Gastrointestinal absorption of cadmium and metallothionein

Intestinal uptake and transport of cadmium (Cd) to different organs were studied in control and oral zinc pretreated rats using an in situ intestinal loop model. Intestinal loop was incubated with either CdCl2 or Cd-metallothionein (Cd-MT) for 30 and 60 min in rats under anesthesia. Induction of MT by oral Zn pretreatment had little effect on intestinal uptake of Cd ion. However, when intestinal loop was incubated with exogenous Cd-MT, the uptake of Cd was significantly smaller than that from CdCl2 incubation. About 50% of the Cd in the intestine of control rat after CdCl2 incubation was recovered in the cytosol fraction and bound to high-molecular-weight (greater than 60 kDa) proteins. In both Zn pretreated rats incubated with CdCl2 and control rats incubated with Cd-MT, Cd was mostly recovered in the intestinal cytosol fraction (75-85%) and was mainly bound to MT. After 60 min incubation of control intestinal loop with CdCl2. Cd was detected mainly in liver with small amounts in kidney and pancreas: with Cd-MT incubation, Cd was detected only in the kidney. The deposition of Cd in the liver was markedly decreased by Zn pretreatment. Both the uptake of Cd-MT by intestine and the induction of MT synthesis in the intestine by Zn pretreatment were demonstrated by immunohistochemistry using a specific antibody to rat liver MT. The results suggest a slow uptake of exogenous Cd-MT from the intestine and transport to kidney in contrast to deposition of Cd in the liver from CdCl2. Although the intracellular presence of MT does not affect the uptake of Cd from lumen, it may decrease both the release of Cd from the intestine and its deposition in liver.     

Effects of mucosal metallothionein in small intestine on tissue distribution of cadmium after oral administration of cadmium compounds.

The effect of mucosal metallothionein (MT) preinduced by zinc (Zn) on tissue distribution of cadmium (Cd) after administration of Cd with several chelating agents was studied in rats. After Cd-cysteine (Cd-Cys) was incubated with intestinal Zn-MT in vitro, all the Cd dissociated from Cys and exchanged the Zn bound to MT. However, dissociation of Cd bound to EDTA (Cd-EDTA) was not observed in the incubation mixture containing intestinal Zn-MT. The concentration of Cd in intestinal mucosa reached a maximum 16 hr after oral administration of Cd-Cys. The Cd level in the intestine was higher than that in the liver and kidney and was similar to that occurring after oral administration of CdCl2. The amount of Cd distributed to the liver and kidney after Cd-EDTA administration was about 30% of the level after CdCl2 administration. Even at 15 mg Cd/kg Cd-EDTA, the Cd level in the intestinal mucosa reached a plateau after 2-4 hr, as it did in the liver and kidney. When Cd-Cys was administered po to control or to Zn-pretreated rats, it was found that Zn pretreatment increased the concentration of Cd in the kidney, as was the case after oral administration of CdCl2. This effect of Zn pretreatment was not observed after oral administration of Cd-EDTA. When Cd-MT was injected into the duodenum, the intestinal absorption of Cd was 60% of that after CdCl2 administration. After the duodenal administration of Cd-MT, at all doses, the concentration of Cd in the kidney was higher than that in the liver. These results suggest that mucosal MT in the small intestine might trap Cd absorbed from the intestinal lumen and transport it to the kidney.     

Degradation of cadmium metallothionein in vitro by lysosomal proteases.

