 ppm Se. The use of selenium for the histochemical demonstration of endogenous zinc versus exogenous metals is discussed. Also, consideration is given to the question of which part of the total tissue zinc that is histochemically reactive.     

Increase of Metallothionein-immunopositive Chloride Cells in the Gills of Brown Trout and Rainbow Trout After Exposure to Sewage Treatment Plant Effluents

Metallothionein, a biomarker of exposure and toxicity of heavy metals, has been detected in the gills of brown trout (Salmo trutta fario L.) and rainbow trout (Oncorhynchus mykiss Richardson) by means of immunohistochemistry. A very prominent labelling of chloride cells was found after exposure to diluted sewage plant effluents. No significant increase was observed in either the number of labelled cells or their labelling intensity after exposure to water of a polluted river compared to fish kept in tap water. These results do not correlate with findings of a histopathological study, suggesting that the metal levels at the sewage treatment plant were too low to produce gross histopathology. A comparison between the species indicated that the rainbow trout showed a generally higher metallothionein expression than the brown trout.     

Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn

In this work we have studied the accumulation of heavy metals in two brown trout (Salmo trutta) populations in their natural environment and the participation of metal binding to metallothionein (MT) in this process. Cd, Cu and Zn concentrations, total MT (including Cu MT) and Cd/Zn MT were measured in the gills, liver and kidney of trout inhabiting two rivers, one Cu-contaminated and the other Cd/Zn-contaminated, located at Røros, Central Norway. In both populations, high levels of Cu were found in the liver, whereas Cd was accumulated in liver and particularly in the kidney. The proportions of Cd/Zn MT and Cu MT in liver and kidney, but not in gills, reflected the accumulated and the environmental concentrations of these metals. The total Cu MT concentrations in the investigated tissues, however, were highest in trout from the river with the lowest ambient Cu concentration. It is suggested that MTs are of less importance in Cu-acclimated trout. The data also suggest that acclimation to a Cu-rich environment involves reduced Cu accumulation or increased Cu elimination. In trout from the Cd-rich environment, this metal was mainly bound to MT, whereas in trout from the Cu-rich environment Cd was also associated with non-MT proteins. These findings emphasize the importance to determine both Cd/Zn MT and Cu MT levels, when the participation of this protein in metal handling in trout tissues is investigated.     

Comparison of dietary and waterborne exposure to benzo a pyrene: bioavailability,tissue disposition and CYP1A1 induction in rainbow trout Oncorhynchus mykiss

Absorption and tissue distribution of benzo\[ a ]pyrene (BaP)-derived radioactivity were studied in juvenile rainbow trout following dietary or waterborne exposure. In order to compare the bioavailability of BaP, the fish were exposed to 1.5 mCi 3H-BaP kg-1 fish, either in the diet or in the water as a 2 days static exposure. Furthermore, tissue levels of BaP-derived radioactivity bound to macromolecules in different tissues were studied in non-induced fish, and in fish induced by additional treatment with unlabelled BaP (corresponding to 5 mg kg-1 fish) in the water. Absorption and tissue distribution of 3H BaP were studied by liquid scintillation counting and whole-body autoradiography. BaPderived radioactivity bound to macromolecules in different tissues was studied by autoradiography of solvent-extracted whole-body sections. The hepatic CYP1A induction was measured as EROD activity. Exposure to unlabelled BaP resulted in a marked induction of hepatic EROD activity in rainbow trout 2 days after the start of the exposure. Significant higher concentrations of radiolabelled compound were observed in waterborne-exposed fish, in contrast to dietary-exposed fish. High concentrations of radiolabelling were observed in the gills, liver, bile, intestines, olfactory organ, kidney and the skin of the waterborne-exposed fish. In the dietary-exposed fish, high levels of radioactivity were observed in the intestines and the bile, whereas lower concentrations were present in the liver. Only traces of radioactive compound were observed in the gills. In contrast to waterborne-exposed fish, no radioactivity was detected in the olfactory organ or skin. In autoradiograms of sections extracted with a series of polar and non-polar solvents, a large fraction of radioactivity was still present in the gills, olfactory organ, liver, kidney, skin and intestinal mucosa of the waterborne-exposed fish, indicating that reactive BaP intermediates formed by CYP1A-mediated metabolism were bound to macromolecules in these tissues.     

