Induction and metal speciation of metallothionein in wood mice (Apodemus sylvaticus) along a metal pollution gradient

We investigated the binding of Cd, Cu, and Zn to metallothionein (MT) and other metal-binding proteins in free-living wood mice (Apodemus sylvaticus L.) captured in four areas along a metal pollution gradient. We measured total and cytosolic Cd, Cu, and Zn concentrations in mouse liver and kidney by means of inductively coupled plasma-mass spectrometry (ICP-MS). Total (Cu, Cd, Zn)-MT levels were determined in the same tissues by means of the cadmium thiomolybdate saturation assay. Metal speciation of metalloproteins was studied by means of size-exclusion high-performance liquid chromatography-ICP-MS. Liver and kidney of wood mice from the site adjacent to the pollution source showed the highest Cd and Zn concentrations (total and cytosolic) and (Cu, Cd, Zn)-MT levels compared to the other sites farther away from the pollution source. No or only small site differences in tissue Cu concentrations were observed. Almost all the variation (85-95%) in hepatic and renal (Cu, Cd, Zn)-MT levels was explained by the total or cytosolic hepatic Zn and Cd concentration or the renal Cd concentration, respectively. An analysis of the cytosolic metal speciation showed that the Cd-MT, Cu-MT, and Zn-MT fractions in liver and kidney increased significantly with increasing cytosolic metal concentrations. Metals associated with the other cytosolic protein fractions did not increase with increasing exposure. These results illustrate the important role of MT in metal homeostasis and detoxification processes. We conclude that MT is a useful biomarker for environmental metal contamination in free-living wood mice.     

Variations in metallothionein, Zn, Cu, and Fe concentrations and ceruloplasmin activity in pregnant rat dams and their fetuses

Growing rat fetuses have great need for Zn, Cu, and Fe. In fetal livers (FL) large accumulations of Zn and Cu connected with increased metallothionein (MT) synthesis take place. In dams, serum changes in metals concentrations with increased ceruloplasmin (Cp) activity are observed. The aim of this study was to determine (1) mutual relationships in the accumulation of MT, Zn, Cu, and Fe in fetal livers; (2) changes in Zn, Cu, and Fe concentrations in dam serum; and (3) the day with the maximum Cp activity. Sections of rat dams were taken on 16th-21st day of gestation, twice a day, and MT, Zn, Cu, and Fe concentrations in liver, spleen, kidneys, and placenta of dams and in liver and brain in fetuses were determined. In fetal livers high correlations between MT and Zn and between Zn and Cu were obtained. The investigated Cp activity was always high, reaching its maximum on the 20th day and minimum on the 21st day. Significant correlation between Cp activity and Cu concentration in dam serum was also revealed. In conclusion it is suggested that Zn accumulation in FL is strictly connected with MT synthesis but Cu content in FL is rather dependent on Cp activity in dam serum. Iron accumulation in fetal livers is connected with the diminution of iron concentrations in dam serum.     

Metal distribution and metallothionein induction after cadmium exposure in the terrestrial snail Helix aspersa (Gastropoda, Pulmonata)

The aim of the present work was to study the effect of Cd2+ exposure on metallothionein (MT) induction and on the distribution of metals (Cd, Cu, and Zn) in the terrestrial pulmonate Helix aspersa. In particular, the soluble and nonsoluble pools of the accumulated metals and their tissue distribution in uncontaminated and contaminated edible snails were investigated after a two-week exposure to Cd2+. In the soluble cytosolic pool of the midgut gland of H. aspersa, three metal-specific putative MT isoforms were separated following a fractionation protocol with diethylaminoethyl cellulose, size-exclusion chromatography, ultrafiltration, and reversed-phase high-performance liquid chromatography (RP-HPLC). Interestingly, one of the above isoforms seems to bind both Cd and Cu, which may in addition mobilize, after induction by Cd2+, some of the intracellular Cu and, thus, perhaps increase the Cu pool in the cytosolic fraction. The cDNA and its translated amino acid sequence of a Cd2+-binding MT isoform from the snail midgut gland was characterized and attributed to one of the putative MT isoforms obtained by RP-HPLC. The amino acid sequence of this Cd-MT isoform of H. aspersa differed from similar sequences described in other terrestrial pulmonates, such as Helix pomatia or Arianta arbustorum, by only a few amino acids (n = 4 and 8, respectively). That the identified Cd-MT from H. aspersa is inducible by Cd2+ also was shown, chromatographic evidence aside, by a specific polymerase chain reaction protocol on a cDNA basis, which included a noninducible housekeeping gene as a control.     

