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.     

Effect of cadmium on membrane potential in isolated rat hepatocytes

The effect of cadmium (Cd) on rat hepatocytes upon short term exposure was studied by focusing on the integrity of mitochondria and on the possible consequences of its disturbance, such as alterations in plasma membrane potential and loss of cell viability. Changes in the potential of mitochondrion and plasma membranes were monitored using [3H]triphenylmethylphosphonium (TPMP+) and [14C]SCN- probes, respectively. Isolated rat hepatocytes were exposed to increasing CdCl2 concentrations for short time periods (30-120 min). Cd measurement by atomic absorption showed that the cells efficiently accumulated Cd, as did mitochondria in situ. In CdCl2-treated cultures, it was observed that the release of TPMP+, which revealed a drop in the mitochondrial membrane potential, was time- and concentration-dependent, and that the first significant efflux was caused by a 30-min exposure to 89 microM CdCl2. No significant change in plasma membrane potential, as judged from the increase in the uptake of SCN-, was detected after 30 min, suggesting the greater precocity of the mitochondrial attack. Finally, the release of lactate dehydrogenase (LDH) occurred only after 2 h of exposure, reflecting ultimate stages of cell injury induced by Cd. These results suggest that Cd induces an alteration in mitochondrial function in hepatocytes which may lead to the loss of plasma membrane potential and cell viability. The study therefore adds further evidence of the role of mitochondria as primary targets in Cd-induced cytotoxicity.     

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.     

Induction of metallothionein by cadmium-metallothionein in rat liver: a proposed mechanism.

Distribution of Cd to various organs following iv administration of CdCl2 (3.5 mg Cd/kg) resulted in more than 43% of total tissue Cd accumulating in the liver. In contrast, after CdMT administration (0.5 mg Cd/kg), only 1% of the Cd was found in liver. Rats administered CdCl2 (1.0 mg Cd/kg) had hepatic MT values 30-fold greater than controls and a hepatic Cd concentration of 17 micrograms/g. In comparison, rats treated with CdMT (0.4 mg Cd/kg) had hepatic MT concentrations 7-fold greater than controls and a hepatic Cd concentration of 0.80 micrograms/g. However, when hepatic MT levels were normalized to tissue Cd concentrations, induction of MT by CdMT was 5-fold greater than by CdCl2. Northern and slot-blot analyses of mRNA showed that both CdCl2 and CdMT coordinately increased MT mRNA. These data suggest that both CdMT and CdCl2 increase hepatic MT by similar mechanisms. A dose-response increase in MT produced by CdCl2 indicated a biphasic response, with low doses producing relatively more hepatic MT than higher doses. In addition, the amount of MT produced per unit Cd after CdMT treatment was similar to those observed after low doses of CdCl2 in the dose-response experiment. These data provide strong evidence to support the conclusion that the apparent potency of CdMT observed here and in previous studies is most likely due to the small amount of Cd distributed to the liver, which is relatively more effective in inducing MT than are higher concentrations.     

Distribution of cadmium chloride and cadmium-metallothionein to liver parenchymal, Kupffer, and endothelial cells: their relative ability to express metallothionein.

Acute exposure to cadmium (Cd) salts results in liver toxicity, while administration of cadmium-metallothionein (CdMT) iv, causes renal damage. When CdMT is administered iv there is a rapid accumulation of Cd in the proximal tubule cells of the kidney. In comparison, only small amounts of Cd accumulate in the liver following administration of CdMT. Thus, in order to better understand the regulation of MT as well as the toxicity of Cd, the present study has examined the ability of each of the three primary liver cells, parenchymal (PC), Kupffer (KC), and endothelial (EC), to accrue Cd after administration of either inorganic or organic forms of Cd. In addition, the relative ability of each cell type to express metallothionein (MT) mRNA and protein was examined. Following CdCl2 (3.5 mg Cd/kg) treatment, Cd concentrations increased to about the same degree in PC and KC, but EC had about 2-fold more than PC. After administration of CdCl2 (1.0 mg Cd/kg) each cell responded to the presence of Cd by increasing intracellular MT mRNA and protein. However, PC showed the greatest response, with a 30-fold increase in mRNA and a 21-fold increase in protein. Interestingly, KC and EC possessed intracellular Cd concentrations equal to or greater than that of PC, but contained less MT than would have been expected on the basis of their intracellular Cd concentrations. Thus, KC had a 7-fold increase in MT mRNA and a 2-fold increase in protein, while EC increased mRNA 3-fold and protein 2-fold over control values. In contrast, following CdMT (0.5 mg Cd/kg) administration, only low levels of Cd were detected, with similar concentrations in each cell type. After administration of CdMT (0.4 mg Cd/kg), PC again showed the greatest response, with a 3-fold increase in mRNA and a 6-fold increase in MT protein. Only slight changes were observed in KC and EC. In conclusion, the present study has shown the following: (1) Endogenous levels of MT in KC and EC are higher than those in PC. (2) Cd is readily accumulated by all three cell types, when administered as CdCl2, but not when given as CdMT. (3) PC, KC, and EC are capable of responding to intracellular Cd by increasing MT.     

