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.     

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.     

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.     

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

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

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.     

Cadmium-metallothionein interactions in the olfactory pathways of rats and pikes.

Deposition of cadmium onto the olfactory epithelium results in transport of the metal along the primary olfactory neurons to the olfactory bulbs of the brain. The present investigation was undertaken to determine the intracellular ligand binding of cadmium during this process. (109)Cd(2+) was applied on the olfactory epithelium of rats and pikes, and the subcellular distribution of the metal in the olfactory pathways was then examined. Two groups of rats were used: one pretreated with intranasal instillations of nonlabeled cadmium and the other given physiological saline (controls). Cellular fractionations showed that the (109)Cd(2+) was predominantly present in the cytosol of all samples, both in the rats and the pikes. Gel filtrations of the olfactory epithelium of control rats killed 2 h after the (109)Cd(2+) instillation showed that the metal was recovered in two peaks with elution volumes corresponding to metallothionein (MT) and glutathione (GSH)-the latter peak being the predominant one. However, in the epithelium of the cadmium-pretreated rats killed at 2 h, (109)Cd(2+) was recovered in one peak corresponding to MT. In the olfactory epithelium and bulbs of both groups of rats killed at 48 h, as well as in the olfactory epithelium, nerves, and bulbs of pikes killed at this interval, (109)Cd(2+) was recovered in one peak corresponding to MT. Immunohistochemistry of the olfactory system of rats given cadmium in the right nasal cavity showed induction of MT in the neuronal, sustentacular, and basal cells of the right olfactory epithelium, in the nerve fascicles in the lamina propria of the right olfactory mucosa, and in the olfactory nerve layer of the right olfactory bulb. On the left side, the immunoreactivity was low in these structures. MT immunoreactivity was observed in the glomeruli of both the right and the left olfactory bulbs. However, the staining was homogeneously distributed within the entire glomeruli of the right bulb, whereas it showed a mesh-like pattern corresponding to the localization of astrocytes in the glomeruli of the left bulb. We conclude that exposure of the olfactory epithelium to cadmium results in induction of MT in the primary olfactory neurons and a transport of the metal in these neurons as a cadmium-metallothionein (CdMT) complex. Our results further indicate that GSH is a ligand that can interact with cadmium before the metal binds to MT.     

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.     

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.     

Cadmium-metallothionein nephrotoxicity in the rat: transient calcuria and proteinuria

After a s.c. injection of 0.4 mg Cd/kg as cadmium-metallothionein (CdMT) in rats, a marked increase in urinary protein concentration appeared at 16-40 h. There was a peak of urinary Cd content during the first 4 h after the treatment. Urinary Ca was increased at 8 h after the CdMT injection and returned to normal level at 32 h. Luminal and basolateral renal membrane vesicles were isolated from both control group and CdMT (0.4 mg Cd/kg) group at 24 h after the injection. Calcium uptake and binding of both fractions were decreased in the group treated with CdMT. Cd, Zn and MT concentrations in the kidney cortex were increased, but Ca concentration was not significantly changed. Since injected CdMT is probably only partly reabsorbed by tubular cells at the dose level of 0.4 mg Cd/kg as CdMT, excessive plasma CdMT is rapidly excreted in urine, explaining the increased Cd excretion during the first few hours observed in the present experiment. Decreased Ca binding in the luminal membranes as observed in vitro could be one of the mechanisms of production of calcuria if occurring in vivo. Another possible explanation of calcuria is that Cd ions released from CdMT into the cytoplasm of the tubular cell, may exert ionic interference with Ca transport across the luminal membranes and produce decreased Ca reabsorption. It is known that a disturbance of Ca metabolism could influence the membrane stability and such a change may contribute to explaining the proteinuria characteristic of CdMT nephrotoxicity. The reversibility of the proteinuria observed after a single dose of CdMT may be related to the induction of metallothionein synthesis in the renal cells.     

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.     