Combined effects of methylmercury,lead, and cadmium on hepatic metallothionein and metal concentrations in the pekin duck

This study was initiated to determine the effects of combined treatments of methylmercuric chloride (MeHg), lead acetate (Pb) and cadmium chloride (Cd) administered orally on metallothionein (MT) and metal levels in the liver of the pekin duck (Anas platyrynchos). Methylmercury and Pb induced a two-fold increase in MT while Cd caused a seven-fold increase over controls. Concurrent administration of MeHg and Pb caused no increase in MT over single treatments. The hepatic MT content of the MeHg + Cd and MeHg + Pb + Cd groups was not different from the Cdalone group. Hepatic lead levels were lowest followed by Hg and Cd. Lead increased Hg levels when given simultaneously with MeHg, and further addition of Cd increased this effect. In groups given Pb, MeHg increased hepatic levels of this element, but when MeHg was also given with Cd, the combination produced a reduction in levels of Pb. Lead or MeHg had no effect on Cd levels. Methylmercury, Pb, and their combination elevated hepatic Cu over controls. All treatments significantly raised hepatic Zn levels above controls. Combinations of Cd with either MeHg or Pb lowered Zn levels below that caused by Cd alone. The simultaneous administration of all three metals did not produce Zn levels which were additive. The liver content of iron was not significantly altered.     

Variation of metal and metallothionein concentrations in a natural population of Ruditapes decussatus

The spatial and seasonal variation of total and subcellular distribution of Cd, Cu, and Zn was followed in different tissues (gills, digestive gland, and remaining tissues) of the clam Ruditapes decussatus collected along a metal contamination gradient in the Ria Formosa lagoon (southern Portugal) and compared with metallothionein (MT) concentrations.Total metal concentrations decreased according to the sequence digestive gland > gills > remaining tissues for Cd, digestive gland approximately gills > remaining tissues for Cu and gills > digestive gland > remaining tissues for Zn. MT concentrations in these tissues decreased according to the same sequence observed for Cd. In all the tissues, the highest subcellular concentration was in the cytosol for Cd and Cu and in the pellet for Zn. Among the three metals, Cd concentrations showed the most evident spatial variation. In all tissues, total and subcellular Cd concentrations decreased from the inner parts of the lagoon toward the ocean. However, no significant spatial or seasonal variation occurred in clam tissues for the other two metals, though marginal elevated Cu concentrations were observed in the inner parts of the lagoon. Therefore, Cu subcellular distribution in clam tissues was not significantly altered by Cu changes in the lagoon and are the baseline levels for normal metabolism of this clam population. The fact that total Zn concentrations remained unchanged both spatial and seasonal suggested that these clams regulate Zn in their tissues.In the three tissues, MT bind most significantly to Cd and Cu, while Zn, although binding to MT, is preferably bound to other ligands. MT concentrations showed the same spatial and seasonal variation of Cd and were significantly related with total and heat-treated cytosolic Cd in all tissues. For Cu a significant relationship between MT and total or cytosolic Cu was only observed in the remaining tissues. No relationship was observed between MT and total or cytosolic Zn concentrations. Metals and MT concentrations increased with the increase in the condition index for the gills and the digestive gland and decreased from the remaining tissues.Cd concentrations in the gills increased only in the heat-treated cytosolic fraction while Zn in this fraction decreased. Thus Cd concentrations in this tissue displaced Zn from the MT-fraction, leading to a modification of the soluble/insoluble Zn ratio once total Zn concentrations remained unchanged. This modification reflects a perturbation in the normal metabolism in this tissue due to the excess of Cd present. With the exception of the gills, Zn subcellular distribution in the other two tissues was similar among sites and season.The model that describes the relationship between MT, metals, and weight in the gills, digestive gland and remaining tissues also indicates that Cd was the only metal that influence MT synthesis significantly in all the tissues. The induced and/or existent MT was sufficient to bind free Cd ions present in the cells, preventing any damage to cellular metabolism in this clam population. Therefore, MT in the gills and digestive gland of R. decussatus can be used as an early warning signal for Cd exposure and are a useful biomarker to assess the toxicological status of this population in the Ria Formosa lagoon.     