The effects of various protease inhibitors on the degradation of cadmium metallothionein (Cd-MT) by lysosomal proteases were studied in vitro. Degradation of Cd-MT was observed after incubation with the lysosomal extracts, but not after incubation with the cytosol or heat-treated lysosomal extracts. After incubation of [35S]-Cd-MT or 109Cd-MT with lysosomal extracts, 35S and 109Cd radioactivity in the MT fraction decreased, while the low molecular weight (LM) fraction increased with time (half life; 3 hr). When EDTA was added to this incubation mixture, most of the MT was degraded within 30 min. Cd in the LM fraction, produced after the incubation of Cd-MT with the lysosomal extracts, was moved to the high molecular weight fraction by the addition of cytosol. Both leupeptin and E-64, which reduced cathepsin B (cysteine protease) activity, inhibited the degradation of Cd-MT by the lysosomal extracts. But pepstatin A, a specific inhibitor of cathepsin D, did not inhibit this degradation. E-64 inhibited degradation, as well as inhibiting cathepsin B activity, in accordance with its concentration in the incubation mixture. The incubation of Cd-MT with purified cathepsin B resulted in its degradation which was inhibited by E-64. These results suggest that Cd-MT may be broken down by the cysteine protease in lysosomes and that the released Cd bound low molecular weight fragment(s) was subsequently transferred to the high molecular weight protein in cytosol.     

Influence of cadmium-metallothionein pretreatment on tolerance of rat kidney cortical cells to cadmium toxicity in vitro and in vivo

Kidney cells were isolated from rats pretreated by daily subcutaneous doses of cadmium metallothionein (CdMT: 0.05-0.2 mg Cd/kg X 5) and from non-pretreated rats. Upon exposure to CdCl2 in vitro (0-200 micrograms Cd/ml), a concentration dependent decrease in viability was observed in the non-pretreated cells, while no such decrease occurred in the pretreated cells indicating that these cells were more resistant to the toxic action of cadmium. There was a higher in vitro uptake of Cd+2 and an increased metallothionein (MT) concentration in the pretreated cells (compared to non-pretreated cells). Subcellular distribution studies revealed that Cd was mainly recovered in the "cytosol" fraction. The higher total cadmium uptake in pretreated cells corresponded to an increase of Cd in "cytosol" and "nuclear" fractions. This observation may be explained by MT-binding of Cd in the cells and is in accordance with a possible protective effect of induced MT in the pretreated cells. In order to assess whether pretreatment-induced tolerance to cadmium toxicity--indicated by the cellular studies--could also be observed in vivo, some whole animal experiments were also performed. A dose-related proteinuria was observed in non-pretreated rats after a single subcutaneous administration of 109Cd-MT at doses of 0.05 and 0.4 mg Cd/kg. Urinary total Cd, 109Cd and MT was also increased in a dose-related fashion. Cadmium concentrations in kidney were dose related and reached 19 micrograms/g wet weight. In contrast, in animals repeatedly pretreated with CdMT according to 1), no proteinuria was observed after administration of the same single doses of 109CdMT. Total Cd. 109Cd and particularly MT-concentrations in urine were lower in such pretreated animals than in in non-pretreated ones in spite of the accumulation of higher tissue concentrations of total Cd (up to 80 micrograms/g). The pretreatment was thus shown to prevent some of the acute nephrotoxicity of CdMT, possibly by means of induction of MT synthesis.     

Effect of cadmium chloride and Cd-metallothionein on the nervous tissue culture

Influence of metallothionein (MT) isolated from rat liver on rat cerebellum in culture was investigated by comparison with that of CdCl2. Cd-MT at 0.2 X 10(-5) M as Cd significantly depressed the outgrowth of nerve fibers, fibroblasts and glial cells as compared to the control culture. In the range from 0.2 X 10(-5) M to 2.7 X 10(-5) M Cd, the toxicity of Cd-MT was the same as that of CdCl2. Above 5 X 10(-5) M Cd, however, the toxicity of Cd-MT was less than that of CdCl2. Cadmium added as CdCl2 was perfectly recovered at a region of higher Mr than MT on the Sephadex G-75 column. Cadmium added as Cd-MT was detected in part at the higher Mr region and in part at the MT region, depending on incubation time and Cd concentration in the medium. The toxic action of Cd-MT was proportional to the recovery level of Cd at the higher Mr region.     