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.     

Comparative toxicity and accumulation of cadmium chloride and cadmium-metallothionein in primary cells and cell lines of rat intestine, liver and kidney

The protective role of metallothionein (Mt) in the toxicity of cadmium (Cd) is controversial, since Cd bound to Mt is more nephrotoxic than ionic Cd after parenteral exposure and less hepatotoxic than ionic Cd after oral exposure. This study compared the uptake and toxicity in vitro of CdCl₂ and two isoforms of rat cadmium-metallothionein (CdMt-1 and CdMt-2) using primary rat kidney cortex cells, primary rat hepatocytes, liver hepatoma cell line H-35, kidney epithelial cell line NRK52-E and intestinal epithelial cell line IEC-18. The molar ratio of Cd was 2.1 and 1.4 mol Cd/mol Mt for CdMt-1 and CdMt-2, respectively. Monolayer cultures were incubated for 22 hr with CdCl₂, CdMt-1 or CdMt-2 and Cd accumulation was examined at Cd levels of 0.25–10 μM-Cd. Cells exposed to CdCl₂ accumulated more Cd in 22 hr than cells exposed to an equimolar amount of CdMt. For CdCl₂ the Cd accumulation is directly related to the Cd concentration in the medium; however, for CdMt an increase in Cd concentration in the medium above 2 μM had no effect on the Cd accumulation in the cells. At Cd concentrations above 2 μM, therefore, the difference in Cd accumulation between CdCl₂ and CdMt was greater (5–6 times) than at concentrations below 2 μM (1–2 times). Cytotoxicity was examined in the Cd-concentration range from 0.25 to 100 μM by determining the lactate dehydrogenase (LDH) release in the medium and the neutral red uptake in the cells. Under these culture conditions CdCl₂ was at least 100 times more toxic than CdMt-1 or CdMt-2 in all cell types tested. Primary hepatocyte cultures were 10 times more sensitive (50% LDH release at 1–2 μM) to CdCl₂ intoxication than primary cultures of renal cortical cells or the intestinal cell line (50% LDH release at 10–20 μM). Hepatic and renal cell lines were less sensitive (50% LDH release at 20–35 μM) than the corresponding primary cultures. No difference in sensitivity towards CdMt-1 or CdMt-2 was found for the various cell types tested. To investigate the influence of the molar Cd ratio of CdMt on cytotoxicity, the Cd content of CdMt-1 (2.1 mol Cd/mol Mt) was artificially raised in vitro to 5 mol/mol Mt. Compared with native CdMt, CdMt with a high molar Cd ratio in primary renal cultures showed a 15% increase in LDH release at a Cd concentration of 1500 μM in the medium. In conclusion, exogenous CdMt is far less toxic than CdCl₂ to cell cultures in a serum-free medium. Whereas CdCl₂ in all cases showed dose-dependent Cd accumulation, Cd accumulation due to CdMt exposure in all cell types tested reached a plateau at medium Cd concentrations of 2 μM. The low cellular Cd uptake of CdMt and the corresponding low cytotoxicity supports previously reported results in vivo, showing that the difference in toxicity between CdMt and CdCl₂ is associated with a difference in Cd distribution.     

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.     

Cadmium uptake kinetics in rat hepatocytes: correction for albumin binding.