Variation of Metal and Metallothionein Concentrations in a Natural Population of <Emphasis Type="Italic">Ruditapes decussatus</Emphasis>

The spatial and seasonal variation of total and subcellular distribution of Cd, Cu, and Zn was followed in different tissues (gills, digestive gland, and remaining tissues) of the clam Ruditapes decussatus collected along a metal contamination gradient in the Ria Formosa lagoon (southern Portugal) and compared with metallothionein (MT) concentrations. Total metal concentrations decreased according to the sequence digestive gland > gills > remaining tissues for Cd, digestive gland ≈ gills > remaining tissues for Cu and gills > digestive gland > remaining tissues for Zn. MT concentrations in these tissues decreased according to the same sequence observed for Cd. In all the tissues, the highest subcellular concentration was in the cytosol for Cd and Cu and in the pellet for Zn. Among the three metals, Cd concentrations showed the most evident spatial variation. In all tissues, total and subcellular Cd concentrations decreased from the inner parts of the lagoon toward the ocean. However, no significant spatial or seasonal variation occurred in clam tissues for the other two metals, though marginal elevated Cu concentrations were observed in the inner parts of the lagoon. Therefore, Cu subcellular distribution in clam tissues was not significantly altered by Cu changes in the lagoon and are the baseline levels for normal metabolism of this clam population. The fact that total Zn concentrations remained unchanged both spatial and seasonal suggested that these clams regulate Zn in their tissues. In the three tissues, MT bind most significantly to Cd and Cu, while Zn, although binding to MT, is preferably bound to other ligands. MT concentrations showed the same spatial and seasonal variation of Cd and were significantly related with total and heat-treated cytosolic Cd in all tissues. For Cu a significant relationship between MT and total or cytosolic Cu was only observed in the remaining tissues. No relationship was observed between MT and total or cytosolic Zn concentrations. Metals and MT concentrations increased with the increase in the condition index for the gills and the digestive gland and decreased from the remaining tissues. Cd concentrations in the gills increased only in the heat-treated cytosolic fraction while Zn in this fraction decreased. Thus Cd concentrations in this tissue displaced Zn from the MT-fraction, leading to a modification of the soluble/insoluble Zn ratio once total Zn concentrations remained unchanged. This modification reflects a perturbation in the normal metabolism in this tissue due to the excess of Cd present. With the exception of the gills, Zn subcellular distribution in the other two tissues was similar among sites and season. The model that describes the relationship between MT, metals, and weight in the gills, digestive gland and remaining tissues also indicates that Cd was the only metal that influence MT synthesis significantly in all the tissues. The induced and/or existent MT was sufficient to bind free Cd ions present in the cells, preventing any damage to cellular metabolism in this clam population. Therefore, MT in the gills and digestive gland of R. decussatus can be used as an early warning signal for Cd exposure and are a useful biomarker to assess the toxicological status of this population in the Ria Formosa lagoon. Received: 31 January 2002/Accepted: 29 July 2002     

The effects of zinc deficiency on turnover of cadmium-metallothionein in rat liver