The Effects of Acute Waterborne Exposure to Sublethal Concentrations of Molybdenum on the Stress Response in Rainbow Trout,Oncorhynchus mykiss

To determine if molybdenum (Mo) is a chemical stressor, fingerling and juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne sodium molybdate (0, 2, 20, or 1,000 mg l^-1 of Mo) and components of the physiological (plasma cortisol, blood glucose, and hematocrit) and cellular (heat shock protein [hsp] 72, hsp73, and hsp90 in the liver, gills, heart, and erythrocytes and metallothionein [MT] in the liver and gills) stress responses were measured prior to initiation of exposure and at 8, 24, and 96 h. During the acute exposure, plasma cortisol, blood glucose, and hematocrit levels remained unchanged in all treatments. Heat shock protein 72 was not induced as a result of exposure and there were no detectable changes in total hsp70 (72 and 73), hsp90, and MT levels in any of the tissues relative to controls. Both fingerling and juvenile fish responded with similar lack of apparent sensitivity to Mo exposure. These experiments demonstrate that exposure to waterborne Mo of up to 1,000 mg l^-1 did not activate a physiological or cellular stress response in fish. Information from this study suggests that Mo water quality guidelines for the protection of aquatic life are highly protective of freshwater fish, namely rainbow trout.     

Morphological organ alterations and infectious diseases in brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss exposed to polluted river water

Poor water quality is discussed as a major factor causing a decline of brown trout populations in Swiss rivers. For our study we have chosen a river in the Swiss midlands, where the brown trout population has decreased dramatically during the last 10 yr and where feral fish have shown distinctive pathological alterations. The objective of our study was to investigate whether river water may be responsible for impaired fish health leading to an increased mortality in the river. In an active monitoring program, groups of brown and rainbow trout were exposed to polluted river water for 24 mo. Fish held in tap water served as a reference. Mortality, macroscopic and histopathologic changes, and infectious agents were investigated. Compared with the reference group, high mortality rates and severe pathological alterations of the inner organs were observed in fish held in river water. Especially gills, liver and kidney of these fish showed significantly higher changes than fish from tap water. These changes were dominated by degenerative and inflammatory reactions. Additionally, several infectious agents were diagnosed in fish exposed to river water. The most important findings were furunculosis and proliferative kidney disease. Brown trout seemed to be more sensitive than rainbow trout to environmental stress and infectious agents.     

In vivo and in vitro effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius Ham. (rosy barb).

1. Enzyme modulation by cadmium in selected organs of the fish, Barbus conchonius (rosy barb), was investigated in vivo (48 hr exposure to 12.6 mg/l cadmium chloride) and in vitro (10(-6) M cadmium chloride). 2. The acetylcholinesterase (AchE) activity was depressed in the gills but stimulated in the skeletal muscles and brain in vivo. The hepatic, branchial, and renal acid phosphatase (AcP) activity decreased marginally in vivo but it was significantly increased in the gut and ovary. In vitro, except for the liver, the AcP activity was depressed in the selected organs. Collaterally, gut alkaline phosphatase (AlP) was significantly inhibited but a pronounced stimulation was noted in the kidneys and ovary in vivo. In vitro, the AlP activity was conspicuously elevated in the kidneys and gut, and moderately in the gills. 3. Cadmium inhibited the glutamate-oxaloacetate and glutamate-pyruvate transaminases (GOT and GPT) in the liver, gills and kidneys in vivo. In vitro, the GOT and GPT activities were decreased in the liver, gills and kidneys. The lactic dehydrogenase (LDH) was significantly stimulated by Cd in the heart in vivo but in vitro the metal inhibited the enzyme in the gills. 4. Enzymes in the liver, followed by those in the kidneys and gills seem to be most seriously affected by Cd poisoning in this fish.     

Selenium-induced autometallographic demonstration of endogenous zinc in organs of the rainbow trout,Salmo gairdneri

Summary Autometallographic (AMG) silver enhancement of endogenous zinc was studied in seven organs of the rainbow trout Salmo gairdneri. Groups of trout were injected intraperitoneally with sodium selenite in doses ranging from 0.08 to 25 ppm, administered 1 h before being killed. The concentration of selenium obtained by each organ was determined by gamma-spectrometry, and compared with the autometallographic deposition of silver grains. The relative accumulation of selenium in the organs was: liver > spleen > kidney > intestine > gills > brain > muscle. In the fish labelled with 10 and 25 ppm Se, AMG-deposits were found (1) within lysosomes of liver cells, (2) within the granules and on the nuclear membrane of melanophores in the spleen, (3) on the microvilli and in the apical cytoplasm of renal proximal tubular cells, (4) within the granules and along the plasma membrane of intestinal eosinophilic granule cells, and in the apical portion of the intestinal epithelium, and (5) in the gills, within granule cells and on the surface of the ionocytes. In the trouts injected with 5 ppm Se, silver grains were still observed in the liver, the intestine, and the gills, whereas, no such grains were found in preparations from fish having received 1 ppm Se. The use of selenium for the histochemical demonstration of endogenous zinc versus exogenous metals is discussed. Also, consideration is given to the question of which part of the total tissue zinc that is histochemically reactive.     