The relationship between cytotoxicity and kinetics of cadmium uptake was investigated in primary rat hepatocyte cultures. Primary rat hepatocytes were exposed to cadmium concentrations ranging from 1.0 to 80 micro M in albumin-free buffer or 32 to 8,000 microM in buffer containing physiological concentrations of bovine serum albumin (600 micro M) for 1 h, and cellular toxicity was observed at 23 h postexposure. Hepatocytes exposed to cadmium in the presence of albumin appeared less sensitive to cadmium toxicity when compared to cells exposed in the absence of albumin. The experimentally derived 23-h postexposure EC(50)s for hepatocytes exposed to cadmium in both presence and absence of albumin was 65.5 +/- 2.4 and 14.3 +/- 3.9 microM, respectively. A Scatchard plot of cadmium binding to albumin suggested two high-affinity binding sites. The observed uptake of cadmium by hepatocytes in the absence and presence of albumin consisted of a composite fast uptake rate and cell membrane association (Component I), and a slow, sustained uptake rate (Component II). Cadmium uptake rates in hepatocytes, based on total medium cadmium concentrations, indicated that Component II uptake rates were four times faster under albumin-free exposure conditions. However, when uptake rates were evaluated, based on the calculated equilibrium concentration of free cadmium in the exposure buffer, uptake rates in hepatocytes exposed in the presence of albumin were two times as fast. This faster cadmium uptake in the presence of albumin may result from diffusion-limited, nonequilibrium conditions occurring at the cell surface.     

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.     

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 cytotoxic effects of cadmium chloride and mercuric chloride mixtures in rat primary hepatocyte cultures

The toxic effects of Cd and Hg mixtures were studied using primary monolayer cultures of rat hepatocytes. Cytotoxicity was assessed by measuring the release of lactic dehydrogenase from the cells. Cytotoxic and non-cytotoxic metal levels were used. At the higher exposure concentrations (0.2 micrograms Cd.ml-1 and 2.0 micrograms Hg.ml-1), Cd was very toxic to hepatocytes whereas Hg was only marginally toxic. The combination of Cd and Hg was more toxic than predicted by summation of the individual metal toxicities. The incorporation of [35S]cysteine into protein of the cytosol and insoluble cell fraction was increased in response to Cd or Hg exposure and was directly related to cell 35S accumulation. Combinations of Cd and Hg significantly increased the proportion of total 35S which was incorporated in cell protein, an effect that was attributed to the accumulation of protein in the insoluble cell fraction. Cd uptake by hepatocytes was related to exposure concentration but was lower when Hg was also present in the incubation medium. Gel chromatography of the cytosol from Cd-exposed cells showed 3 Cd containing fractions which corresponded to the elution positions of high Mr proteins, metallothionein (MT) and low Mr molecules. When hepatocytes were exposed to Hg in combination with Cd, the MT-like fraction was no longer evident and Cd in the low Mr fraction was greatly reduced. Regardless of the presence or absence of Cd in the exposure medium, 98% of cytosol Hg in Hg-exposed cells was found to elute after the low Mr fraction, at a position equivalent to inorganic salts. This indicates that the enhanced cytotoxicity of Cd and Hg may be related to a decrease in the MT-like protein in the cytosol and not due to a direct competitive binding interaction in relation to the protein.     

Metallothionein protects against the nephrotoxicity produced by chronic CdMT exposure.

Metallothionein (MT) is a low-molecular-weight, cysteine-rich, metal-binding protein. Induction of MT has been proposed to be an important adaptive mechanism in decreasing Cd toxicity. MT has been shown to protect against CdCl2-induced lethality and hepatotoxicity; however, MT does not protect against acute CdMT-induced nephrotoxicity. This study was aimed at clarifying the role of metallothionein in chronic CdMT-induced renal injury. Wild type and MT-I/II knockout (MT-null) mice were therefore given sc injections of CdMT (25 and 100 microg Cd/kg) or saline daily, 6 times/week for 6 weeks, and renal injury was evaluated. Multiple injections of CdMT to wild-type mice resulted in renal Cd concentrations up to 120 microg/g kidney, along with a 100-fold increase in renal MT (450 microg/g kidney). In contrast, renal Cd concentration in MT-null mice administered multiple injections of CdMT reached a much lower level than in wild-type mice (<10 microg/g kidney). Although less Cd accumulated in their kidneys, MT-null mice were more susceptible than wild-type mice to CdMT-induced nephrotoxicity, as indicated by increased urinary excretion of protein and N-acetyl-beta-D-glucosaminidase, as well as by elevated blood urea nitrogen levels. At the higher daily dose of CdMT (100 microg Cd/kg), kidneys of MT-null mice were enlarged. Chronic CdMT administration eventually damaged the entire kidney, which included glomerular swelling, interstitial inflammation, edema, tubular cell degeneration, and atrophy. In contrast to a single injection of CdMT that produces proximal tubular necrosis, chronic injection of CdMT results in tubular cell apoptosis in both wild-type and MT-null mice. These data indicate that chronic CdMT administration produces similar renal injury to that observed after chronic CdCl2 administration, and that intracellular MT protects against nephrotoxicity produced by chronic CdMT administration.     

