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.     

Cadmium toxicity in kidney cells. Resistance induced by short term pretreatment in vitro and in vivo

Epithelial cells from the kidney were freshly isolated from rats pretreated by daily subcutaneous doses of CdCl2 in vivo (0.5-2 mg Cd/kg X 5). Such cells were incubated in vitro in media with different concentrations of cadmium chloride (0-200 micrograms Cd/ml). There was no inhibition of cell growth in such cells. However, in cells isolated from non-treated rats, in vitro exposure to the same concentrations of CdCl2 caused a dose dependent decrease in viability. When cells, isolated from non-treated rats were pretreated in vitro with CdCl2 (10 micrograms/ml) and subsequently exposed to cadmium chloride (0-200 micrograms/ml), a protective effect was observed, which was similar to the one observed in cells isolated from animals pretreated with CdCl2. The concentration of metallothionein in the cells treated with cadmium was increased. A lower uptake of cadmium chloride, in vitro has been observed in kidney cells pretreated in vivo or in vitro compared to nonpretreated cells. Subcellular distribution studies indicate that Cd-distribution was similar in pretreated and non-pretreated cells, but concentrations were generally lower in the pretreated cells. The decreased uptake of Cd by pretreated kidney cells is a sign of Cd-interference with cellular function. These changes are suggested as a contributing mechanism to the prevention of acute toxic effects of cadmium on the kidney.     

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.     

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.     

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.     

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.     

Modulation of calciuria by cadmium pretreatment in rats with cadmium-metallothionein-induced nephrotoxicity.

One group of male Wistar rats (Group B) was pretreated by a daily subcutaneous injection with CdCl2 during 5 days with increasing doses (0.5, 1, 1, 2 and 2 mg Cd/kg). Another group of rats (Group A) was daily given normal saline subcutaneously for 5 days. On the second day after the last injection, a single s.c. injection of 109Cd-metallothionein (CdMT, 0.4 mg Cd/kg) was given to each animal in both groups. Urinary calcium, protein, metallothionein (MT), N-acetyl-beta-D-glucosaminidase (NAG) and gamma glutamyltransferase (gamma-GT) were measured. In Group A, calciuria, proteinuria, metallothioneinuria and enzymuria was induced by CdMT. Calciuria reached a peak during 0-6 h after the administration of CdMT, thus appearing earlier than other effects. Enzymuria was displayed at 6-12 h for gamma-GT and 12-24 h for NAG. A prominent increase of proteinuria appeared at 24-48 h after the challenge of CdMT. In Group B, no significant increase of urinary calcium, protein, or NAG was observed after the CdMT injection and urinary gamma-GT was only slightly elevated, thus demonstrating the protective action of pretreatment. This study demonstrates for the first time that calciuria, one of the signs of cadmium nephrotoxicity, can be prevented by cadmium pretreatment. Urinary MT increased slightly during the 4-5 days of CdCl2 pretreatment. This is in accordance with previous observations that cadmium pretreatment induces new synthesis of MT which is likely to constitute the background for the resistance to the CdMT challenge to the kidney.     

Fate of cadmium in rat renal tubules: a microinjection study

109Cd was injected into the lumen of superficial proximal or distal tubules of rat kidneys, and recovery in the pelvic urine from the ipsilateral kidney was measured. Fractional recovery of labeled inulin always exceeded 90%. About 70% of injected inorganic Cd (CdCl2) was taken up by the epithelium of proximal tubules, while more than 90% of the injected amount was recovered after distal microinjection. The proximal fractional Cd uptake of a 1:1 (molar) Cd-L-cysteine complex was 82%, but was below 60% for a 5-10:1 molar ratio of cysteine:Cd. The chelate Cd-pentetic acid was recovered in final urine nearly quantitatively after proximal or distal microinjection. Fractional uptake of 109Cd from a Cd-metallothionein (Mt) complex, following proximal microinjection, ranged between 17 (Cd-Mt 0.19 mM) and 8% (Cd-Mt 1.5 mM). It is concluded that luminal Cd uptake by the tubular epithelium depends markedly on the chemical form of Cd and, when present, occurs mostly or exclusively in proximal tubules.     

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.     

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.     

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.     

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     

Comparative effects of three chelating agents on distribution and excretion of cadmium in rats

Sodium N-benzyl-D-glucamine dithiocarbamate (NBG-DTC), which was newly synthesized, 2,3-dimercaptopropanol (BAL), and N-methyl-D-glucamine dithiocarbamate (NMG-DTC) were compared for their relative efficacies in the distribution and excretion of cadmium in rats exposed to cadmium. Rats were injected ip with 109CdCl2 (1 mg Cd and 10 microCi 109Cd/kg) and 3 days later, they were treated with the chelating agents (400 mumol/kg) every other day for 2 weeks. These chelating agents were effective in removing cadmium from the body without increasing the amount of cadmium in the kidney. After treatment with these chelating agents, cadmium was excreted mainly in the feces through the bile and the fecal excretion of cadmium by NBG-DTC was significantly larger than that by BAL or NMG-DTC. The hepatic cadmium content after treatment with NBG-DTC was much more decreased than that with BAL or NMG-DTC. The renal cadmium content was decreased only after treatment with NBG-DTC. These chelating agents did not result in the redistribution of cadmium to brain, testes, and heart. The growth of rats was little retarded by treatment with NBG-DTC and NMG-DTC, but was retarded by treatment with BAL. The treatment with NBG-DTC decreased the tissue amounts of Zn, Fe, and Mn to a small extent as compared with the treatment with cadmium alone. The results of this study reveal that the injection of NBG-DTC to rats pretreated with cadmium can more effectively remove cadmium from the body without the mobilization of cadmium to the kidney, the critical organ in cadmium toxicity, and without redistribution of cadmium to other tissues such as brain, testes, and heart, than injection of BAL and NMG-DTC.     

