Naturally-occurring heavy metal binding proteins in invertebrates

Low molecular weight cadmium, copper, and zinc binding protein has been isolated from the musselMytilus edulis in the cadmium polluted areas of Corio Bay adjacent to Melbourne. Its molecular weight and other properties suggest that it belongs to the metallothionein family of metal binding proteins. Its synthesis may have been induced in the cyctoplasmic solutions of the cells as a regulatory mechanism to avoid intoxication by excess uptake of any of the metals.     

Copper content in cadmium-exposed animal kidney metallothioneins

Cadmium chloride was injected intraperitoneally into five animals (mouse, hamster, rat, guinea-pig, and rabbit) with the same amount of cadmium per body weight. The concentrations of three metals (Cd, Zn, and Cu) in the 105,000 g supernatant and in the heat-treated supernatant of livers and kidneys of the cadmium-exposed animals were determined by atomic absorption analyses. The supernatants of both organs were applied to a Sephadex G-75 column and the distribution profiles of the three metals were analyzed to show the metal contents in the metallothionein fraction. The results indicated that the amount of retained cadmium and the increased amount of zinc in both organs were different from animal to animal and that the metals were bound to metallothionein. The most significant result was that the five animals were divided into two groups according to changes of increased copper in the renal metallothionein of cadmium-exposed animals. One group had a dramatic increase in copper content while the other group had little or no increase in copper content for the renal metallothionein after exposure to cadmium. Rat and guinea-pig belonged to the former group and mouse, hamster, and rabbit belonged to the latter group.     

Zinc and cadmium body burdens in terrestrial oligochaetes: use and significance in environmental risk assessment

Uptake and elimination kinetics of zinc and cadmium were studied in the oligochaetes Enchytraeus albidus and Eisenia fetida. Even at the highest exposure concentrations where E. fetida survived, the internal zinc concentration was still regulated to a constant level. Enchytraeus albidus could not regulate the internal zinc concentration as well, and the body burden increased after exposure to high zinc concentrations. After transfer to clean soil, the internal zinc concentration dropped back to the control level within a few weeks. For both species, internal cadmium concentrations did not reach equilibrium during the uptake experiments. The internal concentrations causing 50% reduction in cocoon production for E. fetida exposed to cadmium varied between different soil types, indicating that no fixed critical body burdens exist. For both zinc and cadmium, bioaccumulation factors decreased with increasing soil metal concentrations. Bioaccumulation factors may therefore be poor indicators of environmental risk. Their dependence on the total soil concentration makes bioaccumulation factors also unsuitable for assessing the influence of soil characteristics on the bioavailability of metals in contaminated field soils. For the same reason, uptake rate constants are probably not suited for this purpose.     

Toxicity of metal mixtures to a tropical freshwater alga (Chlorella sp): the effect of interactions between copper,cadmium, and zinc on metal cell binding and uptake

The individual and combined effects of copper, cadmium, and zinc on the cell division rate of the tropical freshwater alga Chlorella sp. were determined over 48 to 72 h. Metal mixtures were prepared based on multiples of their single-metal median effective concentration (EC50) values, i.e., toxic units (TU) using a triangular mixture design with five toxicant levels (0, 0.75, 1.0, 1.25, and 1.5 TU). Single-metal EC50 values after a 72-h exposure were 0.11, 0.85, and 1.4 microM for copper, cadmium, and zinc, respectively. Significant interactions were observed for all metal combinations after 48 and 72 h. An equitoxic mixture of Cu + Cd was more than concentration additive (synergistic) to the growth of Chlorella sp., while combinations of Cu + Zn, Cd + Zn, and Cu + Cd + Zn were all less than concentration additive or were antagonistic. To determine the effect of each metal on the uptake of the other, extracellular (membrane-bound) and intracellular metal concentrations, both alone and in mixtures, were compared. The increased growth inhibition observed for mixtures of Cu + Cd was due to higher concentrations of cell-bound and intracellular copper in the presence of cadmium compared with copper alone (i.e., cadmium-enhanced copper uptake). In contrast, both extra- and intracellular cadmium concentrations were reduced in the presence of copper. In mixtures of Cu + Zn, copper also inhibited the binding and cellular uptake of zinc, which resulted in decreased toxicity. Zinc had no appreciable effect on the uptake of copper by Chlorella sp. Our results suggest that all three metals share some common uptake and transport sites on Chlorella cells and that copper out competes both cadmium and zinc for cell binding. Determination of metal cell distribution coefficients (K(d)) confirmed that K(d) values for cadmium and zinc in single-metal exposures decreased in the presence of copper.     