The object of this experiment was to determine the effects of Zn deficiency on the turnover of Cd-induced metallothionein (MT) in rat liver. Male rats were fed a purified Zn-deficient or Zn-adequate diet. After 13 days, the rats were given three daily injections of Cd2+ totaling 1.5 or 3.0 (Zn-deficient) and 3.0 or 6.0 (Zn-adequate) mg Cd/kg body weight. The MT was labeled by injecting the rats with [35S]cystine 2 hours after the final Cd injection. One, 3 or 5 days after labeling, the rats were killed, and their livers were assayed for MT 35S and metal content. The metal composition of MT (mole %) was 41-42% Cd, 51-54% Zn and 4-7% Cu in the Zn-adequate groups and 64% Cd, 27-31% Zn and 6-9% Cu in the Zn-deficient groups. The half-lives of Cd-induced MT in the Zn-deficient rats were 2.6 days (1.5 mg Cd/kg) and 2.8 days (3.0 mg Cd/kg). In the Zn-adequate rats, the half-lives were 3.6 days (3.0 mg Cd/kg) and 3.1 days (6.0 mg Cd/kg). The half-lives of general, soluble hepatic proteins were 4.1 to 4.3 days in all groups. Despite the stabilizing effect of the higher Cd content, the half-life of hepatic MT in the Zn-deficient rats was significantly shorter than in the Zn-adequate rats. These results indicate that hepatic MT degradation is faster in Zn-deficient animals.     

The effect of low doses of dietary cadmium oxide on the disposition of trace elements (zinc,copper, iron), hematological parameters,and liver function in rats

The Zn, Cu, and Fe concentrations in selected organs, tissues and in the excreta of male Wistar rats, exposed to dietary CdO (2.80 and 7.15 ppm) for 40 and 60 days, were investigated. Concomitantly hematological values and serum enzyme activities were recorded. Zn levels of spleen, kidney, testes, muscle and feces were not affected by the Cd treatment. After 40 days of Cd exposure, the Zn concentration in the hair decreased, while the Zn level of the lung slightly increased. During the feeding trial, Cd produced an increase of liver Zn by about 24%. Femur Zn decreased and Zn in urine increased after 60 days of exposure to 7.15 ppm Cd. Except in hair and urine, the Cu concentration in all organs and tissues investigated remained unchanged. Hair Cu levels decreased after 40 days of Cd exposure but increased after 60 days. At this time, elevated Cu concentration in urine was recorded. After exposure to 7.15 ppm Cd, the Fe content of the lung, liver, spleen, testes, muscle, femur and blood decreased significantly. After 60 days on the 7.15 ppm diet, highest reductions (30%–60%) of Fe content were recorded in the lung, liver, and spleen. Similar to Zn and Cu, the Fe concentration in urine increased at that time. No changes were found in the Fe content of hair, kidney, and feces. In spite of a disturbed Fe metabolism, an anaemic response of Cd-treated rats was not observed. Hematocrit and hemoglobin remained unchanged and the number of red blood cells slightly increased after 40 days of Cd exposure. While the activity of serum alkaline phosphatase was not affected by dietary Cd, the activities of serum-GOT and serum-GPT increased after exposure to 7.15 ppm Cd indicating disturbed hepatic functions.     

Assimilation and bioconcentration of Ag and Cd by the marine black bream after waterborne and dietary metal exposure

We determined the aqueous uptake and dietary assimilation of Cd and Ag by the marine black bream Acanthopagrus schlegeli following one to four weeks' exposure (or conditioning) to waterborne or dietary Cd or Ag at different concentrations. The concentrations of metals and metallothioneins (MT) in different tissues also were determined. The viscera contained the highest Ag, Cd, and MT concentrations after metal exposure. After exposure to waterborne metals, the metal and MT concentrations in the gills were higher than those in the remaining tissues (mainly muscles and bones), but this pattern was reversed following exposure to dietary metals. The assimilation efficiencies (AEs) of Cd and Ag ranged from 6 to 24% and 15 to 30%, respectively. The rate constant of uptake from the dissolved phase (ku) of Cd and Ag ranged from 2.2 to 7.5 and 8.0 to 31.7 L kg(-1) d(-l), respectively. In all the exposure experiments, the ku and AE increased with induced MT concentration and tissue metal concentration. Increasing metal accumulation may have been due to the increased available binding sites following the induction of MT in the fish. Furthermore, the MT induced by either Cd or Ag was not specific, but was able to bind with both metals and enhance bioaccumulation. Exposure to dissolved and dietary metals may increase metal accumulation, which potentially may lead to metal toxicity, although the fish may develop a tolerance to metals due to the apparent induction of MT.     