The origin of metallothionein in red blood cells

The origin of metallothionein (MT) in red blood cells (RBCs) from a mouse given cadmium was studied in connection with RBC kinetics. Plasma Cd concentration rapidly decreased 3 hr following 109CdCl2 (2 mg/kg, sc) administration, whereas RBC Cd increased from 2 to 4 days, followed by a gradual decrease. RBC Cd was found to be distributed more in the high-molecular-weight fraction than in the MT fraction 12 hr after administration. But, thereafter, Cd increased rapidly in the MT fraction to show changes with time similar to Cd level in RBCs. Hepatic damage induced in a mouse given 21 injections of Cd, with resultant marked elevation of plasma MT concentrations, did not cause any change in RBC Cd concentration. MT was hardly transferred to RBC when a mouse RBC suspension was incubated with mouse hepatic MT. To examine the relationship of Cd-MT and erythropoietic function, mice in the normal group, the phenylhydrazine-induced anemia group (PH), the transfusion-induced plethora group (TR), and the erythropoietin administered plethora group (TR + EP) were given 109CdCl2. Three days after administration, Cd concentration in its RBCs and its MT fraction remarkably increased in the PH group, and was greatly decreased in the TR group. A significant increase was noted in the TR + EP group as compared with the TR group. These results indicate that MT in the RBCs is formed in erythroblasts.     

Response of rat hepatocyte cultures to cadmium chloride and cadmium-diethyldithiocarbamate

Cellular effects of cadmium (Cd) were studied in primary cultures of rat hepatocytes incubated with cadmium chloride (CdCl2) or cadmium-diethyldithiocarbamate (Cd(DTC)2), labelled with 109Cd. The lipid-soluble complex Cd(DTC)2 was rapidly taken up into the cells and a maximal concentration was reached after 4 h incubation. On the other hand, incubation with CdCl2 resulted in a slow, continuous accumulation of Cd for up to 20 h. Cd was found to be associated with proteins to a higher extent when added to the incubation medium as CdCl2 than when added as Cd(DTC)2, which in addition to differences in lipophilicity of the Cd compounds partly explains the differences in Cd uptake. Subcellular distribution studies showed that a significantly higher proportion of Cd was associated with the total particulates fraction in cells after incubation with Cd(DTC)2 compared to CdCl2 (32 and 19%, respectively). The activities of glutathione reductase and succinic dehydrogenase were inhibited to a similar extent by the 2 Cd compounds. Alcohol dehydrogenase was more strongly affected by CdCl2 than by Cd(DTC)2, although the uptake of Cd was 3-4 times higher in cells incubated with Cd(DTC)2 than in those incubated with CdCl2. The results from the present study show that DTC can increase the transport of Cd into the cell by complex formation with Cd. Compared to CdCl2 the Cd(DTC)2 complex was less toxic as indicated by the biochemical parameters used.     

Fate of erythrocyte Cd-metallothionein in mice

Degradation of metallothionein (MT), which appears in erythrocytes following cadmium (Cd) administration, was investigated in mice. Cd-MT underwent only slight decomposition by hemolysate in an in vitro experiment unlike an 800g supernatant fraction of the liver homogenate. In an in vivo study, [3H]diisopropylfluorophosphate was given to mice which had received 109CdCl2 to investigate the relationship between the decay of 109Cd-MT in the erythrocyte and the life span of the erythrocyte. A similar reduction pattern of radioactivity of 109Cd and 3H was observed. Erythrocytes containing 109Cd-MT obtained from mice preadministered with 109CdCl2 was transfused to normal mice. The 109Cd radioactivity of erythrocytes decreased in a manner similar to Cd in erythrocytes of 109CdCl2-administered mice. Contrary to this decrease of erythrocyte Cd in the transfused mice, Cd concentration of the spleen increased markedly. Cd increased also in the liver. These results indicate that erythrocyte MT degrades along with the erythrocyte. The Cd from this MT is deposited in the spleen and liver where blood cells are catabolized.     

Exogenous metallothionein and renal toxicity of cadmium and mercury in rats.

