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.     

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.     

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.     

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.     

Detection of cadmium exposure in rats by induction of lymphocyte metallothionein gene expression.

The induction of metallothionein (MT) gene expression in lymphocytes of rats was determined in order to detect exposure in vivo to cadmium. Both acute and chronic CdCl2 exposures resulted in the induction of the MT-1 gene in lymphocytes as measured by standard RNA Northern blot analysis. Twenty-four hours following an ip injection of 3.4 mg/kg CdCl2, a ninefold increase in MT gene expression was observed in lymphocytes, as well as five- and sevenfold increases in liver and kidney, respectively. Oral exposure of rats to 1-100 ppm CdCl2 via drinking water resulted in an approximate twofold enhanced MT signal in lymphocytes after 6 wk, and a threefold increase after 13 wk of exposure to 100 ppm Cd. No increases in lymphocyte MT gene expression were observed after 3 wk of Cd exposure. Liver MT gene expression was substantially induced following chronic Cd exposure, while kidney was not, although this organ had a higher basal expression of the MT-1 gene. Analysis of tissue Cd burdens demonstrated a dose-response Cd accumulation in liver and kidney, but only kidney burdens increased substantially with prolonged Cd exposure. These results demonstrate the utility of lymphocyte gene expression assays to detect in vivo toxicant exposure, and thus their applicability as molecular biomarker assays for human exposure assessment.     

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.     

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.     

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.     

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.     

Metallothionein-I/II null mice are sensitive to chronic oral cadmium-induced nephrotoxicity.

Chronic exposure to cadmium (Cd) via food and drinking water is a major human health concern. We have previously shown that metallothionein (MT), a metal-binding protein, plays an important role in protecting against Cd toxicity produced by repeated sc injections. However, it is unclear whether MT protects against Cd-induced nephrotoxicity following chronic oral exposure, a route with obvious human relevance. To clarify this issue, MT-I/II knockout (MT-null) and background-matched wild-type (WT) mice were allowed free access to drinking water containing CdCl(2) (30, 100, and 300 ppm Cd), or feed containing CdCl(2) (100 ppm Cd) for 6 months, and the resultant nephrotoxicity was examined. Chronic oral Cd exposure produced a dose-dependent accumulation of Cd in liver and kidney of WT mice, reaching levels up to 50 microg Cd/g tissue. Immunohistological localization of renal MT indicated that chronic oral Cd exposure in WT mice greatly increased MT in the proximal tubules and the medulla, with cellular localization in both the cytoplasm and nuclei. As expected, no MT was detected in kidneys of MT-null mice. After 6 months of Cd exposure, tissue Cd concentrations in MT-null mice were only about one-fifth of that in WT mice. Even though the renal Cd concentrations were much lower in the MT-null mice, they were more sensitive than WT mice to Cd-induced renal injury, as evidenced by more severe nephropathic lesions, increased urinary excretion of gamma-glutamyl-transferase and glucose, and elevated blood urea nitrogen. Six months of Cd exposure to MT-null animals resulted in greater increases in renal caspase-3 activity, an indicator of apoptosis, than to WT mice. In conclusion, this study demonstrates that lack of MT renders MT-null mice vulnerable to Cd-induced nephrotoxicity after chronic oral exposure, the primary route of human Cd exposure.     

Metallothionein-null mice are more sensitive than wild-type mice to liver injury induced by repeated exposure to cadmium.

