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Changes in metallothionein (MT) levels in liver and kidney of juvenile rainbow trout (Oncorhynchus mykiss) were followed for 8 months, as the water temperature was allowed to follow the natural temperature decrease in Tvaren, a bay in the Baltic Sea. The temperature dropped from 15 °C to 2 °C during the experimental period. The liver and kidney were analysed for MT, zinc, copper and cadmium. Both the hepatic and renal MT levels were found to increase in the control group during the first 4 months of the experiment, as the water temperature dropped to 4 °C. Once the temperature had stabilized at 2 °C there was a noticeable decrease in both hepatic and renal MT levels. One group of fish was exposed to water-borne cadmium (100 μg Cd/litre) for the duration of the experiment. Cadmium accumulated in both liver and kidney, and reached the highest levels in the kidney. Exposure of the fish to cadmium resulted in marked elevations of kidney MT levels. In the liver the MT levels did not increase above the control levels as the temperature dropped to 2 °C after 6 months. However, once the temperature stabilized at 2 °C (after 8 months) the hepatic MT levels were significantly higher in cadmium-exposed fish than in the control fish.

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, metalbinding protein found in all vertebrates. The primary structure of the protein is evolutionary conserved, especially the location of cysteine-residues. There are nonetheless significant differences between MTs from different vertebrate classes, notably in major antigenic epitopes. Previous work has shown that MT from different fish species also differ. The objective of the present work was to characterize MT from temperate, sub-tropical and tropical fish species by their relative reactivities with two anti-fish MT antisera. One or both antisera cross-reacted with MT from all fish species investigated. Surprisingly, the relative reactivities of the two antisera with MT from species belonging to different taxonomic groups appeared to reflect differences at the level of family rather than differences between major phylogenetic lineages.

Induction of metallothionein (MT) in the hepatopancreas of the tropical, commercially-farmed shrimp (Penaeus vannamei,) was studied in the laboratory after exposure to waterborne cadmium. MT was quantified with differential pulse polarography. Two laboratory experiments were performed: a dose-response and a time-course experiment. MT was significantly induced in a dose-dependent way in response to waterborne CdCl2. In the time-course experiment a significant correlation was found both between induced MT and accumulated cadmium in the hepatopancreas, and between depuration of MT and cadmium. Final value of MT after 9 days of exposure to 1.50 mg Cd/litre was 5600 μg MT/g wet weight hepatopancreas, which is an induction of 180%.

Two methods to quantify metallothionein (MT), differential pulse polarography (DPP) and radioimmunoassay (RIA), were compared for MT analysis of livers from ZnCd-injected perch ( Perca fluviatilis ). There was a strong agreement in the results obtained by the two methods. The regression line for the plot MT RIA versus MT DPP is described by the formula: MT RIA = (MT DPP × 0·99)—0·048 (r = 0·92). When the MT value of each 200 mg tissue sample was divided by the value of corresponding 100 mg tissue sample, the quotient was 1·70±0·78 for DPP and 1·86±0·34 for RIA (mean±SD). MT values, from individual liver samples, plotted against the Cd and Zn content of the corresponding samples provided a slightly lower correlation coefficient for MT values measured by DPP ( r = 0·87), than by RIA ( r = 0·92).

Effects of cadmium exposure on plasma levels of calcitonin and free and protein-bound calcium were studied in vitellogenic female rainbow trout kept in brackish water (7%(.)). Fish were exposed to 100 μg cadmium litre−1 for four weeks. Exposure of female rainbow trout in the stage of vitellogenesis, with increased total plasma levels of calcium, resulted in a complex hypocalcemic response. Thus, hypocalcemia was found to be due to three different processes: (1) a decrease in the free plasma calcium, and a reduction in protein-bound calcium; due both to (2) decreased plasma levels of vitellogenin; and (3) a reduced binding of calcium to vitellogenin. These findings support the concept of an interference of cadmium with ionregulating tissues as a mechanism for hypocalcemia in rainbow trout. A direct effect on the vitellogenin-binding of calcium was also observed and reproductive function in the females was affected by decreased plasma levels of vitellogenin. In spite of the marked changes of plasma calcium in exposed fish, no significant effects on plasma calcitonin were observed, indicating a lack of a direct relationship between plasma calcium and calcitonin levels in rainbow trout.

The secondary or physiological stress responses occurring immediately after capture were measured in a wild fish population of perch (Perca fluviatilis) and during a recovery period of eight days. Capture induced blood hyperglycemia and hyperlactemia, a depletion of liver and muscle glycogen, a reduced number of circulating lymphocytes, and an increased number of circulating neutrophilic granulocytes. Further, the activity of the enzyme ALA-D in blood, the plasma levels of magnesium and inorganic phosphate, and the muscle water content were increased after capture, while the liver size appeared to be reduced. No consistent effects were observed in hematocrit values, blood hemoglobin content, red blood cell count, plasma sodium, chloride and total protein content, or in liver water content. Parameters affected by capture were found to recover and stabilize within two to four days after capture. The experimental set-up involving a careful handling of fish captured with gill nets and including a subsequent recovery in fish chests was found to be a useful approach for the assessment of the physiology of wild fish populations.