Effects of calcium and magnesium acetates on tissue distribution of carcinogenic doses of cadmium chloride in Wistar rats

Previous studies have shown that magnesium, unlike calcium, prevents cadmium carcinogenesis at the subcutaneous injection site, and that neither magnesium nor calcium has any significant influence on the production of testicular tumors by cadmium in rats. The present investigation attempts to disclose the nature of those different effects by comparing the results of administration of both physiological metals on the uptake and distribution of carcinogenic doses of cadmium in rats. Male Wistar rats received a single subcutaneous (s.c.) injection of 109CdCl2 (0.02 mmol/kg or 0.04 mmol/kg) and s.c. injections (one daily) of calcium acetate (CaAcet; 0.16 mmol/kg), or magnesium acetate (MgAcet; 4 mmol/kg), or saline on the day before, the day of and the day after the 109CdCl2 dosing. The concentration of cadmium in tissues was determined by gamma-counting on the 4th, the 15th and the 45th day after the 109CdCl2 injection. The concentration of cadmium in tissues on day 4 was ranked as follows: liver greater than kidney greater than the injection site skin greater than pancreas greater than spleen greater than heart greater than lung greater than distant skin greater than testes greater than blood. Administration of CaAcet increased by over 20% and that of MgAcet decreased by over 30% the initial uptake of both cadmium doses at the injection site. Thus MgAcet may prevent cadmium carcinogenesis by inhibiting the uptake of cadmium by the injection site tissues. In the testis and in all other tissues investigated, except kidney, the effects of the physiological metals were reversed, CaAcet tended to decrease and MgAcet tended to increase the uptake of cadmium. CaAcet and MgAcet exerted no noticeable effects on the uptake of cadmium by the kidney. The observed results of CaAcet and MgAcet administration on the concentration of cadmium in distal tissues seem to depend on the alterations in cadmium uptake at the injection site.     

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.     

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.     

Renal cadmium deposition and injury as a result of accumulation of cadmium-metallothionein (CdMT) by the proximal convoluted tubules--A light microscopic autoradiography study with 109CdMT.

Chronic, but not acute, exposure to inorganic Cd produces renal damage. However, a single injection of cadmium bound to metallothionein (CdMT) produces renal injury. It is hypothesized that an interorgan redistribution of Cd as CdMT is responsible for the chronic nephrotoxic effect of Cd. To better understand the mechanism(s) of CdMT-induced nephrotoxicity, the intrarenal distribution of 109CdMT was examined. 109CdMT isolated from rat liver was injected into mice at a nonnephrotoxic dose (0.1 mg Cd/kg, iv). The radioactivity in the kidney reached a maximum level (85% of the dose) as early as 30 min following administration and remained essentially constant for up to 7 days after injection. Within the kidney, 109Cd distributed almost entirely to the cortex. Light microscopic autoradiography of the kidney showed that, within the cortex, 109Cd distributed preferentially to the S1 and S2 segments of the proximal convoluted tubules. Within the S1 and S2 segments, the concentration of 109Cd in the basal and apical parts of the cells was similar to that after the nonnephrotoxic dose of CdMT, but after a nephrotoxic dose (0.3 mg Cd/kg) the radioactivity distributed preferentially to the apical portion of the cells. In contrast, light microscopic autoradiography studies with 109CdCl2 revealed that 109Cd was more evenly distributed throughout the proximal tubules. Moreover, after administration of a large dose of inorganic Cd (3 mg Cd/kg), a similar concentration of Cd was found in the convoluted and straight proximal tubules. These data support the hypothesis that CdMT-induced nephrotoxicity might be due, at least in part, to its preferential uptake of CdMT into the S1 and S2 segments of the proximal tubules, the site of Cd-induced nephrotoxicity.     

Parathyroid hormone-stimulated cadmium accumulation in Madin-Darby canine kidney cells.