Liver is a major target organ of cadmium (Cd) toxicity following acute and chronic exposure. Metallothionein (MT), a low-molecular-weight, cysteine-rich, metal-binding protein has been shown to play an important role in protection against acute Cd-induced liver injury. This study investigates the role of MT in liver injury induced by repeated exposure to Cd. Wild-type and MT-I/II knockout (MT I/II-null) mice were injected sc with a wide range of CdCl(2) doses, 6 times/week, for up to 10 weeks, and their hepatic Cd content, hepatic MT concentration, and liver injury were examined. Repeated administration of CdCl(2) produced acute and nonspecific chronic inflammation in the parenchyma and portal tracts and around central veins. Higher doses produced granulomatous inflammation and proliferating nodules in liver parenchyma. Apoptosis and mitosis occurred concomitantly in liver following repeated Cd exposure, whereas necrosis was mild. As a result, significant elevation of serum enzyme levels was not observed. In wild-type mice, hepatic Cd concentration increased in a dose- and time-dependent manner, reaching 400 microgram/g liver, along with 150-fold increases in hepatic MT concentrations, the latter reaching 1200 microgram/g liver. In contrast, in MT I/II-null mice, hepatic Cd concentrations were about 10 microgram/g liver. Despite the lower accumulation of Cd in livers of MT I/II-null mice, the maximum tolerated dose of Cd was one-eighth lower than that for wild-type mice at 10 weeks, and liver injury was more pronounced in the MT I/II-null mice, as evidenced by increases in liver/body weight ratios and histopathological analyses. In conclusion, these data indicate that (1) nonspecific chronic inflammation, granulomatous inflammation, apoptosis, liver cell regeneration, and presumably, preneoplastic proliferating nodules are major features of liver injury induced by repeated Cd exposure, and (2) intracellular MT is an important protein protecting against this Cd-induced liver injury.     

Protection by Zinc-Metallothionein (ZnMT) against Cadmium-Metallothionein-Induced Nephrotoxicity

In contrast to inorganic Cd, acute iv administration of Cd boundto metallothionein (CdMT) concentrates in renal tissue. Thisuptake of CdMT produces functional and morphological changesin kidneys, similar to those observed after chronic exposureto inorganic Cd. In order to examine the importance of the metalcomponent of MT in the renal uptake of MT, the renal concentrationof ³⁵S after administration of [³⁵S]ZnMT and [³⁵S]CdMT was compared.Renal uptake of ³⁵S from both CdMT and ZnMT was very rapid,with peak concentrations observed 15–30 min after administration.³⁵S in kidneys increased in a dose-dependent manner after administrationof various doses of [³⁵S]ZnMT, up to 1.3 µmole MT/kg;however, higher doses did not further increase renal ³⁵S concentrations.A similar saturation of ³⁵S reabsorption was observed for therenal uptake of [³⁵S]CdMT. CdMT produced renal injury with dosesas low as 0.26 µmol MT/kg (0.2 mg Cd/kg). In contrast,with a dose of ZnMT as high as 5.12 µmol MT/kg (2 mg Zn/kg),no histopathological changes were observed. Therefore, ZnMTappears to be nontoxic even though ZnMT delivers more MT tothe kidney than does CdMT. Because ZnMT and CdMT are apparentlyhandled by the same renal transport mechanism, the effects ofZnMT on ¹⁰⁹CdMT renal uptake and nephrotoxicity were determined.One group of mice was given a nephrotoxic dose of ¹⁰⁹CdMT (0.51µmol MT/kg containing 0.4 mg Cd/kg, iv), and the othergroup received an equimolar dose of unlabeled ZnMT 1 min before¹⁰⁹CdMT administration. Renal function was evaluated by measuringurinary glucose and protein excretion, as well as histopathology.Marked renal toxicity was observed 24 hr after ¹⁰⁹CdMT administration.In contrast, renal function appeared normal in mice receivingZnMT before ¹⁰⁹CdMT. However, a similar concentration of ¹⁰⁹Cdwas found in kidneys of both groups. The present results demonstratethat ZnMT is not only nontoxic to the kidney at a dose as highas 5 µmol MT/kg, but can also protect against the nephrotoxiceffect of CdMT without decreasing renal Cd concentration.     

Accumulation and degradation of the protein moiety of cadmium-metallothionein (CdMT) in the mouse kidney.