Cadmium accumulation in herbivorous and carnivorous small mammals: meta-analysis of field data and validation of the bioaccumulation model Optimal Modeling for Ecotoxicological Applications

Environmental risk assessment procedures often use bioaccumulation as a criterion for hazard identification of a polluted location. Field studies regarding metal concentrations in food chains, however, have provided widely different information, because accumulation is shown to vary between the extremes of bioreduction and biomagnification. Bioaccumulation models provide insight regarding species-specific uptake and elimination kinetics of metals and assist in the interpretation of field data. Here, we use the bioaccumulation model OMEGA (Optimal Modeling for Ecotoxicological Applications) to estimate cadmium accumulation in herbivorous voles and carnivorous shrews. In addition to model validation, a meta-analysis of cadmium accumulation data is performed, because earlier studies generally have focused on relationships between cadmium concentrations in either specific tissues (kidney and liver) or whole-body concentrations and total soil levels. Additionally, we included the food-small mammal relationship. Our results show that cadmium whole-body concentrations are significantly related to cadmium levels in food items such as earthworms and plants. In addition, a significant relationship is found between cadmium accumulation in the liver and kidney of small mammals and total soil levels. Cadmium concentrations in shrews typically are an order of magnitude higher than metal levels in voles as a result of higher metal accumulation in earthworms compared to plants. Model predictions for both voles and shrews are in good agreement with field observations; deviations generally are within a factor of five. Small mammals prevent cadmium toxicity by binding this metal to metallothionein, which likely results in low elimination rates. Comparison with empirical elimination rates shows that rate constants of loss are accurately predicted assuming that cadmium is only released via growth dilution.     

Bioconcentration and depuration of copper, cadmium, and zinc mixtures by the freshwater amphipod Hyalella azteca

The uptake and elimination of copper (Cu), cadmium (Cd), and zinc (Zn) by the amphipod Hyalella azteca during exposure to the metals singly and in various combinations was examined in controlled laboratory experiments. In single metal exposures the accumulation of all metals was rapid and increased with exposure time. Copper elimination was slower compared to that for zinc and for cadmium no elimination was detected after 5 days in clean water. In the two-metal mixtures it appears that the presence of one metal influenced the bioconcentration of the other, since the bioconcentration factor (BCF) for copper was higher in the presence of cadmium than in the presence of zinc and in the case of cadmium, the decrease of K(1) values from cadmium single exposure to the binary and tertiary mixtures suggests possible inhibition of cadmium uptake by the other metals. In the case of the three-metal mixture the situation is less clear, with both increased and decreased BCFs recorded, in comparison to single-metal and two-metal mixtures, suggesting both stimulation and inhibition of metal accumulation.     

Relationships between heavy metal and metallothionein concentrations in lesser black-backed gulls, Larus fuscus, and Cory's shearwater, Calonectris diomedea

