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.     

Uptake of cadmium in isolated kidney cells--influence of binding form and in vivo pretreatment

Uptake of cadmium as 109CdCl2, 109Cd-cysteine, 109Cd-albumin and 109Cd-metallothionein was studied in isolated kidney cells from rat. Cd as 109CdCl2 and 109Cd-albumin was taken up at similar rates. The uptake of cadmium as 109Cd-cysteine was greater and that of 109Cd-metallothionein lower compared with that of the other substances. These observations were made on non-pretreated cells. In cells taken from rats pretreated with CdCl2 in vivo, the uptake of cadmium as 109CdCl2, 109Cd-cysteine and 109Cd-albumin was lower than in cells from non-pretreated rats. However, the uptake of 109Cd-metallothionein was considerably enhanced in pretreated cells. In pretreated kidney cells the decreased uptake of Cd (as Cd-albumin) might be related to protection of the kidney against acute Cd toxicity and increased uptake of metallothionein-Cd might contribute to the explanation of renal damage in long-term Cd exposure.     

肝脏损害对染镉大鼠镉分布的影响

大鼠腹腔内注射ＣｄＣｌ２０．５ｍｇＣｄ＾２＋／Ｋｇ体重，每周三次，共１０周。注射ＣｄＣｌ２第４周末，其中一组动物灌胃ＣＣｌ４９００ｍｇ／ｋｇ体重。结果表明ＣｄＣｌ２＋ＣＣｌ４组动物肝脏损害后肝镉浓度明显低于单纯ＣｄＣｌ２组，同时伴随血镉，肾镉水平显著升高。肝、肾中金属硫蛋白浓度也与相应组织中隔浓度呈类似的变化形式。ＣｄＣｌ２＋ＣＣｌ４组动物尿镉和尿金属硫蛋白浓度均明显高于ＣｄＣｌ２组。这些实验     

Toxicity of cadmium oxide instilled into the rat lung. II. Inflammatory responses in broncho-alveolar lavage fluid

Biochemical and cytological responses in the broncho-alveolar lavage fluid were investigated after instillation of cadmium oxide (CdO) or cadmium chloride (CdCl2) into the rat lung. Although biochemical responses of the lung to CdO were similar to the CdCl2-exposed lung, cytological response was more sensitive to CdO than CdCl2. Increases of lactate dehydrogenase, protein content and number of cells in the lavage fluid were proportional to the dose over the range of 0.5-10 micrograms Cd/rat. beta-Glucuronidase activity in the fluid increased with dose at low doses of Cd, but the activity did not continue to increase above 2 micrograms Cd/rat. A dose-response profile of phosphorus content in the lavage fluid, which might indicate amount of surfactant produced by Type II cells was similar to that observed for beta-glucuronidase in CdO-treated rats. Thus, tolerable level of instilled CdO for the rat lung was about 2 micrograms Cd/rat.     

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.     

Relationship between toxicity and cadmium accumulation in rats given low amounts of cadmium chloride or cadmium-polluted rice for 22 months

To clarify toxic effects of long-term oral administration of low dose cadmium (Cd) on the liver and kidney, six groups of female Sprague-Dawley rats were fed a diet containing Cd-polluted rice or CdCl2 at concentrations up to 40 ppm, and killed after 12, 18, and 22 months. With toxicological parameters, including histopathology, there was no evidence of Cd-related hepato-renal toxicity, despite a slight decrease of mean corpuscular volume and mean corpuscular hemoglobin of red blood cells with 40 ppm CdCl2. Dose-dependent accumulation of Cd was observed in the liver and kidneys with peak levels of 130 +/- 42 micrograms/g and 120 +/- 20 micrograms/g, respectively, at 18 months in animals treated with 40 ppm CdCl2. A dose-dependent increase in urinary Cd levels became evident with time. Induction of metallothionein (MT) was also observed in the liver and kidney with a high correlation to the corresponding Cd levels. In the proximal renal tubular epithelia of 40 ppm CdCl2-treated rats at 22 months, prominent accumulation of Cd was observed in secondary lysosomes associated with MT deposits in their exocytotic residual bodies. The results demonstrated that, in contrast to the case with high-dose Cd-administration, renal toxicity is not induced by long-term oral administration of low amounts of Cd, although tissue accumulation does occur. Possible protective mechanisms may be operating.     