Of major concern in Cd toxicity is its ability to produce renal damage after chronic exposure in humans and experimental animals. Renal injury affects predominantly the proximal tubules and more specifically the first segments of these tubules. Similar toxic effects to the kidneys are observed after administration of cadmium bound to metallothionein (CdMT). Therefore, CdMT was used in this study as a model to understand the mechanism(s) of Cd nephrotoxicity. It has been recently demonstrated that Cd from CdMT was preferentially taken up by the proximal convoluted tubules. Therefore, the purpose of these studies was to determine if the organic portion of the complex was also accumulated in these tubules. [35S]CdMT prepared from rat liver was administered intravenously to mice at a nonnephrotoxic dose (0.1 mg Cd/kg). The radioactivity in the kidney showed maximum level (80% of the dose) 15 min after the injection. This preferential renal uptake was also observed after administration of various doses of [35S]CdMT. In contrast to the earlier observed persistency of 109Cd in the kidney after 109CdMT administration, 35S disappeared rapidly (with a half-life of approximately 2 hr), and 24 hr after injection of [35S]CdMT, there was very little 35S left in the kidneys. These observations indicate that the protein portion of CdMT is rapidly degraded after renal uptake of CdMT and the released Cd is retained in the kidney. Within the kidney, 35S distributed mainly to the cortex. Light microscopic autoradiography showed that [35S]CdMT preferentially distributed to the proximal convoluted tubule (S1 and S2), which is the site of nephrotoxicity. Within the S1 and S2 segments, a greater distribution of 35S to the apical portion of the cells was observed after administration of both a nonnephrotoxic (0.1 mg Cd/kg) and a nephrotoxic (0.3 mg Cd/kg) dose. 109Cd administered as 109CdMT also distributed to the apical portion of the S1 and S2 cells. Therefore, both the organic (35S) and inorganic (109Cd) portions of CdMT are rapidly and efficiently taken up by the S1 and S2 cells of the proximal tubules, the site of nephrotoxicity. These observations support the concept that CdMT is readily taken up by the proximal tubular cells as a complex, and then its protein portion is rapidly degraded to release Cd that binds permanently to intracellular sites and produces nephrotoxicity.     

Susceptibility of MT-Null Mice to Chronic CdCl2-Induced Nephrotoxicity Indicates That Renal Injury Is Not Mediated by the CdMT Complex

Chronic human exposure to Cd results in kidney injury. It hasbeen proposed that nephrotoxicity produced by chronic Cd exposureis via the Cd-metallothionein complex (CdMT) and not by inorganicforms of Cd. If this hypothesis is correct, then MT-null mice,which cannot form CdMT, should not develop nephrotoxicity. Controland MT-null mice were injected sc with a wide range of CdCl₂doses, six times/week for up to 10 weeks, and their renal Cdburden, renal MT concentration, and nephrotoxicity were quantified.In control mice, renal Cd burden increased in a dose-and time-dependentmanner, reaching as high as 140 µg Cd/g kidney, alongwith 150-fold increases in renal MT concentrations, reaching800 µg MT/g kidney. In MT-null mice, renal Cd concentration(10 µg/g) was much lower, and renal MT was nonexistent.The maximum tolerated dose of Cd in MT-null mice was approximatelyone-eighth that of controls. MT-null mice were more susceptiblethan controls to Cd-induced renal injury, as evidenced by increasedurinary excretion of protein, glucose, -glutamyl-transferase,and N-acetyl-ß-D-glucosaminidase, as well as by increasedblood urea nitrogen levels. Kidneys of Cd-treated mice wereenlarged and histopathology showed various types of lesions,including proximal tubular degeneration, apoptosis, atrophy,interstitial inflammation, and glomerular swelling. These lesionswere more severe in MT-null than in control mice, mirroringthe biochemical analyses. These data indicate that Cd-inducedrenal injury is not necessarily mediated through the CdMT complexand that MT is an important intracellular protein in protectingagainst chronic Cd nephrotoxicity.     

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.     

Cadmium-induced hepatic and renal injury in chronically exposed rats: likely role of hepatic cadmium-metallothionein in nephrotoxicity