Dosage-dependent absorption of cadmium in the rat intestine measured in situ

Previous studies have shown that the disposition of cadmium (Cd) following oral administration is dosage dependent and may possibly be due to dosage-dependent intestinal absorption of Cd. Though extensively studied, the precise nature of Cd absorption by the intestine remains unclear. Similarly, the role of metallothionein (MT) in the intestinal absorption of Cd remains equivocal. The present study was designed (1) to characterize the intestinal absorption of Cd in the rat, and (2) to determine the role of MT in intestinal Cd absorption. The study has been conducted with an isolated intestinal loop preparation in situ, which allows direct measurement of intestinal absorption under nearly physiological conditions. Under urethane-induced anesthesia. Cd (0.1, 10, 100, 1000, or 10,000 micrograms/kg) was injected intraluminally into the isolated intestinal loop in situ and all mesenteric venous (portal) blood exiting from the loop was collected for 90 min. Absorption of Cd into the portal circulation was low at all dosages studied. The percentage of the dosage absorbed ranged from 0.09% at the 0.1 microgram/kg dosage to 3.4% at the 10,000 micrograms/kg dosage. At low dosages (0.1 and 10 micrograms/kg), little difference was noted in the fractional absorption of Cd (0.09 and 0.14% of the dosage, respectively). However, the fractional absorption of Cd was 10-fold greater in rats administered 100 micrograms Cd/kg (1.1% of the dosage). Administration of higher dosages of Cd (1000 and 10,000 micrograms/kg) further increased the percentage of the dosage absorbed (1.8 and 3.4%, respectively). To evaluate the role of MT in the intestinal absorption of Cd. rats were subcutaneously injected with zinc (Zn) for 4 days (30 mg/kg/day) and the absorption of an intermediate dosage of Cd (100 micrograms/kg) was subsequently assessed in situ. Zn pretreatment increased the endogenous concentration of MT in the intestine 25-fold. Following intraluminal administration. 93% of Cd in intestinal cytosol of Zn-treated rats was bound to MT whereas 40% of the cytosolic Cd was bound to MT in saline-treated (control) rats. Moreover, the amount of Cd in intestinal cytosol was 2-fold greater in Zn-treated rats than in control rats. However, the intestinal absorption of Cd in rats pretreated with Zn demonstrated no difference from that in saline-treated rats. These results indicate that the intestinal absorption of Cd is dosage independent at low dosages of Cd (less than 10 micrograms/kg) and dosage dependent at high dosages (greater than 10 micrograms/kg). Furthermore, saturation of intestinal MT is not a major determinant of the observed dosage-dependent absorption of Cd.     

半胱氨酸，谷胱甘肽和除去金属的金属硫蛋白对含镉金属硫蛋白肾毒性的保护作用

在大鼠不同部位ｓｃ巯基化合物与含镉金属硫蛋白（ＣｄＭＴ），观察除去金属的金属硫蛋白（ＡｐｏＭＴ），Ｌ－半胱氨酸（Ｃｙｓ），还原型谷胱甘肽（ＧＳＨ）对大鼠ＣｄＭＴ损伤肾的保护作用。测定不同时相尿蛋白和尿碱性磷酸酶活性以及肾组织形态学结果表明，与单独给予ＣｄＭＴ比较，肾损伤作用明显减轻。ＡｐｏＭＴ，ＧＳＨ促使大鼠尿Ｃｄ排泄量显著增加，肾组织细胞中金属硫蛋白（ＭＴ）结合Ｃｄ和游离Ｃｄ含量明显降低，以ＧＳＨ尤为明显Ｃｙｓ能增加Ｃｄ在肾细胞内蓄积，提示在Ｃｄ中毒时，除ＭＴ对肾损伤有保护作用外，也有其它巯基化合物的参与，且作用机理不尽相同     

Induction of hepatic metallothionein in the immature rat following administration of cadmium

Hepatic metallothionein (MT) levels, as measured indirectly by total metal-binding capacity, are approximately 8.5-fold higher in the 7-day-old rat than in the 28-day-old rat where levels are barely detectable. To stydy how the presence of MT might influence the toxicity of cadmium, a single subcutaneous injection of cadmium chloride was administered at dose levels of 0, 1, 2, 3, and 6 mg Cd/kg body weight, 48 hr prior to sacrifice on Day 7 or Day 28. In both the 7- and 28-day-old groups, there was a dose-related increase in the amount of cadmium bound to MT. There were no significant age-related differences in the amount of cadmium bound to MT at the various doses, with the exception of the 6 mg/kg dose, where 7 day levels were higher. The 28-day-old rats responded to cadmium exposure with induction of MT and subsequent binding of both cadmium and zinc at all doses. The 7-day-old appeared to have sufficient levels of MT to handle cadmium doses at or below 3 mg/kg without induction; however, at the 6 mg/kg dose induction was observed. Despite the presence and inducibility of hepatic MT at Day 7, 30% of the rats treated with 6 mg Cd/kg died within 48 hr of exposure compared with only 4% at Day 28.     