Dietary iron lowers the intestinal uptake of cadmium-metallothionein in rats.

It has been shown that addition of extra calcium/phosphorus (Ca/P), zinc (Zn) and iron (Fe2+) to the diet results in a significant protection against cadmium (Cd) accumulation and toxicity in rats fed inorganic Cd salt. However, it is not clear whether the presence of these mineral supplements in the diet also protects against the Cd uptake from cadmium-metallothionein. The present study examines the influence of Ca/P, Zn and Fe2+ on the Cd disposition in rats fed diets containing either 1.5 and 8 mg Cd/kg diet as cadmium-metallothionein (CdMt) or as cadmium chloride (CdCl2) for 4 weeks. The feeding of Cd resulted in a dose-dependent increase of Cd in intestine, liver and kidneys. The total Cd uptake in liver and kidneys after exposure to CdMt was lower than after exposure to CdCl2. At the low dietary Cd level and after addition of the mineral supplement, the kidney/liver concentration ratio increased. However, this ratio was always higher with CdMt than with CdCl2, suggesting a selective renal disposition of dietary CdMt. The uptake of Cd from CdCl2 as well as from CdMt was significantly decreased by the presence of a combined mineral supplement of Ca/P, Zn and Fe2+. The protection which could be achieved was 72 and 75% for CdMt and 85 and 92% for CdCl2 after doses of 1.5 mg/kg and 8 mg/kg respectively. In a following experiment it was shown that the protective effect of the mineral mixture against CdMt was mainly due to the presence of Fe2+. It seems clear that Cd speciation and the mineral status of the diet have a considerable impact on the extent of Cd uptake in rats.     

Indirect and lactation-associated changes in renal alkaline phosphatase of newborn rats prenatally exposed to cadmium chloride.

Pregnant Sprague-Dawley rats were intraperitoneally injected with physiological saline solution (vehicle) or cadmium chloride (CdCl2) at 2.5 mg kg-1 body wt. on days 8, 10, 12 and 14 of gestation. Offspring were examined for renal alkaline phosphatase activity (ALP) on postnatal days (PND) 3 and 12, and for kidney metallothionein (MTh) and for liver, kidney and entire gastrointestinal tract 109Cd content at birth and on PND 3 and 12. No effects were observed on neonatal survival or on body, liver and kidney weights of pups up to PND 12. Newborns born and fed by mothers exposed to CdCl2 during pregnancy exhibited a significant decrease in ALP activity on PND 3. Conversely, no significant changes were observed in newborns lactated by surrogate non-treated mothers. Renal MTh increased with age but was not influenced by maternal treatment. Traces of 109Cd were present in the liver at birth (5-7 ng). Thereafter, 109Cd was mainly found in the gastrointestinal tract of newborns lactated by their biological mothers (610-690 ng on PND 12), with a marginal uptake in the liver (10-12 ng on PND 12). 109Cd was not detectable in the kidneys at any age (less than 4 ng). These results show that prenatal exposure to Cd cannot be the sole aetiological agent in the induction of the subtle and transitory changes in renal biochemistry observed in offspring born and fed by female rats intraperitoneally injected with 2.5 mg CdCl2 kg-1 body wt. on days 8, 10, 12 and 14 of gestation. The results also contradict the role of a direct effect on the kidney.     

Resistance to acute nephrotoxicity induced by cadmium-metallothionein dependence on pretreatment with cadmium chloride