Although most renal cadmium transport occurs in proximal tubules indirect evidence suggests that distal tubules may also transport this heavy metal. Since the distal nephron is the site at which parathyroid hormone (PTH) regulates calcium absorption, we evaluated the effects of PTH on Cd2+ accumulation in Madin-Darby canine kidney (MDCK) cells. MDCK cells express a distal-like phenotype including PTH-sensitive adenylyl cyclase and stimulation of calcium transport. MDCK cells were grown to confluence in phenol red-free Dulbecco's modified Eagle's medium. PTH increased 109CdCl2 accumulation in a concentration-dependent manner over the range of 10(-11)-10(-9) M bPTH[1-34]. At 10(-9) M, PTH increased Cd2+ accumulation maximally by 205%. The PTH antagonist, bPTH[3-34], failed to augment 109Cd2+ accumulation. The dihydropyridine agonist, Bay k 8644, in the presence of PTH, increased 109Cd2+ uptake by 200% over vehicle-treated controls and by approximately 100% over PTH or Bay k 8644 alone. The apparent Km for Bay k 8644 activation was 1.3 microM. Bay k 8644-augmented 109Cd2+ uptake was competitively inhibited by the calcium channel antagonist nifedipine. No voltage dependence of Bay k 8644-amplified 109Cd2+ uptake could be detected. Based on these observations we conclude: (1) MDCK cells accumulate Cd2+; (2) PTH increases Cd2+ uptake into MDCK cells; and (3) Cd2+ entry in kidney epithelial cells is mediated, at least in part, by dihydropyridine-sensitive calcium channels.     

Cadmium in milk and mammary gland in rats and mice

The purpose of the present investigation was to study the uptake of cadmium in mammary tissue, effects on milk secretion and composition, and lactational transport of cadmium to the sucklings. Cadmium exposure during lactation resulted in retention of cadmium in the mammary tissue in mice and rats. The uptake of cadmium in the mammary tissue was rapid, as shown in lactating mice by whole-body autoradiography 4 h after an intravenous injection of a tracer dose of (109)CdCl(2). Retention of cadmium in kidneys of suckling pups was observed in the autoradiograms at 7 days after exposure of the dams. Lactating rats were intravenously infused with (109)CdCl(2) in 0.9% saline via osmotic minipumps from day 3 to day 16 after parturition. The cadmium dose given was 0, 8.8, 62 and 300 microg Cd/kg body wt. per day. Plasma and milk were collected at day 10 and 16 after parturition. Plasma cadmium levels in dams increased from day 10 to day 16. Cadmium levels were higher in milk than in plasma, with milk/plasma ratios varying from 2 to 6. Zinc levels in milk were positively correlated to cadmium levels in milk (r(2)=0.26; P=0. 03). In milk, (109)Cd was distributed in fat (46-52%), casein fraction (40-46%), and whey fraction (6-8%). There was a high correlation between cadmium concentrations in pups' kidney and cadmium concentrations in dam's milk (r(2)=0.98; P < 0.001). Of the cadmium dose given to the dams <0.05% was retained in the litters on day 16 of lactation. No effects were observed due to cadmium exposure on body weight in pups or dams. Cadmium treatment did not cause any effect on the lactose or protein concentration in milk, the concentrations of DNA, RNA or the ratio RNA/DNA in the mammary gland. Histological evaluation of mammary tissue did not reveal any abnormalities at any dose level. (109)Cd was bound to metallothionein in mammary tissue. The fraction of radiolabelled cadmium bound to metallothionein increased in a dose-dependent manner in both the liver (88-98%) and mammary tissue (57-80%). The present results indicate a low transfer of cadmium to the suckling pup, which might be due to binding of cadmium to metallothionein in the mammary tissue. However, during the susceptible developmental period even a low cadmium exposure may be of concern.     

Cadmium absorption and its relationship to divalent metal transporter-1 in the pregnant rat

Increased intestinal absorption of essential nutrients is characteristic of pregnancy as the maternal gastrointestinal tract undergoes physiological and biochemical changes to accommodate the increased demand for essential nutrients by the fetus. Divalent metal transporter-1 (DMT-1) is primarily responsible for dietary iron uptake in the duodenum but also recognizes nonessential metals such as cadmium (Cd). Increased absorption of Cd has been reported in pregnant compared with nonpregnant mice; however, the mechanism is not understood. The purpose of this work was to determine whether Cd absorption is increased in pregnant compared with nonpregnant rats and whether this correlates with a time-dependent up-regulation of DMT-1 expression. Timed pregnant and nonpregnant female Sprague-Dawley rats were administered (109)Cd-labeled CdCl(2) by oral gavage on gestation day (gd) 19. Tissues were collected on gd 20 for (109)Cd assessment (values expressed as pmol Cd). Greater accumulation of (109)Cd was observed in duodenum than in jejunum and ileum in both pregnant and nonpregnant rats. However, the amount of Cd in small intestine was higher in pregnant than nonpregnant rats. Additionally, more Cd accumulated in the liver and kidney of pregnant than nonpregnant rats. DMT-1 mRNA levels were determined in duodenum, placenta, liver, and kidney with branched DNA signal amplification. DMT-1 mRNA levels were about sixfold higher in duodenum of (gd 21) pregnant than nonpregnant rats and the levels in pregnant rats increased from gd 15 through gd 21. The correlation between Cd absorption and DMT-1 expression observed in pregnant rats suggests a role for DMT-1 in the increased absorption of Cd during pregnancy.