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.     

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.     

Cellular response after mobilization of metals by diethyldithiocarbamate in rat hepatocyte cultures

Cellular effects and mobilization of metals by diethyldithiocarbamate (DTC) were studied in primary cultures of rat hepatocytes incubated with lead acetate (PbAc), lead-diethyldithiocarbamate (Pb(DTC)2), cadmium chloride (CdCl2), cadmium-diethyldithiocarbamate (Cd(DTC)2), mercuric acetate (HgAc) and methylmercuric chloride (MeHgCl). In cells pretreated with inorganic Pb, Cd and Hg, the cellular levels of Cd and Pb were somewhat decreased or unchanged after incubation with DTC, while the cellular concentration of Hg was increased. Cells preincubated with MeHgCl showed a marked decrease in cellular Hg concentration upon DTC treatment. In Pb(DTC)2-treated cells the Pb concentration was increased when incubated with DTC, while DTC caused a decrease in Cd concentrations of cells preincubated with Cd(DTC)2. The activity of alcohol dehydrogenase (ADH) in cells incubated with CdCl2, Cd(DTC)2 and HgAc was significantly decreased. In Hg-treated cells the ADH activity was further decreased after incubation with DTC, whereas the activity in Cd-treated cells recovered gradually after incubation with increasing concentrations of DTC. The activity of the enzyme delta-aminolevulinic acid dehydratase (ALAD) was significantly inhibited in cells treated with PbAc and Pb(DTC)2, but could be restored to 80% and to almost 100%, respectively, of control activity after incubation with DTC. It is suggested that, in the absence of excess DTC, a decomposition of Pb(DTC)2 takes place after penetration into the cell, resulting in inhibition of ALAD by the released Pb. In the presence of excessive amounts of DTC, the equilibrium is shifted towards Pb(DTC)2 and Pb in the complex form is not available for ALAD. These studies suggest that DTC decreases cellular effects of Pb and Cd despite unchanged or even increased cellular concentrations of the metals, while the antidotal efficacy of DTC on inorganic Hg toxicity seems to be of low value.     

Renal cortical mitochondrial dysfunction upon cadmium metallothionein administration to Sprague-Dawley rats

A bolus dose of cadmium metallothionein (CdMT) produces renal proximal tubular dysfunction because it accumulates in the tubular epithelial cells and undergoes rapid degradation, releasing Cd. Morphologically, mitochondria appear to be the target organelle. The present study examined changes in renal cortical mitochondrial function following CdMT administration and investigated whether some of these effects could be ascribed to Cd2+ accumulation in the mitochondria. Sprague-Dawley rats were injected ip with 0.3 mg Cd as CdMT/kg and the animals were sacrificed after 6, 8, or 12 h. Two- to threefold increases in urinary protein excretion and LDH activity were evident at 8 h, with marked elevations (11- and 29-fold) thereafter. Renal cortical mitochondria were swollen and rounded at 12 h. The mitochondrial Cd level was 399 pmol/mg protein at 6 h and did not change significantly during the next 6 h; however, mitochondrial respiratory function declined with time. At 12 h, state 3 oxygen consumption, respiratory control ratio (RCR), and ADP:O (P/O) ratio were 48, 49, and 76% of control values, respectively, indicating inhibition of electron transfer and oxidative phosphorylation. The direct effect of Cd on mitochondrial function was examined by incubating mitochondria from untreated rats with 0.1-2 microM CdCl2. Rapid uptake of Cd resulted in concentration-dependent effects on respiration. After 1 min of incubation with 2 microM Cd, the mitochondria contained 262 microgCd/mg protein and state 3 respiration and RCR values were 75 and 33% of control levels, respectively. Thus, renal proximal tubular cell damage following a bolus dose of CdMT involves perturbations in mitochondrial respiration, brought on by the accumulation of Cd.     