Three groups of rats (B-D) were given various daily doses of CdCl2 (0.5-2 mg Cd/kg) continuously or in intervals during time periods of 1-8 weeks. Another group of animals (A) were kept untreated. At the end of the period, selected subgroups of groups A-D were given a single subcutaneous injection of 109Cd-metallothionein (109CdMT) 0.05 or 0.4 mg Cd/kg ("challenge dose"). Subsequently, urinary creatinine, protein, Cd, 109Cd and MT and kidney cortex Cd, 109Cd and MT were determined. In group A (no long term pretreatment), an increased proteinuria was observed after the rats had received the lower of the challenge doses of 109CdMT, and an even greater increase after the higher challenge dose of 109CdMT. No such increase appeared in group B, C and D (repeatedly pretreated with CdCl2) at either of the challenge doses. Higher metallothionein concentrations in kidney cortex observed in the pretreated groups constitute a plausible explanation of the protective effects of pretreatment against the development of increased proteinuria after challenge dosing. It is likely that increasing Cd concentrations, gradually accumulating in the renal cortex (22-226 micrograms/g wet wt.) as a result of the pretreatment, served to induce the synthesis of metallothionein in the renal cortical cells, thus making them resistant to the challenge from 109CdMT.     

Toxic effects of cadmium on LHRH-induced LH release and ovulation in rats.

CFY rats were given 5 or 10 mg/kg bw cadmium chloride (CdCl2) or 0.9% NaCl solution (1 mL/kg) subcutaneously on the day of diestrus II. Six days later (proestrus) at 1200 h they were anesthetized with pentobarbital, 0.5 or 2 micrograms/kg LHRH was injected intravenously at 1400 h, and blood was collected for LH determination. A second group of animals pretreated with 10 mg/kg bw CdCl2 and treated with 2 or 4 micrograms/kg LHRH was allowed to recover from the anesthesia and checked for ovulation the next day (estrus). In rats treated with 10 mg/kg of CdCl2, the LH content of pituitary gland diminished, but no significant difference was found in the LH response to LHRH. In controls (ovulation blocked by anesthesia) 2 as well as 4 micrograms/kg of LHRH completely restored ovulation, while after Cd pretreatment, ovulation recovered depending on the dose of LHRH. It is concluded that Cd-induced anovulation is related to altered function of the pituitary gland and ovary, which can be restored by excess LHRH.     

Toxicity of cadmium oxide instilled into the rat lung. I. Metabolism of cadmium oxide in the lung and its effects on essential elements

The metabolism of cadmium oxide (CdO, insoluble form) and cadmium chloride (CdCl2, soluble form) instilled intratracheally into the rat lung was investigated. CdO might be solubilized rapidly in the lung and consequently pulmonary clearance rate of CdO was not so different from that of CdCl2. At a dose of 5 micrograms Cd/rat about 20% of the dose was translocated to the liver within 12 h, whereas gradual and consistent accumulation of Cd was observed in the kidney up to 7 days. Both pulmonary clearance and translocation of Cd to the liver were accelerated with the dose of instilled CdO, however, Cd accumulated in the kidney was proportional to the dose. Lung weight was increased by the instillation of CdO. Lung essential elements such as S, P, Mg, Zn and Mn were not affected in the inflammatory-reparative proliferative process, but Cu content of unit lung weight was slightly decreased.     

Kidney synthesizes less metallothionein than liver in response to cadmium chloride and cadmium-metallothionein

Acute exposure to Cd produces liver injury, whereas chronic exposure results in kidney injury. Tolerance to the hepatotoxicity is observed during chronic exposure to Cd due to the induction of metallothionein (MT). The nephrotoxicity produced by chronic Cd exposure purportedly results from renal uptake of Cd-metallothionein (CdMT) synthesized in liver. The change in target organ from liver to kidney might be due to a lower amount of MT synthesized in the kidney in response to CdMT. Therefore, the purpose of the present study was to quantitate hepatic and renal MT induced by CdCl2 and CdMT. MT levels in mice were quantitated using the Cd-heme assay 24 hr after administration of CdCl2 (0.5-3.0 mg Cd/kg) and CdMT (0.1-0.5 mg Cd/kg). In both liver and kidney, MT reached higher levels following administration of CdCl2 (220 and 60 micrograms/g, respectively) than of CdMT (25 and 35 micrograms/g, respectively), probably because higher dosages of CdCl2 than CdMT are tolerated. CdMT produced 19 and 3 micrograms MT/micrograms Cd in liver and kidney, respectively, while CdCl2 produced 11 and 6 micrograms MT/micrograms Cd, respectively. In conclusion, induction of MT occurs in both the liver and kidney after administration of CdCl2 and CdMT. However, the kidney is less responsive than the liver to the induction of MT by both forms of Cd, which may contribute to making the kidney the target organ of toxicity during chronic Cd exposure.     