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.     

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

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

镉致大鼠肾损害时尿酶变化及其意义

我们报道镉中毒大鼠尿液主要生化指标和肾皮质组织病理改变，结果表明，尿总蛋白、葡萄糖、ｒ－谷氨酰转移酶（ｒ－ＧＴ）、碱性磷酸酶（ＡＬＰ）和Ｎ－乙酰－Ｄ－氨基葡萄糖苷酶（ＮＡＧ）排泄均明显增加，光镜和电镜下见近曲小管细胞浊肿、水样变性，微绒毛稀疏脱落，线粒体肿胀变性及胞浆空泡形成。提示Ｃａ２＋是ＣｄＭＴ的毒作用形式，尿ｒ－ＧＴ、ＮＡＧ是镉性肾损害的理想监测指标。     

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.     

Altered subcellular distribution of cadmium following cadmium pretreatment: possible mechanism of tolerance to cadmium-induced lethality

Animals pretreated with cadmium (Cd) subsequently develop tolerance to an otherwise lethal dose of Cd. Possible mechanisms for this tolerance include reduced absorption, an altered tissue distribution, and an altered subcellular distribution of Cd. Male rats received a single Cd pretreatment (2.0 mg/kg, sc) 24 hr prior to administration of a typically lethal challenge dose of Cd (3.9 mg/kg, iv). Tolerance was evident because no mortality was observed in Cd-pretreated rats. Since Cd induces synthesis of hepatic metallothionein (MT), a higher percentage of the challenge dose might be sequestered in the liver of Cd-pretreated animals with less distributed to target organs of toxicity. At 2 and 24 hr following Cd challenge, no marked changes in organ distribution of the challenge dose of Cd were observed as a result of Cd pretreatment. However, isolation of hepatic subcellular fractions 2 hr following injection of the challenge dose revealed less Cd in nuclei, mitochondria, and endoplasmic reticulum, and more in cytosol as a result of Cd pretreatment. The increased cytosolic Cd was bound primarily to MT which had been induced markedly by Cd pretreatment. These data indicate that differences in absorption or tissue distribution of Cd are unlikely explanations for development of tolerance to Cd. Rather, tolerance appears to result from an MT-related change in the hepatic subcellular distribution of Cd, evidenced by lower concentrations of Cd in nuclei, mitochondria, endoplasmic reticulum, and cytosolic high-molecular-weight proteins and higher concentrations bound to MT in cytosol.     

Influence of chemical and environmental stressors on acute cadmium toxicity

Previous investigations have demonstrated that the cytosolic protein metallothionein (MT) is induced not only by exposure to certain heavy metals but also by a variety of other factors, including environmental stress. While MT synthesis has been observed with exposure to cold temperatures, there is a paucity of data concerning the influence of cold on heavy-metal toxicity. The present investigation focused on the influence of metal and cold pretreatments on the acute toxicity of cadmium. Mortalities of 80% and 100% were observed for mice orally administered challenge doses of 100 mg Cd/kg and 150 mg Cd/kg, respectively. To determine a protective cadmium pretreatment dose, animals were administered 2.5, 5, 10, 20, 25, and 50 mg Cd/kg 24 h prior to cadmium challenge. In animals pretreated with 10 mg Cd/kg, mortalities of 20% and 70% were observed with the respective challenge doses. Immediately following cold stress (4 degrees C, 12 h), mortalities of 30% and 90% were observed with cadmium challenge doses of 100 and 150 mg Cd/kg, respectively. Significant correlations were demonstrated between induced hepatic MT concentrations and cadmium pretreatment (r = 0.99), as well as cold pretreatment (r = 0.87). Results of this investigation indicate that stressors, such as cold, influence the acute toxicity of cadmium to the same magnitude as metal pretreatment. This induced tolerance to cadmium was attributed, in part, to the induction of MT synthesis. Furthermore, the induced levels of MT resulting from cold stress may confound the simplistic approach of using MT as a biological monitor of occupational exposure to cadmium.     

Protective role of renal metallothionein against Cd nephropathy in rats

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