Cytotoxicity related changes in biochemical cell function following in vitro cadmium treatment

In an effort to examine cellular responses to cadmium insult a bovine kidney cell line was used to monitor select cell functions for toxicity related alterations. Cadmium concentrations used ranged between 0.2 and 2.5 microM CdCl2 and elicited 0-85% cytotoxicity (cell attachment); 24-h incubations were used for all studies. Toxicity related inhibition of leucine incorporation into cellular protein and thymidine incorporation into DNA was noted. Decreases in protein synthesis activity closely paralleled the cytotoxicity profile; DNA synthesis was a less sensitive indicator to toxicity. K+-dependent phosphatase (KP), acid phosphatase (AP) and succinate dehydrogenase (SDH) were monitored in surviving cells and in a cell-free system. Significant inhibitions were detected for all enzyme activities following a 24 h culture with cadmium. KP and AP were most sensitive. In the cell-free system KP was significantly inhibited with 0.1 microM cadmium; AP and SDH were either unchanged or sensitive only at concentrations of 100 microM cadmium or greater. Reduced glutathione (GSH) concentration in surviving cells was elevated up to 7-fold over control cultures. The elevation occurred in a progressive toxicity-related manner.     

Changes in expression of fibrotic markers and histopathological alterations in kidneys of mice chronically exposed to low and high Cd doses

The main target organ for cadmium (Cd) is the kidney, and more specifically the proximal tubular cells. Little is known about the effects of a long-term Cd exposure on the ultrastructure of the kidney and the involvement in tubulointerstitial fibrosis. Therefore, mice were exposed to Cd concentrations varying from 10 to 500 mg CdCl(2)/l in the drinking water during 4, 16 and 23 weeks. Ultrastructural changes were studied by means of light- and electron microscopical analyses. Furthermore, the expression of the extracellular matrix (ECM) proteins collagen I and fibronectin, and the myofibroblast/epithelial-to-mesenchymal transition (EMT) marker alfa-smooth muscle actin (alpha-SMA) were studied by means of immunohistochemistry. The histopathological changes caused by Cd varied considerably from one animal to another, and from one individual cell to another. An exposure to Cd concentrations up to 100mg CdCl(2)/l elicited only minor changes that were restricted to increasing amounts of lysosomes and vacuolisation. When higher Cd concentrations were applied, the changes became more pronounced and featured mitochondrial damage, cellular swelling and loss of basal invaginations. An overproduction of the interstitial matrix component fibronectin and the expression of the myofibroblasts/EMT marker alpha-SMA in kidneys of mice exposed to 100mg CdCl(2)/l clearly indicated that an exposure to relatively low Cd doses might lead ultimately to renal fibrosis. Increasing the Cd dose (up to 500 mg CdCl(2)/l) evoked an increased immunoreactivity for fibrotic markers. In conclusion we may state that concentrations up to 100mg CdCl(2)/l evoked minor changes, although the expression of fibrotic markers was increased. Changes became more pronounced when exposing to higher Cd concentrations.     

Effect of cadmium and colcemide on the mitoses of a human epithelial cell line with high content of cytoplasmic metallothionein

Cultures of cell strains with and without metallothionein were exposed to CdCl2 in doses ranging from 10 mu mol/l to 200 mu mol/l . Cell growth parameters were monitored by flow cytometric DNA-measurements, cell counts and counting of mitoses during the first two days after exposure. CdCl2 inhibited cell growth in a dose dependent way. The cadmium resistant cells were inhibited with concentrations above 100 mu mol/l, the concentration which the metallothionein-containing cells had previously been adjusted to. Microscopy of the cell cultures showed a dose dependent accumulation of cells in the mitotic prophase, whereas the other phases of the cell cycle were unaffected as measured by flow cytometry. When exposed to colcemide, however, the two cell strains showed identical responsiveness.