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.     

Cadmium accumulation and metallothionein concentrations after 4-week dietary exposure to cadmium chloride or cadmium-metallothionein in rats.

The distribution of cadmium was examined in rats fed diets containing either cadmium-metallothionein (CdMt) or cadmium chloride (CdCl2) for 4 weeks. The test diets contained 3, 10, or 30 mg Cd/kg diet (3, 10, or 30 ppm) as CdMt or 30 mg Cd/kg diet (30 ppm) as CdCl2. A second study was performed to establish the Cd content in liver and kidneys after exposure to low doses of both CdMt and CdCl2 (1.5 and 8 ppm Cd). The feeding of CdMt resulted in a dose- and time-dependent increase of the Cd concentration in liver, kidneys, and intestinal mucosa. Rats fed 30 ppm CdMt consistently showed less Cd accumulation in liver and intestinal mucosa than did rats fed 30 ppm CdCl2. However, renal accumulation in rats fed 30 ppm was similar until Day 28 regardless of Cd form. At lower dietary Cd levels (1.5 and 8 ppm), relatively more Cd is deposited in the kidneys, although even at these doses the kidney/liver ratio of Cd is still higher with CdMt than with CdCl2. Tissue metallothionein (Mt) levels in the intestinal mucosa were relatively constant but always higher after CdCl2 exposure than after CdMt exposure. Mt levels in both liver and kidney increased after CdCl2 or CdMt exposure during the course of study. Although Mt levels in liver were higher after CdCl2 intake (30 ppm) than after CdMt intake (30 ppm), renal Mt concentrations were the same for both groups. In fact on Day 7, CdMt administration resulted in slightly higher Mt levels than CdCl2 administration, suggesting a direct accumulation of exogenous CdMt in the kidneys. In conclusion, after oral exposure to CdMt in the diet there is a relatively higher Cd accumulation in the kidneys. However, the indirect renal accumulation via redistribution of Cd from the liver might be lower than after CdCl2 exposure. Which of these two phenomena is decisive in the eventual level of renal toxicity of Cd after long-term oral intake could determine the toxicological risk of the chronic intake of biologically incorporated Cd.     

Role of trace elements in cadmium chloride uptake in hepatoma cell lines

Cadmium coexists with other metals in various products. Releases of cadmium in the environment occur in parallel to the release of other metals including copper, iron and zinc which also have an essential role in human homeostasis as they participate in various biochemical pathways. We studied the interaction of iron, copper, zinc and calcium channel blockers (nifedipine and verapamil) with cadmium chloride in two hepatoma cell lines (HepG2 and HTC cells) in order to determine if these trace elements can affect CdCl(2) uptake and interfere with its toxicity. Both cell lines were initially exposed to CdCl(2) (0-200 microM) for 2h and the uptake of the metal was determined. Cadmium chloride uptake by HepG2 and HTC cells shows an increase with increasing doses of the metal. Cells were also pretreated with 100 uM of FeCl(2) or ZnCl(2) or CuCl(2) or with a nifedipine/verapamil (100 uM) mixture for 2h and then exposed to 200 uM CdCl(2) for 1h in the presence of the trace elements. The uptake of CdCl(2) was determined as well as the membrane integrity (LDH leakage assay), the cell viability (neutral red assay) and cell proliferation (protein assay). Zinc and calcium channel blockers inhibited the uptake of cadmium chloride by both cell lines. On the other hand iron loading resulted in increased uptake of CdCl(2) by both cell lines whereas copper loading increased the uptake of cadmium chloride from HTC cells and inhibited the uptake by HepG2 cells. These findings are of importance when the effects of cadmium on living organisms are examined since co-exposure to cadmium and other metals can occur.     

Reciprocal inhibition of Cd and Ca uptake in isolated head kidney cells of rainbow trout (Oncorhynchus mykiss).