Metallothionein, cadmium, zinc, copper, and mercury concentrations were measured in adult lesser black-backed gulls, Larus fuscus; and metallothionein, cadmium, zinc, and copper concentrations were measured in fledgling Cory's shearwaters, Calonectris diomedea. In gulls, metallothionein was positively correlated with cadmium (kidney r=0.83, liver r=0.46), zinc (kidney r=0.46, liver r=0.37), and copper (kidney r=0.28, liver r=0.34). Mercury levels in lesser black-backed gulls showed no correlations with metallothionein or with any other metal. In shearwaters metallothionein was positively correlated with cadmium in the kidney (r=0.41) but not in liver, zinc in kidney (r=0.43) and liver (r=0.52), and copper in kidney (r=0.55) but not in liver. Cadmium levels were the most important factor determining tissue metallothionein concentrations in adult lesser black-backed gulls demonstrating the role of metallothionein in heavy metal detoxification. In fledgling Cory's shearwaters, the most important factor in determining metallothionein concentrations in kidney was copper concentrations, and in liver, zinc concentrations. During the latter phases of chick growth high levels of zinc are required for feather development, and at this time the binding of cadmium may be masked by the presence of a large amount of zinc- and copper-bound metallothionein. These results illustrate disparate roles of metallothionein, the levels of which will be in a state of flux both seasonally and annually.     

The influence of zinc on the uptake and loss of cadmium and lead in the woodlouse, Porcellio scaber (Isopoda, Oniscidea)

Uptake of cadmium, lead, and zinc was studied in juvenile Porcellio scaber in feeding experiments over 5 months. The metals were offered separately and in different combinations and concentrations in the food. The ability of P. scaber to eliminate the accumulated metals was studied subsequently for 3 months on uncontaminated food. Characteristic patterns of accumulation are described for the three metals. The combination of lead and zinc resulted in only minor differences in these patterns. On the other hand, the combination of zinc and cadmium at high concentrations completely changed the accumulation patterns for both metals. Not only cadmium but also zinc was excreted by P. scaber exclusively when the animals had been contaminated with both metals. In contrast both metals were stored permanently when offered separately. Possible reasons for the interactions of cadmium and zinc are discussed.     

Native and induced rat kidney metallothioneins and their relation to cadmium toxicity

The concentrations of zinc, copper, iron, manganese, magnesium, and phosphorus in the kidneys were determined for control female rats of five different ages (4, 8, 12, 16, and 20 weeks old). The concentration of copper increased with age and it was attributed to three metallothionein peaks on a SW 3000 column. Parenteral administration of Cd-thionein caused swelling of kidneys to different degrees, depending on the age of the rats; the youngest rats were affected most. The concentrations of cadmium and the six metals in the kidneys at 12 hr and 7 days after injection were determined and compared with controls. The distribution among protein fractions of cadmium, zinc, and copper, which are related to metallothionein, were examined by gel permeation chromatography. The elution profiles of the three metals changed not only with the ages of the rats but also with time after injection. The changes were correlated with the copper content in the metallothioneins and with swelling of the kidneys. The changes also correlated with native metallothionein levels.     

Cadmium, zinc, and copper in rabbit kidney metallothionein--relation to kidney toxicity

Twenty-two rabbits were given repeated subcutaneous injection of cadmium chloride. The cumulative cadmium dose given ranged from 13 to 214 mumole/kg body weight. Five rabbits served as controls. The treatment resulted in cadmium concentrations in kidney cortex that ranged from 0.3 to 3.2 mmole Cd/kg and a subsequent production of metallothionein. The molar ratio of cadmium, zinc, and copper in metallothionein fractions from kidneys with different concentrations of cadmium was determined. At low concentrations of cadmium in rabbit kidneys, zinc was the dominating metal bound to metallothionein (70-90%). At high concentrations of cadmium in kidneys, cadmium was the dominating metal in metallothionein. Evidence of kidney toxicity, in the form of beta2-microglobulinuria, was seen when cadmium constituted 85% of the metal ions recovered from metallothionein fractions. The remaining 15% was zinc. This indicates that at most six of the seven metal-binding sites in mammalian metallothionein are occupied by cadmium and that the remaining site is occupied by zinc. Our data provide further support for the hypothesis that chronic cadmium nephrotoxicity develops when there is a lack of metal-binding sites available for cadmium in metallothionein.     