The relative tissue distribution and toxicity of cadmium (Cd) and mercury (Hg) in the liver and kidneys of rats when the metals are administered as either inorganic salts or complexed with MT were studied. Male Sprague-Dawley rats were injected (i.v.) with Cd or Hg inorganic salt of chloride or in a complex of MT at a dose of 0.3 mg/kg body weight. The concentration of MT and metals in plasma and urine was monitored for 7 days, at the end of which the rats were killed. Injection of both HgCl2 and Hg-MT induced the synthesis of MT only in the kidney but not in the liver, whereas CdCl2 and Cd-MT injections induced MT synthesis in both liver and kidney, respectively. Plasma MT levels increased 3 days after CdCl2 but not after HgCl2 injection, suggesting that hepatic MT may be an important source of plasma MT under our experimental conditions. Renal toxicity was observed morphologically and by an increase in blood urea nitrogen, plasma creatinine, proteinuria in rats injected with Cd-MT and both forms of Hg. Urinary MT excretion was significantly elevated in Cd-MT injected rats compared with those injected with CdCl2. However, HgCl2 and Hg-MT injected rats showed no significant difference in urinary MT excretion. The magnitude in the renal accumulation of Hg is similar after the administration of Hg-MT or HgCl2, but our findings suggest that the site of epithelial injury may be different. Injury effects of Hg-MT localized mainly in the terminal portions of the proximal convoluted tubule and the initial portions of the proximal straight tubule whereas inorganic Hg caused necrosis in pars recta segments of the proximal tubule.     

Absorption and distribution of cadmium (Cd), copper and zinc following oral subchronic low level administration to rats of different binding forms of cadmium (Cd-acetate, Cd-metallothionein, Cd-glutathione)

Male Wistar rats received by gavage saline or about 25 micrograms cadmium (Cd)/kg/day as Cd-acetate (Cd-Ac), Cd-metallothionein (Cd-MT) or Cd-glutathione (Cd-GSH) 5 times per week for 28 times. At all treatments 0.2-0.3% of the totally administered Cd dose was found in liver, kidneys, small intestine and pancreas, whereas none of the Cd forms applied resulted in a Cd accumulation in testes. Cd in small intestine was not increased by Cd-MT. However, it was raised by Cd-Ac and even more by Cd-GSH. A smaller increase in hepatic and renal Cd resulted from Cd-GSH than from Cd-Ac or Cd-MT. Cd in pancreas increased after Cd-GSH but not after Cd-Ac or Cd-MT. Copper (Cu) rose in small intestine and testes but decreased in kidneys independent of either Cd treatment. Concomitantly, zinc (Zn) was decreased in small intestine and testes. The tissue concentration of metallothionein (MT) was only marginally increased by all treatments. The highest value (80%) above controls) was found in small intestine after Cd-GSH. Intestinal Cd as well as testicular Cu were related to the tissue MT. Therefore, the distribution of Cd between various organs depends on the Cd form applied. There is some relationship to the distribution of Cu and Zn.     

Protective role of renal metallothionein against Cd nephropathy in rats

Rats were treated with four types of Cd compound: CdCl2, Cd bound (Cd-peptide), and Cd bound to metallothionein (Cd-MT). This treatment caused no nephropathy. Subsequently, toxic doses of Cd compounds were administered to these pretreated rats and their effects on renal function were examined. When 1.4 mg Cd/kg as Cd-Cys was administered, marked increases in urinary protein, glucose, and amino acid were observed. However, when the animals were pretreated with 1 mg Cd/kg/day as CdCl2 for 3 days, and 1.4 mg Cd/kg as Cd-Cys was administered 24 hr later, no renal damage was observed. Such a protective effect against the nephrotoxic action of Cd-Cys was also shown by pretreatment with Cd-Cys, Cd-peptide, or Cd-MT. Furthermore such a phenomenon was also observed when the nephropathy was caused by Cd-peptide or Cd-MT. The efficacy of pretreatment depended on the time before subsequent administration of Cd and the dose used for pretreatment. Incorporation of Cd into the liver and the kidney was not altered by the pretreatment. No matter in which form the nephrotoxic dose of Cd was administered, the incorporated Cd was distributed between particulates and cytosol; 3 hr after administration, cytosolic Cd was present in almost equal amounts in the high-molecular-weight and the MT fractions in the nonpretreated rats. However, after pretreatment, more of the Cd subsequently administered was found in the MT fraction. These results suggest that MT participates in the detoxication mechanism against Cd in the kidney, as it does in the liver.     