A putative beta2-adrenoceptor from the rainbow trout (Oncorhynuchus mykiss). Molecular characterization and pharmacology.

Extensive molecular characterization of mammalian beta-adrenoceptors has revealed complex modes of regulation and interaction. Relatively little attention, however, has focused on adrenoceptors from early branching vertebrates such as fish. Using an RT-PCR approach we have cloned a rainbow trout beta2-adrenoceptor gene that codes for a 409-amino-acid protein with the same seven transmembrane domain structure as its mammalian counterparts. This rainbow trout beta2-adrenoceptor shares a high degree of amino-acid sequence conservation with other vertebrate beta2-adrenoceptors. The conclusion that this sequence is a rainbow trout beta2-adrenoceptor is further supported by phylogenetic analysis of vertebrate beta-adrenoceptor sequences and competitive pharmacological binding data. RNase protection assays demonstrate that the rainbow trout beta2-adrenoceptor gene is highly expressed in the liver and red and white muscle, with lower levels of expression in the gills, heart, kidney and spleen of the rainbow trout. The lack of regulatory phosphorylation sites within the G-protein-binding domain of the rainbow trout beta2-adrenoceptor sequence suggests that the in vivo control of trout beta2-adrenoceptor signaling differs substantially from that of mammals.     

The distribution kinetics of waterborne silver-110m in juvenile rainbow trout

Juvenile rainbow trout (Oncorhynchus mykiss) were subjected to a 2-day radioactive pulse of 110mAg at 11.9 microg/l (as AgNO3), followed by a 19-day post-tracer exposure to non-radioactive Ag(I) (3.8 microg/l). The distribution of 110mAg in the gills, liver, intestine, kidney, brain and remaining carcass was investigated over a 19-day post-tracer period. Initially, the intestine contained the highest proportion of the 110mAg burden (34%), however, by day 8, less than 5% of the total radioactivity remained in this tissue. The majority of the 110mAg eliminated from the intestine appeared to distribute to the liver. Eventually, the 110mAg content in the liver accounted for as much as 65% of the total radioactivity in the fish. Apart from the liver and intestine, only the gills and carcass contained any appreciable amount (>5%) of the total body 110mAg content. Liver and gill samples were fractionated using differential centrifugation techniques to discern the subcellular distribution of 110mAg in these tissues. In the liver, the 110mAg levels in the cytosolic fraction increased from 35% to 72% of the total cellular burden between days 8 and 19, respectively. The radioactive pulse in the gills was predominantly found in a membrane compartment termed the nuclear fraction ( approximately 60% of the total). Little change was observed over time (day 8 to day 19) to the subcellular distribution of Ag in the gills. Using size-exclusion chromatography, most ( approximately 70%) of the 110mAg content in the liver cytosol eluted at a molecular weight characteristic of metallothionein. The cytosolic distribution of 110mAg in gills was quite diffuse, occurring primarily in the heavy molecular weight fractions.     

Antioxidative stress proteins and their gene expression in brown trout (Salmo trutta) from three rivers with different heavy metal levels

Three populations of brown trout (Salmo trutta) exposed to different metal levels in their natural environments, were studied with respect to antioxidants metallothionein (MT), superoxide dismutase (SOD) and catalase (CAT) as well as for corresponding mRNA levels. In addition, mRNA levels were studied for glutathione peroxidase (GPx) and glutathione reductase (GR). The Cd/Zn-exposed trout (Naustebekken River) had higher accumulated levels of Cd, Cu and Zn in gills, and higher levels of MT (both protein and mRNA) in liver and kidney as well as in gills compared to the Cu-exposed trout (Rugla River) and trout from an uncontaminated reference river (Stribekken River). Less MT found in the Cu-exposed trout may increase susceptibility to oxidative stress, but no higher levels of antioxidant mRNAs were found in gills of these trouts. The data indicated that chronic exposures of brown trout to Cd, Zn and/or Cu did not involve maintenance of high activities of SOD and CAT enzymes in gills, although SOD mRNA levels were higher in the Cd/Zn-exposed trout. In livers, mRNA levels of SOD, CAT and GPx were higher in the metal-exposed trout, but in the case of GR this was only seen in kidneys of Cd/Zn-exposed trout. However, both metal-exposed groups had higher activities of SOD enzyme in liver compared to the unexposed reference trout, and CAT activity was found to be higher in kidneys of Cu-exposed trout. The Cu-exposed trout did not seem to rely on MT production to avoid Cu toxicity in gills, but rather by keeping the Cu uptake at a low level. A coordinated expression of different stress genes may also be important in chronic metal exposure. It may be concluded that the observed metal effects relies on acclimation rather than on genetic adaptation in the metal exposed populations.