The effects of In Vivo exposure to cadmium, copper and zinc on the activities of gill ATPases in the Norway lobster, Nephrops norvegicus

Norway lobsters, Nephrops norvegicus were exposed for 18 days to combinations of cadmium, copper and zinc at three sublethal concentrations (1, 5, 25 g/L for Cd and Cu, and 8, 40 and 200 g/L for Zn). Male animals were exposed to all three concentrations, while female animals were studied only in one (highest) concentration of the metals. Activities of Na,K-ATPase and both oligomycin-sensitive and insensitive Mg-ATPase were investigated in relation to metal exposure. A group of male Nephrops of different size groups from a single location in the Clyde Sea was also sampled to investigate the relationship between size and ATPase activity.Cadmium concentrations increased significantly with increases in exposure concentrations in both male and female animals, whereas copper and zinc increased significantly only in the highest treatment in males. There was no significant difference in concentrations of the metals in control male and female animals, whereas males accumulated significantly higher levels of copper and zinc in the highest concentrations of the metals. Control males showed higher activity of Na,K-ATPase than control females. Na,K-ATPase activity was significantly inhibited in male animals exposed to metals whereas in female animals there was only a significant inhibition in total Mg-ATPase activity. Na,K-ATPase activity had a negative relationship with copper in males, while in treated females this ATPase had positive relationship with zinc and copper. There were negative relationships between the activities of Na,K-ATPase and oligomycin sensitive Mg-ATPase with carapace length of the animals.     

Effect of short-term ethanol administration on cadmium retention and bioelement metabolism in rats continuously exposed to cadmium

The present study was performed to assess the effect of short-term ethanol administration on cadmium retention and accumulation as well as on bioelement metabolism (zinc, copper, calcium, and magnesium) in rats exposed to an aqueous solution of cadmium chloride for 8 weeks. Intoxication with cadmium led to accumulation of this toxic metal, particularly in the liver and kidney, which was linked to metallothionein synthesis as well as to a disturbance in the metabolism of zinc, copper, and calcium. These effects were dependent on the level of exposure. The administration of ethanol in the final phase of cadmium treatment increased cadmium retention and accumulation in the body with simultaneous elevation in liver and kidney metallothionein concentration. Ethanol alone or with cadmium caused or intensified the cadmium-induced changes in metabolism of zinc and copper. Calcium metabolism disturbed by cadmium was not influenced by ethanol. Neither agents had any effect on magnesium metabolism. We conclude that even short-term ethanol consumption in conditions of exposure to cadmium can increase this heavy metal body burden and lead to more serious disturbances in metabolism of important elements such as zinc and copper. Cadmium- and ethanol-induced changes in the homeostasis of these microelements are probably connected with the ability of both xenobiotics to cause metallothionein induction.     

Interactions Between Toxic and Essential Trace Metals in Cattle from a Region with Low Levels of Pollution

Studies on the impacts of pollutant metals and metalloids on livestock have largely focused on animals with relatively high levels of exposure. The impact of low-level environmental contamination, which is more common on agricultural land, is largely unknown. The principal aim of the present study was to examine the effects of low-level environmental contamination on trace metal metabolism in cattle from the rural and relatively uncontaminated region of Galicia (NW Spain). Correlations between toxic (cadmium, lead, and arsenic) and essential trace elements (copper and zinc) were evaluated in the tissues (liver, kidney, and muscle) and blood of 494 cattle from throughout Galicia. Cadmium was the toxic element that had the greatest influence on copper and zinc homeostasis. There was a significant positive association between renal cadmium and zinc residues and a significant negative correlation between kidney cadmium and copper. These interactions are likely to be the result of cadmium-induced effects on metallothionein synthesis. Lead and zinc were positively associated in the kidney, although the mechanism of this interaction is uncertain. Arsenic and copper concentrations were strongly correlated with each other in the liver and may indicate that the high copper levels in animals from copper-rich areas in Galicia interfere with their arsenic excretion. The essential metals copper and zinc were also significantly associated with each other in calves but not in cows. Received: 24 April 2001/Accepted: 10 September 2001     

Detoxication of zinc and cadmium by the freshwater protozoan Tetrahymena pyriformis: II. Growth experiments and ultrastructural studies on sequestration of heavy metals