Comparisons of the toxicity of CdCl2 and Cd-metallothionein in isolated rat hepatocytes

In the intact animal, inorganic Cd distributes mainly to the liver and produces hepatotoxicity, while Cd-metallothionein (CdMT) distributes primarily to the kidney and produces nephrotoxicity. CdMT has also been demonstrated to be more toxic than Cd in cultured kidney cells, but it is not known if CdMT is more toxic to all cultured cells or if there is a good correlation between in vitro and in vivo toxicity. Therefore, hepatocytes, which were isolated and grown in monolayer culture for 24 h, were incubated with CdCl2 (1-100 microM) or CdMT (3-100 microM Cd). The intracellular K+ content was quantitated 24 h later as an index of toxicity. The K+ concentration of the hepatocytes was decreased 50% by 4 microM CdCl2, whereas 25 microM CdMT was required to produce similar injury. In the intact animal, zinc induces the synthesis of MT and decreases the hepatotoxicity of Cd. ZnCl2 added to the media (100 microM) for 24 h before exposure to Cd or CdMT increased the intracellular MT concentration 700%. This elevation in MT reduced the toxicity of CdCl2 approximately 80% but did not alter the toxicity of CdMT. In summary, CdCl2 is more toxic to cultured hepatocytes than Cd-MT, and MT induction decreases the toxicity of CdCl2 in hepatocytes, as has been observed in the intact animal. This indicates that cultured hepatocytes appear to be an excellent model for examining the hepatotoxicity of Cd.     

Tissue distribution of cadmium and nephropathy after administration of cadmium in several chemical forms

Cadmium (Cd) was administered as CdCl2, Cd-Cys, Cd-partial structural peptide of metallothionein (MT) II, Cd-MT I, and Cd-MT II to rats, and the distribution of and nephropathy caused by the corresponding Cd compounds were examined. Each Cd complex showed dissociation of Cd in vivo and in vitro in the plasma. With Cd-Cys approximately 80% dissociation was observed while Cd-MT showed only 15% dissociation. When the dissociation of the Cd complex in the plasma was less, the distribution of Cd to the liver was decreased but distribution was increased to the kidney and urine. Each Cd complex showed the presence of Cd in the kidney shortly after the administration in the high molecular weight fraction (HM-fr) and also in MT-fr. This was then followed by a decrease in the Cd level in the HM-fr but by an increase in the MT-fr. All Cd compounds except CdCl2 caused some transient renal damage. Renal damage shown by significant increases of urinary protein, glucose, and amino acids were observed at the doses of 1.3-1.7 mg Cd/kg in the Cd-Cys group, at 0.51-0.64 mg Cd/kg in the Cd-peptide group, and at 0.16-0.23 mg/kg in the Cd-MT I and II groups. The Cd level in the kidney of rats with renal damage from these complexes was approximately the same in all the groups, that is, 10 micrograms/g kidney. It is concluded that Cd causes renal damage when its concentration in the kidney is 10 micrograms/g or higher regardless of the type of Cd complex that is administered.     

Induction and binding of Cd, Cu, and Zn to metallothionein in carp (Cyprinus carpio) using HPLC-ICP-TOFMS.