Some environmental pollutants, including cadmium (Cd) and zinc (Zn), can act as endocrine disruptors in fish, either in vivo or through a direct action on steroidogenic cells, as has been demonstrated in vitro. We have previously characterized Cd uptake in head kidney (homologue of mammalian adrenal) cells of rainbow trout (Oncorhynchus mykiss) and have provided evidence for a Cd/Ca interaction. Here, we pursued our investigation of metal competition for uptake. Our results show that inorganic speciation conditions favour Cd uptake with optimal level of accumulation for Cd2+ compared to chlorocomplexes (CdCl(n)(2-n)). Calcium uptake was studied for the first time in the fish head kidney cells and Ca was found to be less efficiently accumulated compared to Cd. A specific saturable mechanism of transport was characterized for Ca uptake but voltage-gated or La-sensitive cationic channels are unlikely to contribute appreciably. A concentration-dependent reciprocal inhibition was observed between Ca and Cd, whereas, Zn proved to inhibit Cd uptake exclusively. Additive inhibitory effect on Cd uptake was obtained with co-exposure to Ca and Zn. We conclude that Cd, but not Zn, may decrease Ca availability to the head kidney tissue. Also, Zn may partially protect against Cd toxicity but Zn would not protect against Cd-induced perturbation of Ca homeostasis.     

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.     

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.     

Uptake of cadmium and metallothionein by rat everted intestinal sacs

The gastrointestinal uptake and transport of cadmium (Cd) and the role of metallothionein (MT) were studied in everted sacs of rat intestine (ESRI). When ESRI were incubated for 30 min in a medium containing various Cd concentrations (1-5 x 10(-4) M) as CdCl2, Cd-cysteine (Cd-Cyst) or rat liver Cd-MT-II (Cd-MT), a dose-dependent tissue uptake of Cd was observed. In ESRI incubated with Cd-MT, total Cd uptake was lower than that of CdCl2 (25% of CdCl2). Fractionation of the tissue showed that about 80% of Cd in the tissue was recovered in the particulate fraction after CdCl2 and Cd-Cyst incubation, while that after Cd-MT incubation was present mainly in the cytosol fraction (about 80%). Most of the Cd in the cytosol fraction of Cd-MT-incubated ESRI was associated with a 10,000 molecular weight protein on Sephadex G-75 column fractionation. Similar results were obtained after incubation of ESRI from Zn-pretreated rats with 109CdCl2 solution. In addition to the Cd-MT peak, there was a small peak of Cd associated with a high molecular weight fraction. Only a small percentage of Cd was leaked to serosal fluid in the everted sacs incubated at a low concentration of CdCl2 (0.8%) but this leakage of cadmium was increased at higher concentration and was higher after incubation with Cd-MT. The results suggest that the uptake of Cd from CdCl2 and Cd-MT is different. Although Cd-MT was taken up intact by everted sacs, the uptake was slow as compared to Cd salts. The intracellular presence of MT had little effect on the uptake of CdCl2 but the Cd was sequestered by MT in the intestine.     

Mercaptalbumin as a selective cadmium-binding protein in rat serum

Cadmium-binding proteins in blood serum were determined on a gel permeation column by high-performance liquid chromatography-inductively coupled argon plasma-atomic emission spectrometry. Cadmium chloride was administered iv to female rats of the Wistar strain in a single dose of 0.4 mg Cd/kg body wt and the rats were killed 1, 2, 3, 5, 10, 20, and 30 min after the injection. The blood serum was separated on an Asahipak GS-520 column and cadmium concentration in the eluate was monitored continuously along with sulfur, zinc, copper, iron, and phosphorus concentrations. Cadmium was selectively bound to mercaptalbumin and the metal was eliminated from blood serum within 30 min in a biphasic mode. Addition in vitro of cadmium chloride into fresh blood serum revealed that cadmium is bound selectively to mercaptalbumin up to approximately 14 micrograms Cd/ml serum. Excess cadmium in blood serum was found in the lower molecular weight fraction. Nonmercaptalbumin produced by oxidative disulfide bond formation between albumin and glutathione or cysteine was not able to bind cadmium. Sulfur and other element profiles were helpful in characterizing metal-binding proteins.