Sublethal concentrations of zinc and cadmium limited the growth of batch cultures of the ciliate Tetrahymena pyriformis. Electron microscopy of cultured populations revealed many electron-dense cytoplasmic granules in cells exposed to zinc (60 ppm), but fewer in cells grown with less zinc (6 ppm) or cadmium (2 ppm). Ultrastructural abnormalities were observed only in cells grown with cadmium. These contained autophagic vacuoles and had damaged nuclei. Zinc was shown to exert a protective effect against cadmium toxicity both in terms of growth and ultrastructural appearance. Electron probe X-ray microanalysis revealed both zinc and cadmium within the cytoplasmic granules of cells exposed to these metals. Since the granules are normally composed predominantly of calcium and magnesium, these ions are implicated in the intracellular detoxication of zinc and cadmium. Zinc was detected only in the granules, while cadmium was also found in autophagic vacuoles and damaged nuclei.     

Adaptation to soil pollution by cadmium excretion in natural populations of Orchesella cincta (L.) (Collembola)

Population differentiation in Orchesella cincta (L.) (Collembola) populations, from various heavy metal contaminated sites, was studied by comparing cadmium excretion efficiency in first generation (F₁) laboratory individuals. Animals from sites with high metal concentrations in the litter and with a long history of contamination showed significantly higher excretion efficiencies than animals from low pollution, or reference sites. Differences found in the F₁ laboratory animals suggest evidence for genetic differences between the populations.Beneficial and detrimental effects of cadmium excretion were studied in relation to body growth and cadmium concentrations. In chronically exposed animals from an unpolluted site, no physiological acclimation was observed. Excretion efficiency was negatively correlated with body concentrations of cadmium. No detrimental effects were found.Whole-body equilibrium concentrations of cadmium and lead were similar in F₁ animals from the reference site and polluted sites. Significant differences in excretion efficiencies imply that the distribution of toxic metals over body compartments differs, tolerant populations having a higher proportion deposited in the gut. Body concentrations of zinc were consistently higher in animals from the polluted site, during both cadmium and zinc exposure. No detrimental effects of increased cadmium excretion on body concentrations of zinc were observed. Population comparisons of cadmium excretion efficiency data and growth reduction in F₁ laboratory animals showed that both parameters were inversely related. Cadmium and lead contamination were not the sole factors determining tolerance differentiation.     

The relationship of the increased level of metallothionein with heavy metal levels in the tissue of the harbor seal (Phoca vitulina)

The relationship between age, heavy metal levels, and a heavy-metal binding protein, metallothionein (MT), in the liver and kidney of the harbor seal (Phoca vitulina) was studied. The cadmium (Cd) level in the liver and the Cd, inorganic mercury (I-Hg), and zinc (Zn) levels in the kidney were increased with body length, suggesting an age-related accumulation of these metals. The MT levels determined by an MT radioimmunoassay showed concentrations of 240 +/- 139 micrograms/g in the liver and 343 +/- 219 micrograms/g in the kidney. These MT levels were shown to be correlated with age. The statistically significant relationship of the MT levels with the levels of Cd and Zn in the liver and those of Cd, Zn, and I-Hg in the kidney suggested that the protein is responsible for the sequestration of these metals as already observed in terrestrial animals.     

Metal Kinetics and Respiration Rates in F1 Generation of Carabid Beetles (Pterostichus oblongopunctatus F.) Originating From Metal-Contaminated and Reference Areas

We investigated resistance to metals in carabid beetles inhabiting metal-polluted and reference areas. Chronic multigeneration exposure to toxic metal concentrations may potentially result in adaptation through decreased metal uptake rate and/or increased excretion rate. The cost of resistance to pollution could be associated with increased metabolic rate. To test these predictions, laboratory cultured F₁-generation beetles originating from metal-polluted and reference sites were exposed to food contaminated with zinc and/or cadmium for 10 weeks. After that, uncontaminated food was offered to the animals for another 3 weeks. During the experiment, internal concentrations of Cd and Zn were measured as were respiration rates of the animals. The results obtained show no significant differences in metal accumulation and excretion patterns or respiration rates between the populations. This may suggest that adaptation has not occurred in the beetles chronically exposed to toxic metal concentrations. The possible explanations for the lack of differences between the populations are discussed.     