Cadmium Interaction with Essential Metals (Zn, Cu, Fe), Metabolism Metallothionein, and Ceruloplasmin in Pregnant Rats and Fetuses

Cadmium administered to rats per os is accumulated in the duodenal mucosa in the form of metallothionein (MT). Therefore, this toxic metal can influence the efficiency of essential metal absorption, especially their concentration in the maternal organism, which plays an essential role during fetal development. The aim of this study is to elucidate the mechanism of the origin of Zn, Cu, and Fe deficiency in fetal rat livers after maternal exposure to cadmium in drinking water and to investigate the roles of MT and ceruloplasmin (Cp) in this phenomenon. Cadmium was given to pregnant dams exposed for 0–20 days of gestation in drinking water at concentrations of 6.25–100 μg Cd/ml. After cessation of exposure, at the lowest dose, a decrease in Cu and Fe concentrations in the duodenal mucosa was found. Simultaneously, diminution in concentration of two cited metals and Cp activity in serum of dam blood was noted. The lowest dose of cadmium developed a drop tendency in microsomal fetal liver iron. Significant correlations were observed between fetal liver Cu contents and Cp activity in serum of dams and Cu concentrations in serum of dams. Diminished Cp activity in serum of dams is related to reduced availability of Cu and Fe in fetuses. In conclusion, it is suggested that the mechanism of Cu and Fe deficit content in fetuses is based on the diminution of absorption of these metals by dam intestines exposed to cadmium on the reduction of metal concentrations in blood serum and, in consequence, their decreasing availability in fetuses.     

Influence of diethyldithiocarbamate on nickel-induced hepatic metallothionein in rats

The influence of sodium diethyldithiocarbamate (DDC) on 63nickel chloride-induced metallothionein (MT) and alterations in essential metal levels in the liver of rats was investigated. The induction of MT, Zn and Cu levels of the hepatic cytosolic "heat stable fraction" (HSF) by DDC increased with time up to 24 hr. Although MT, Zn and Cu were significantly greater at 17 hr than those at 6.5 hr after 63Ni administration, the Ni level decreased. The treatment with DDC at 6 hr, but not at 10 min, prior to 63Ni administration increased significantly the MT, Zn and Cu contents 17 hr after 63Ni administration over that caused by 63Ni alone at 17 hr., showing a synergistic effect. The induction of hepatic MT by 63Ni or DDC alone was prevented by cycloheximide but not by actinomycin D and seemed to be regulated at the protein synthesis level.     

Metallothionein levels in liver and kidney of Canadians--a potential indicator of environmental exposure to cadmium

Liver and kidney cortex samples were obtained from 89 human autopsies for the determination of metallothionein (MT), cadmium (Cd), zinc (Zn), and copper (Cu). An age-dependent increase of Cd, Zn, and Cd/Zn was observed in the kidney; increases peaked at mid-age and were followed by a decline. Cadmium was present at a low constant level in the liver at all ages. The mean concentrations of Cd and Zn were 18.0 and 40.1 micrograms/g wet weight, respectively, in the kidney, and for the liver the values for Cd and Zn were 2.0 and 79.0 micrograms/g wet weight, respectively. A positive linear relationship was observed between Cd or Zn and MT in the kidney and between Zn and MT in the liver. No other correlation was found between Cu and MT in either organs. Similar values were obtained for MT when estimated by Ag-hem and Cd-hem methods.     