The binding of Cd, Cu, and Zn to metallothionein in carp was studied under control and acute Cd exposure scenarios. Carp were exposed to different Cd concentrations for 96 h. Total (Cu, Cd, Zn)-MT levels were determined by the cadmium thiomolybdate saturation assay. Total tissue and cytosolic Cd, Cu, and Zn concentrations were determined by ICP-MS. The cytosolic metal speciation was determined by high pressure liquid chromatography (size-exclusion [SE] in combination with anion exchange [AE]) directly coupled to an inductively coupled plasma time of flight mass spectrometer (ICP-TOFMS). This coupled technique allows the chromatographic separation and online determination of the metals associated to the protein fractions separated. Very strong differences in the tissue compartmentalization and cytosolic speciation of the metals were observed. For example, over 30% of cytosolic zinc was bound to MT in liver while this was only 2% in the kidneys although total cytosolic levels were considerably higher. Induction of metallothionein during cadmium exposure was also tissue specific, displaying different response patterns in gills, liver, and kidney. Cadmium accumulated much stronger in liver and kidney compared to the gills and the latter also showed much lower MT levels. The renal MT-induction was more sensitive to Cd exposure than the hepatic MT induction since a significant increase of Cd-MT and total MT levels occurred at lower tissue Cd concentrations in the kidney in comparison to the liver, except for the highest Cd exposure level where a drastic 10-fold increase in hepatic Cd-MT was observed. At this Cd exposure level also an apparent spill over of zinc to the high molecular weight fraction was observed in the kidneys.     

The dose-dependent deposition of cadmium into organs of Japanese quail following oral administration

The accumulation and disposition of Cd2+ as CdCl2 administered orally to Japanese quail (Coturnix coturnix) was investigated. Birds received 0.01, 0.10, 1.0, 50, 500, 5000, or 50,000 micrograms Cd/kg/day for 4 consecutive days by gastric tube, and were killed 4 days after the final dose. The percentage of the total administered dose recovered in liver + kidneys + duodenum was 0.7% or less in all but the highest dose, for which recovery was approximately 2%. Only at the highest dose did the hepatic Cd concentration exceed that of the kidney, and only at this dose was there any appreciable increase in metallothionein (MT) concentrations in the liver and kidney. Duodenal cytosol was found to contain high levels (300-1300 micrograms/g) of endogenous MT-like proteins, probably due to the relatively high Zn concentration (approximately 185 ppm) of the commercial diet eaten by the quail. In the small intestine, Cd2+ taken up after trace doses of oral 109Cd2+ was found to be exclusively bound to these 10,000-MW, or lower MW, ligands. In the liver, MT synthesis was accompanied by increased concentrations of Cd and Zn (but not Cu) associated with the MT fractions, whereas in the kidney, all three metals were elevated in response to Cd-induced MT synthesis. A major conclusion of the present study is that, in response to environmentally relevant (less than 10 micrograms/kg/day po) doses of Cd2+, absorbed Cd is transported in blood primarily in a form which enhances deposition in the kidney. This behavior is consistent with the pharmacokinetics of Cd-MT.     

Gastrointestinal absorption of Cd-metallothionein and cadmium chloride in mice

CdCl₂ or Cd-metallothionein (MT) (6 g Cd with 2.25 Ci (83.25 KBq)¹⁰⁹Cd) was given orally to mice, which were sacrificed at 30 min and 2 h after intubation. Although¹⁰⁹Cd in Cd-MT was excreted rapidly into the urine, its absorption was found to be significantly less than that of CdCl₂. The poor absorption was due to a decrease of Cd-MT uptake into the intestine. Cadmium chloride taken up into the mucosa could stimulate MT synthesis even 30 min after its intubation. However, the percentage of MT-bound Cd in the Cd of intestinal supernatants was lower with CdCl₂ (62% at 30 min and 2 h) than with Cd-MT (78% and 84% at 30 min and 2 h, respectively). These results suggest that the transport mode of lumenal Cd-MT to mucosal cells is different from that of lumenal CdCl₂. Lumenal Cd-MT is probably internalized into intestinal cells in an intact form. Furthermore, the Cd-MT may pass through the basolateral membrane in this form. This hypothesis was supported by the different distributions of Cd in the liver and kidney after Cd-MT and CdCl₂ intubations.     