Cadmium,zinc, copper and metallothionein levels in human liver

The concentrations of cadmium, zinc, copper and metallothionein in the autopsy samples of liver among the inhabitants of Lód? (Poland) were determined. The cadmium levels were low in the range of 1.5 to 5.8 micrograms/g. The concentration of metallothionein determined by the Hg-method was high (0.160-1.665 mumol Hg/g); it was mainly a Zn-thionein. The percentage of hepatic zinc bound in the MT-fraction increased with the overall content of zinc in the liver. The elevation of zinc in the liver occurs in the proportion required for the saturation of metal-binding ligands of metallothionein. The role of cadmium remains less clear. Our results suggest that the metallothionein level in the liver increase significantly in response to elevated cadmium concentrations. This response, however, is in high excess to the demand which is justified stoichiometrically.     

Comparative toxicity of cadmium, zinc, and mixtures of cadmium and zinc to daphnids

Investigations were conducted to determine acute (48-h) effects of cadmium and zinc presented individually and in combination on Ceriodaphnia dubia, Daphnia magna, Daphnia ambigua, and Daphnia pulex. Toxicity tests were conducted with single metals to determine lethal effects concentrations (lethal concentrations predicted for a given percent [x] of a population, LCx value). These were used to derive metal combinations that spanned a range of effects and included mixtures of LC15, LC50, and LC85 values calculated for each metal and species. In single-metal tests, 48-h LC50 values ranged from 0.09 to 0.9 micromol/L and 4 to 12.54 micromol/L for cadmium and zinc, respectively. For each metal, D. magna was most tolerant and showed a different pattern of response from all others as determined by slope of concentration-response curves. In the combined metal treatments, all daphnids showed a similar pattern of response when LC15 concentrations were combined. This trend continued with few exceptions when LC15 concentrations of cadmium were combined with LC50 or LC85 values for zinc. However, when this treatment was reversed (LC15, zinc + LC50 or LC85, cadmium), responses of all species except D. magna indicated less-than-additive effects. For C. dubia, a near complete reduction in toxicity was observed when the LC15 for zinc was combined with LC85 for cadmium. Multimetal tests with D. magna did not differ from additive. Collectively, these studies suggest that D. magna may not be representative of other cladocerans.     

Binding of endogenous copper and zinc to cadmium-induced metal-binding proteins in various tissues of Perna viridis

Green lipped mussels, Perna viridis, were exposed to cadmium chloride (CdCl2; 0.52 and 1 g/ml) in water for 4 days. The concentrations of cadmium (Cd), copper (Cu) and zinc (Zn) were measured in the viscera, gill, gonads, mantle, and muscle. There was a significant increase (p<0.05) in Cd concentration in all tissues studied. Results from Sephadex G-75 chromatography indicated that most Cd was bound to a fraction of heat-stable proteins similar to the metal-binding protein (MBP) metallothionein. After exposure to Cd, there was no significant change in Cu concentration in total tissue proteins or in total cytosolic proteins. A significant increase (p<0.05) in Cu, however, was detected in heat-stable proteins bound to the Cd-induced MBP in both viscera and gill. Copper bound to MBP also occurred in the gonad, mantle, and muscle, but to a much lesser extent. These results showed that Cd-induced MBP can also bind endogenous Cu. Zinc concentration in total heat-stable protein was increased only in gill and the muscle. Unlike Cu, a small amount of Zn binding to Cd-induced MBP was detected only in these tissues. Considering that both Cu and Zn exist intracellularly in dynamic equilibrium, the binding of Cu, but not Zn, to MBP may be explained by the kinetic reactivity of the two different metals to protein. The results of this study support the thesis that induction of intracellular MBP may also bind endogenous Cu and Zn.