Structure and expression of chicken metallothionein

The chicken metallothionein (MT) cDNA and gene were cloned, and their nucleotide sequences determined. The cDNA clones encode a cysteine-rich protein of 63 amino acids which shares extensive structural homology with the mammalian MTs. Southern blot analyses of total genomic DNA, and cloned chicken DNA indicated that the MT gene is a unique gene sequence. The chicken MT gene is structurally homologous with the mammalian MT genes; consisting of three exons separated by two intervening sequences. The placement of the intervening sequences in the chicken gene is nearly identical with that in the mammalian MT genes. Levels of hepatic MT mRNA were rapidly induced by metal ions (Cd2+, Zn2+, Cu2+), glucocorticoids and lipopolysaccharide (LPS). MT mRNA was present in low levels in embryonic liver, but was inducible in ovo by injection of metal ions, glucocorticoids or LPS. Hepatic MT mRNA increased to high levels soon after hatching, before decreasing again to the basal levels found in adult liver. Levels of hepatic MT mRNA in hatched chicks were influenced by dietary metals. The results establish that the structure of the MT protein and gene has been highly conserved between birds and mammals, which suggests a functionally important role(s) for this protein.     

Association of heavy metals with metallothionein and other proteins in hepatic cytosol of marine mammals and seabirds

Distribution of Cu, Zn, Cd, Ag, Hg, and Se were determined in hepatocytosol of northern fur seals (Callorhinus ursinus), black-footed albatrosses (Diomedea nigripes), and Dall's porpoises (Phocoenoides dalli). Copper, Zn, and Cd were accumulated preferentially in metallothionein (MT) fraction and their contents in MT fraction increased with the amounts in the hepatocytosol. Silver was bound to both high-molecular-weight substances (HMWS) and MT in the hepatocytosol for all three species, whereas the distribution of Ag in the cytosol was different among the three species. In northern fur seals, Ag mainly was bound to MT, whereas it mainly was associated with HMWS in Dall's porpoises. In contrast, Ag was distributed almost equally in both HMWS and MT for black-footed albatrosses. Mercury content in HMWS and Se content in HMWS and low-molecular-weight substances (LMWS) increased with their contents in hepatocytosol for all the three species. A significant positive correlation was found between Se and Hg contents in high-molecular weight (HMW) fraction in cytosol. The molar ratio of Hg and Se was close to unity in HMW fraction of the specimens with high Hg concentration in cytosol, implying that the Hg-Se complex was bound to the HMWS. Analysis of metals in the hepatocytosol by high-performance liquid chromatography/inductively coupled plasma-mass spectrometry (HPLC/ICP-MS) suggests that multiple isoforms of MT are present in hepatocytosol of the three species and that the metal profiles in hepatocytosols are different among the species. To our knowledge, this is the first report on the association of Ag with HMWS and MT in hepatocytosol of marine mammals and seabirds. Also, distribution and interaction of Hg and Se were investigated for the first time in hepatocytosol of the higher trophic marine animals.     

Hepatic Metal and Metallothionein Levels in a Potential Sentinel Teleost,Dulesauriga, from a Southeastern Brazilian Estuary

The identification of estuarine sentinel species is of paramount importance. The potential of the species Dules auriga sampled from Guanabara Bay, Brazil regarding metal contamination was assessed. Hepatic metallothionein (MT) and Fe, Zn, Cu, Mn, Cd and Ag concentrations were determined in cytosolic fractions (S50 and HT S50) by polarography and ICP-OES, respectively. HT S50 Fe, Mn and Zn were lower than in the S50 fraction, indicating MT-detoxification. MT was correlated to HT S50 Zn, indicating Zn homeostasis. Zn was negatively correlated to weight (TW) and length (TL), suggesting environmental Zn influence. A moderate negative correlation between HT S50 Cu and the condition factor (CF) was observed, indicating that increasing environmental Cu concentrations may decrease D. auriga CF. Several inter-elemental correlations were observed. No MT, TW and TL correlations were found. Thus, MT variability is probably linked to environmental metal concentrations. D. auriga fulfills most sentinel requisites, and MT may be an adequate metal contamination biomarker in this species.