A sensitive radioimmunoassay (RIA) for metallothionein (MT) from perch (Perca fluviatilis) has been developed. The method described is a double antibody RIA with rabbit anti-perch-MT as first antibody, goat anti-rabbit-immunoglobulin G as second antibody and perch MT conjugated to [125I] Bolton-Hunter reagent as tracer. The antiserum cross-reacts with MT from four fish species tested in addition to perch, but has low avidity for horse kidney MT. At a dilution of 1:2000, the MT antibodies were able to bind 36% of the tracer when no cold ligand was present. The sensitivity of the assay was 15 pg perch MT/tube and the practical working range was found to be 0·15–250 ng perch MT/tube. The RIA allows determination of MT in plasma or lysed blood cells at concentrations as low as 3·0 ng/ml and in tissues at levels over 9·0 ng/g (wet weight). Intra- and interassay coefficients of variation were 6 and 10% respectively.

Hepatic levels of Cu, Zn and metallothionein (MT) in perch, caught in a Cu/Zn gradient from a brassworks, reflected the water concentration of Cu (1·0–9·4 ppb) and Zn (0·56–59 ppb). Significant correlations were found between hepatic Cu and MT levels ( r = 0·72), and between Zn and MT levels ( r = 0·69). There was an increase of the amount of Cu and Zn in the cytosolic fraction of the liver with increased hepatic levels of the metals. When liver samples. from perch caught at the most contaminated location, were run on a gel filtration column (Sephadex G-75) 78% of the cytosolic Cu and 24% of the Zn in the cytosol eluted together with MT.

Induction of metallothionein (MT) in liver of the subtropical fish, bluestriped grunt ( Haemulon sciurus ), has been studied after cadmium injection. Hepatic MT was assayed by a heterologous radio immunoassay (RIA) for bluestriped grunt, using antiserum raised against MT from perch ( Perca fluviatilis ). Two laboratory experiments were performed: a dose-response experiment and a time-course experiment. Hepatic MT was significantly induced in a dose-dependent manner in response to intraperitoneal injection of CdCl 2 . Maximum MT levels were observed in the 2200 μg Cd/kg group, where MT values were three times higher than in the control group. In the time-course experiment, elevated MT levels were found 2 days after injection and the maximum was reached after 6 days. MT values in fish 6 days after treatment were increased five times compared to the control group.

In mammals the metallothioneins are characterized as low molecular weight proteins that bind zinc, cadmium, mercury or copper. These proteins have a high cysteine content, usually around 30~/0, but completely lack aromatic amino acids. Exposure to the metals mentioned results in an induction of the thionein(s). However, the function of the metallothioneins is still a matter of controversy. In teleost fishes the complete isolation and characterization of metallothioneins from several species has recently been accomplished (Overnell & Coombs, 1979: Ridlington et al., 1981~ Kito e t a l . , 1982; Takeda & Shimuzu, 1982; Ley et al., 1983). In the present study, partial isolation and purification of cadmium-binding proteins from rainbow trout liver was achieved utilizing biochemical separation methods in combination with radioactive cadmium as a tracer. Juvenile rainbow trout, Salmo gairdneri, weighing around 100 g, were injected i.p. to yield a total dose of 3 mg Cd/kg body weight. Fish used in the tracer experiments were given an additional i.p. injection of 37 kBq 1°9CDC12. Sampling was performed 7 days after the first injection, and the

During recent years laboratory studies have demonstrated the presence of metallothionein in several species of teleosts.1–3 The role of metallothionein (MT) in protection against cadmium toxicity has been investigated by exposing fish to high levels of cadmium in the water.4–6 So far, however, few field studies have focused on the existence of MT and its role in subcellular distribution of cadmium.7 In the present study the synthesis of MT as a possible mechanism by which perch adapt to cadmium contamination was investigated in a wild population of perch. An increased MT content of perch liver was found to correlate to an increase in cadmium present in the liver.

Rainbow trout were exposed to 200 ppb cadmium in the water during four months at 5°C. The liver, kidney and gills were analyzed for cadmium, copper, zinc and metallothionein. Cadmium accumulated in all three organs and reached the highest concentration in the kidney. The zinc and copper concentrations were not altered during the experiment. The metallothionein concentration was significantly higher in liver of exposed fish than in control fish after three months. The kidneys reached significantly elevated levels of metallothionein in the exposed group after four months. These results demonstrate that metallothionein is induced by cadmium after exposure to the metal via water.