Rats were injected sc with 0.5 mg Cd/kg, 6 days/week, for up to 26 weeks. Hepatic and renal function and tissue Cd and metallothionein (MT) content were determined in tissues and plasma at various times after Cd injection. Cd in liver and kidney increased linearly for the first 10 weeks of treatment, but thereafter hepatic concentrations of Cd decreased by 33% whereas the content of Cd in kidney remained constant. MT in liver and kidney increased linearly during the first 12 weeks of Cd treatment to 4400 and 2300 micrograms MT/g, respectively, but rose only slightly thereafter. Circulating concentrations of MT progressively increased beginning 2 weeks after Cd treatment and were approximately 10 times control values in rats dosed with Cd for 12 or more weeks. Plasma activities of alanine and aspartate aminotransferase exhibited a time course similar to that observed with MT, and were elevated as early as the sixth week of Cd exposure. Sharp increases in activities of these enzymes also occurred after 10 to 12 weeks of dosing. Hepatic microsomal metabolism of benzo[a]pyrene and ethylmorphine was severely attenuated beginning 4 weeks after Cd. Renal injury occurred after hepatic damage, as evidenced by decreased in vitro p-aminohippuric acid uptake beginning 8 weeks after exposure. Urine outflow increased threefold 11 weeks after Cd exposure began, while urinary protein and Cd excretion increased beginning at Week 9. These data indicate the liver is a major target organ of chronic Cd poisoning, and suggest that Cd-induced hepatic injury, via release of Cd-MT, may play an important role in the nephrotoxicity observed in response to long-term exposure to Cd.     

Early changes in the tissue distribution of cadmium after oral but not intravenous cadmium exposure

The kinetics of 109Cd distribution in tissues of male and female mice were measured at intervals of 5 min to 15 days after oral (100 micrograms Cd/kg; by gavage) or intravenous (1 micrograms Cd/kg; i.v.) administration of 109CdCl2. Unexpectedly, the ratio of 109Cd in liver to that in kidneys was greater than or equal to 10 within 1 h after administration by either route. However, after 4 h, route-dependent differences in distribution between liver and kidney became apparent. In mice receiving oral cadmium, the liver:kidney 109Cd ratio decreased with time to approximately 4 at 72 h after gavage. In contrast, in mice receiving IV cadmium, the liver:kidney 109Cd ratio remained high and relatively constant during the same time period. The time-dependent decrease in the liver:kidney 109Cd ratio after oral cadmium administration was caused by a 4-5-fold increase in cadmium content of the kidney that occurred between 30 min and 72 h after oral but not i.v. administration. During this time, there was no change in cadmium distribution in subcellular fractions of either liver or kidney. These results could be explained by the existence of 2 separate pathways for cadmium deposition after oral exposure. Early after exposure, cadmium may leave the intestine, bind to serum albumins or other high molecular weight proteins, and accumulate primarily in liver, as is also observed after IV cadmium administration. With time, cadmium may leave the intestinal mucosa bound to metallothionein and deposit primarily in the kidney. The different pathways of deposition after oral vs. i.v. exposure may in part explain why acute parenteral cadmium exposure causes liver toxicity, but chronic oral exposure causes renal toxicity.     

Distribution of cadmium after oral administration of cadmium-thionein to mice

Distribution of Cd was compared after oral administration of either Cd ions or Cd-thionein (Cd-TH). Mice received 0.5 mg Cd/kg, po as CdCl2 in saline, CdCl2 in control rat liver homogenate, Cd-TH in saline, Cd-TH in liver homogenate, or liver homogenate from Cd-treated rats. In all cases, 85-90% of the Cd dose was present in feces within 24 hr. However, in groups receiving CdCl2, more Cd was found in feces on Days 2 and 3 in comparison to those receiving Cd-TH. All treatments resulted in lower levels of Cd in liver than in kidney. In addition, tissue levels indicate that less Cd was absorbed when rats received Cd-TH in saline than CdCl2 in saline. Cd-TH added to liver homogenate or liver homogenate containing Cd-TH increased the absorption of Cd resulting in renal Cd levels similar to those in mice receiving CdCl2 in saline. The kidney/liver Cd concentration ratio (9) was the same for Cd-TH in all three media. Although Cd-TH gave much higher kidney/liver Cd ratios than CdCl2 (9 vs 2), renal Cd concentrations were the same or lower than after CdCl2 treatments. Results indicate that the high kidney/liver Cd ratio after Cd-TH treatment versus CdCl2 is due to lower concentrations of Cd in liver rather than marked increases in renal Cd levels. Heating of Cd-TH did not result in lower amounts of Cd in kidney. While the chemical form of Cd administered affects the absorption and distribution of Cd, the amount of Cd reaching the kidney after Cd-TH administration is similar to that after CdCl2 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.     

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.