     

Relationship of cadmium accumulation to zinc or copper concentration in horse liver and kidney

The concentrations of Cd, Zn, Cu, and metallothionein (MT) in the liver, renal cortex, and renal medulla were determined in 24 male and 15 female younger thoroughbreds (age 27 to 97 months) and two old male horses (age 154 months and 190 months). High correlations were found between Zn and MT in the liver (partial correlation coefficient 0.836), between Cd and MT in the renal cortex (partial correlation coefficient 0.786), and between Cd and Zn in the renal cortex (partial correlation coefficient 0.675), while the correlation between Cd and MT in the liver was low (partial correlation coefficient 0.124). In the renal medulla, high correlations were found between Cd and Zn (partial correlation coefficient -0.631), between Zn and Cu (partial correlation coefficient 0.881), and between Cd and Cu (partial correlation coefficient 0.785). Therefore, in the liver, the MT concentration is the most highly correlated with the Zn concentration and is not correlated with the CD concentration unless artificially exposed to Cd. In the renal cortex, the MT and Cd concentrations are very highly correlated with each other. The Zn concentration is about 20 micrograms/g when the Cd concentration in the renal cortex is the lowest.     

Accumulation of cadmium in organs of mice by a long-term injection of cadmium and interactions of cadmium with copper, manganese and zinc already present in the animals (author's transl)]

It is well known that the greater part of the administered cadmium is accumulated in liver and kidneys. But, in considering the toxicity of cadmium, it is important to make clear the time pattern of cadmium accumulation not only in liver and kidneys but in the other organs. Male mice were injected subcutaneously 1 mg/kg of cadmium daily for 25 weeks except one day in every week. Five mice at a time were killed in the suitable time during this experiment, and Cd, Cu, Mn, and Zn concentrations in several organs were determined by atomic absorption spectrophotometry. Cadmium content in liver and kidneys increased remarkably during the first 30 days, and it is scarcely increased after that period. Cadmium content in other organs, that is, heart, lungs, spleen, testes and femurs, increased slowly during the first 20 days, and this content increased hardly after that. The results show further that: Cd administered at the early stage accumulated mainly in liver an in kidneys. As Cd concentration in liver and kidneys arrives at saturation, Cd content in other orbans increased remarkably. And, after 30th day, Cd content in all the organs increased little in spite of the continuous injection of Cd. Concentrations of Cu, Mn, and Zn in the organs of mice injected with Cd were as follows: Cu concentration increased significantly in heart, liver, and kidneys, Zn concentration increased in heart, lungs, liver, and kidneys, whereas Mn concentration decreased remarkably in kidneys. Cadmium content in the organs of mice injected with Zn (0.5 mg/kg) or Mn (0.5 mg/kg) together with Cd (1 mg/kg) showed a tendency to increase remarkably compared with single injection of Cd.     

Induction of Cd and Zn-thionein in a clonal osteogenic cell, MC3T3-E1

Clone MC3T3-E1 cells at various differentiation stages were exposed to 0.44-13.3 microM Cd or 50-175 microM Zn in culture medium. After a 2-h culture period, the amount of Zn accumulated in the cells was shown to be larger than that for Cd, but the production of Zn-thionein was much less compared with that of Cd-thionein. After a 24-h incubation period, the synthesis of Zn-thionein increased markedly at levels of 150 microM Zn or greater, Cd induced metallothionein (MT) synthesis in a dose-dependent manner at 0.44 microM Cd or greater. Since calcified cells differentiated into osteoblastic cells also produced MT, osteoblasts were confirmed to have an ability to induce MT synthesis. Initiation of production of Cd-or Zn-thionein in the cells occurred at an accumulation of about 0.4 nmol Cd and 2.5 nmol Zn/mg cytosol protein. The ratios of thionein-binding Cd/Cd accumulated in the cytosol and thionein-binding Zn/Zn accumulated in the cytosol were 0.11 mol/mol and 0.067 mol/mol, respectively. These results show that the concentration of accumulated Zn necessary for initiating production of MT is about six times that of Cd and one molecule of Cd induces thionein about 1.6 times as effectively as one molecule of Zn does.     