Decreased uptake and altered subcellular disposition of testicular cadmium as possible mechanisms of resistance to cadmium-induced testicular necrosis in inbred mice

Mechanisms responsible for resistance to Cd-induced testicular necrosis in inbred mice were investigated using strains resistant (A/J) or susceptible (129/J) to Cd-induced testicular damage. Cadmium accumulation was measured in testes of both strains 15 min, 30 min, 1 h, 2 h, 6 h, 12 h and 24 h after intravenous injection of 1 mumol 109CdCl2/kg. The subcellular disposition of Cd was determined at 15 min, 6 h and 24 h by fractionation of testicular cytosol on Sephadex G-75 Superfine. Testicular accumulation of Cd was 5-6 times less in A/J mice than in 129/J mice at all time points examined. Gel filtration revealed 4 Cd-binding peaks; in both strains testicular Cd was bound to a protein with a relative molecular mass (Mr) of 30 000 and to metallothionein (MT). The fraction of the total testicular Cd bound to the 30 000 Mr protein was similar in both strains after 15 min (13-18%) and declined rapidly to 5-7% by 6 h. A/J testes had a significantly greater fraction of the total Cd bound to MT both 15 min; 38% vs. 24% at 6 h). By 24 h both strains had approximately 43% of the total testicular Cd bound to MT. The results indicate 2 possible mechanisms of resistance to Cd-induced testicular necrosis in inbred mice: decreased testicular Cd uptake and sequestration of a greater fraction of the tissue Cd by MT.     

The modulation by metallothionein of cadmium-induced cytotoxicity in primary hepatocyte cultures

The cytotoxicity of CdCl2 and 2 isoforms of hepatic cadmium-metallothionein (CdMT I and II), was investigated using primary cultures of rat hepatocytes. The cell cultures were exposed to cadmium as CdCl2 or as either isoform of CdMT for a 20-h period at concentrations ranging from 50 to 500 ng Cd X ml-1. Cytotoxicity was assessed by determining the amount of lactic dehydrogenase released from the cells into the incubation medium and the incorporation of [3H] arginine into cell protein. The uptake of Cd by the cells was also measured. Cadmium chloride and both isoforms of CdMT were found to be toxic to hepatocytes although partial protection was afforded by the binding of cadmium to metallothionein (MT). At the higher exposure concentrations and in accordance with the toxicity data, the cells exposed to CdCl2 were found to accumulate more cadmium than those exposed to CdMT. The distribution of cadmium in the culture medium was examined using Sephadex G-75 chromatography. The speciation of cadmium is discussed in relation to its cytotoxicity.     

On the role of metallothionein in cadmium absorption by rat jejunum in situ

The role of metallothionein (MT) in the mechanism of cadmium absorption from the jejunum was studied in 7--9 week-old-male rats exposed to 50 ppm of cadmium in drinking water for 9 days. Exposed animals contained an average of 144 micrograms MT/g of mucosal tissue, compared to 40 micrograms in control anaimls. During jejunal perfusion in situ with 5 mM glucose-saline containing 10--20 nM CdCl2 the increased MT content of mucosa exerted no effect either on cadmium absorption from the lumen (step I), or on its further transport into the body (step II). Immediately after perfusion, essentially all cadmium removed from the lumen was fully recovered in the intestinal mucosa. About 50% of the mucosal cadmium was found in the sediment after homogenization and centrifugation; a large portion of this cadmium may be assigned to the membrane fraction. The binding of freshly absorbed cadmium in the mucosal cytosol was not restricted to low molecular weight protein, although cadmium binding capacity in the MT fraction of controls as well as of exposed animals greatly exceeded actual binding of newly absorbed cadmium. Our results offer no support for the view that MT in the jejunal mucosa serves as determinant of cadmium absorption.