Cadmium turnover and changes of zinc and copper body status of rats continuously exposed to cadmium and ethanol

The effects of continuous exposure to cadmium (Cd) and ethanol on Cd turnover and zinc (Zn) and copper (Cu) body status of male Wistar rats were studied. The animals received an aqueous solution of 10% (w/v) ethanol and/or 50 mg Cd/l as the only drinking fluid for 12 weeks. The concentrations of Zn, Cu and Cd in the serum (or blood), liver, kidneys, spleen, brain, heart, femoral muscle and femur as well as in 24-h urine and faeces specimens were assessed by atomic absorption spectrometry (AAS). Ethanol alone had no effect on Cd accumulation or excretion. By contrast, co-administration of ethanol with Cd influenced the turnover of this toxic metal. Long-term consumption of ethanol alone caused a decrease in femur Zn and liver Cu concentrations. Moreover, the urinary loss of both bioelements decreased, whereas their faecal excretion was increased. Exposure to Cd resulted in an increase in liver and kidney and in a decrease in femur and 24-h urine Zn concentrations. An increase in Cu concentration in the kidney and a decrease in the brain were also noted. Moreover, Cd increased the total pool of Zn in organs (kidneys, liver, spleen, heart and brain), but did not influence that of Cu. Zn concentration in the liver, kidney and spleen of rats co-exposed to Cd and ethanol were increased, but were decreased in the brain and femur, compared to controls. The concentrations of Cu in livers and brains of these rats were decreased, whereas those in kidney, spleen and heart were increased. The urinary excretion of the elements was decreased, whereas their faecal excretion was increased. Moreover, the total amount of Cu in organs decreased below the control value and that of Zn was in the normal range. These changes in Zn and Cu levels could be explained by different effects of both toxic substances, differences in bioelement intakes (due to reduced consumption of drinking solutions and food), and the modifying effect of ethanol on Cd turnover. Our results suggest that alcoholics may be more susceptible to Cd accumulation and its effects on body Zn and Cu.     

Relationships among developmental stage,metamorphic timing,and concentrations of elements in bullfrogs (Rana catesbeiana)

We collected bullfrog (Rana catesbeiana) larvae from a coal combustion waste settling basin to investigate the effects of developmental stage and timing of metamorphosis on concentrations of a series of trace elements in bullfrog tissues. Bullfrogs at four stages of development (from no hind limbs to recently metamorphosed juveniles) and bullfrogs that metamorphosed in the fall or overwintered in the contaminated basin and metamorphosed in the spring were analyzed for whole-body concentrations of Al, V, Cr, Ni, Cu, As, Pb, Cd, Zn, Ag, Sr, and Se. After the effects of dry mass were removed, tissue concentrations of six elements (Al, V, Cr, Ni, Cu, As, and Pb) decreased from the late larval stage through metamorphosis. Decreases in concentrations through metamorphosis ranged from 40% for Cu to 97% for Al. Tissue concentrations of these elements were also similar or higher in spring; Al and Cr concentrations were 34 and 90% higher in the spring, respectively, whereas As, Ni, Cu, and Pb concentrations were <10% higher. Concentrations of Cd, Se, and Ag varied among seasons but not among stages; Cd and Ag concentrations were 40 and 62% lower, respectively, and Se concentrations were 21% higher in spring. Concentrations of Zn varied only among stages; concentrations decreased gradually through late larval stage and then increased through metamorphosis. Concentrations of Sr varied among stages, but this variation was dependent on the season. Concentrations of Sr were higher in larval stages during the spring, but because concentrations of Sr increased 122% through metamorphosis in the fall and only 22% in the spring, concentrations were higher in fall metamorphs when compared with spring metamorphs. Our results indicate that metamorphosis and season of metamorphosis affects trace element concentrations in bullfrogs and may have important implications for the health of juveniles and the transfer of pollutants from the aquatic to the terrestrial environment.