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The effect of dietary zinc (Zn) supplementation on copper (Cu)-induced liver damage was investigated in Long-Evans Cinnamon rats (LEC), a model for Wilson's disease (WD). Four-week-old LEC (N=64) and control Long-Evans (LE) (N=32) female rats were divided into two groups; one group was fed with a Zn-supplemented diet (group I) and the other was given a normal rodent diet (group II). LEC rats were killed at 6, 8, 10, 12, 18, and 20 wk of age; the LE control rats were killed at 6, 12, 18, and 20 wk of age. Cu concentration in the liver was reduced in LEC rats fed the Zn-supplemented diet compared with LEC rats on the normal diet between 6 and 18 wk of age. Metallothionein (MT) concentration in the livers of LEC rats in group I increased between 12 and 20 wk of age, whereas hepatic MT concentration in LEC rats from group II decreased after 12 wk. Hepatocyte apoptosis, as determined by TUNEL, was reduced in Zn-supplemented LEC rats at all ages. Cholangiocellular carcinoma was observed only in LEC rats in group II at wk 20. These results suggest that Zn supplementation can reduce hepatic Cu concentration and delay the onset of clinical and pathological changes of Cu toxicity in LEC rats. Although the actual mechanism of protection is unknown, it could be explained by sequestration of dietary Cu by intestinal MT, induced by high dietary Zn content.

In the present study, the role of endothelin-1 (ET-1) on alterations of hepatic and renal metallothionein (MT) and trace metals (Zn, Cu, and Fe) were investigated in streptozotocin (STZ)- induced diabetic rats. Diabetic rats, age- and sex-matched controls, as well as control and diabetic animals on a dualETA/ETBreceptor blocker, bosentan, were investigated after 6 months of follow-up. MT was measured by cadmium-heme assay. Metals were measured by atomic absorption spectrometer. ET-1 mRNA was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR) technique. Hepatic and renal ET-1 mRNA was increased in diabetic rats as compared to control rats, along with an increase in both hepatic and renal MT proteins. The increased hepatic MT protein level was associated with decreases in hepatic Cu and Fe, whereas increased renal MT was associated with increases in renal Cu and Fe accumulation. Zn levels were unaltered in both organs in diabetic rats. Bosentan treatment partially prevented the increase in MT levels in both liver and kidney, along with reduced serum creatinine and increased urinary creatinine levels. Further bosentan treatment corrected the increased Cu and Fe levels in the kidney in diabetic rats, but reduced hepatic Cu and Fe levels. No significant effects of bosentan treatment on nondiabetic rats were observed. The data suggest that the possible effects of ET antagonism in diabetes may be mediated via changes in MT and trace metals.

The ability of cadmium (Cd) to induce the synthesis of metallothionein (MT) in the developing teeth of the rat was investigated. Rats were given daily intraperitoneal injections of cadmium chloride (1.5 mg Cd/kg) for 7 days. The induction of MT synthesis in incisor teeth after Cd treatment was investigated immunohistochemically using a polyclonal antibody to MT. Immunoreactivity to MT was observed in the papillary layer of epithelial cells of the secretory zone, in one layer of epithelial cells of the presecretory zone and within ameloblasts of the postsecretory zone. Normal control rats did not exhibit MT staining. These results indicate that Cd induces MT synthesis within specific epithelial cells of the enamel organ of the rat. It is proposed that these findings demonstrate an adaptive cellular mechanism that protects these cells from cadmium toxicity.

In the present study, we evaluated the renal and hepatic accumulation of mercury, the intrarenal distribution of mercury and the urinary and fecal excretion of mercury in rats injected intravenously with a non-toxic 0.1 mumol/kg-dose of mercury in the form of mercuric chloride (HgCl2) or a complex of mercury-metallothionein (Hg-MT). Between 6 and 72 h after injection, the concentration of mercury in the kidneys of the rats injected with Hg-MT was significantly greater than that in the rats injected with HgCl2. The greatest difference in the renal concentration of mercury between the two groups of rats was detected 6 h after injection. In the kidneys of both experimental groups of rats, the cortex and the outer stripe of the outer medulla contained the highest concentrations of mercury, with the greatest concentrations found in the renal cortex and outer stripe of the outer medulla of the rats injected with Hg-MT. No differences were found between the two experimental groups with respect to the concentration of mercury in the renal inner stripe of the outer medulla and inner medulla throughout 72 h of study. The content of mercury in the blood and liver decreased over time in both groups of rats, but was always significantly greater in the blood and liver of rats injected with HgCl2. The rats injected with Hg-MT excreted more than eight times the amount of mercury in the urine than the corresponding rats injected with HgCl2 during 72 h. These data indicate that there may be decreased tubular reabsorption of filtered Hg-MT and/or tubular secretion of mercury in the rats injected with Hg-MT. In contrast, the rats injected with HgCl2 excreted significantly more mercury in the feces during the same period of time than the corresponding rats injected with Hg-MT. In conclusion, our data clearly indicate that the renal and hepatic uptake and accumulation of mercury, and the urinary and fecal excretion of mercury, are altered significantly when inorganic mercury is administered intravenously as a complex with metallothionein.

Metallothionein genes (MT) are inducible by a variety of agents, including heavy metals. We report the induction of MT expression by arsenite (As3+) in rat liver in vivo. As3+ (but not arsenate [As5+]) injection increased MT protein and MT-1 and MT-2 mRNA accumulation in liver only, but not in kidney or pancreas. In addition, As3+ enhanced zinc-induced MT protein accumulation in liver without any increase in MT mRNA levels. These data indicate that arsenic may increase MT expression either directly (by inducing MT mRNA accumulation), or indirectly by altering post-transcriptional events. This constitutes an unusual mechanism of enhancement of MT gene expression and appears to be mediated by processes not specifically associated with binding of arsenite to MT in vivo.

The relative tissue distribution and toxicity of cadmium (Cd) and mercury (Hg) in the liver and kidneys of rats when the metals are administered as either inorganic salts or complexed with MT were studied. Male Sprague-Dawley rats were injected (i.v.) with Cd or Hg inorganic salt of chloride or in a complex of MT at a dose of 0.3 mg/kg body weight. The concentration of MT and metals in plasma and urine was monitored for 7 days, at the end of which the rats were killed. Injection of both HgCl 2 and HgMT induced the synthesis of MT only in the kidney but not in the liver, whereaa CdCl 2 and CdMT injections induced MT synthesis in both liver and kidney, respectively. Plasma MT levels increased 3 days after CdCl 2 but not after HgCl 2 injection, suggesting that hepatic MT may be an important source of plasma MT under our experimental conditions. Renal toxicity was observed morphologically and by an increase in blood urea nitrogen, plasma creatinine, proteinuria in rats injected with CdMT and both forms of Hg. Urinary MT excretion was significantly elevated in CdMT injected rats compared with those injected with CdCl 2 . However, HgCl 2 and HgMT injected rats showed no significant difference in urinary MT excretion. The magnitude in the renal accumulation of Hg is similar after the administration of HgMT or HgCl 2 , but our findings suggest that the site of epithelial injury may be different. Injury effects of HgMT localized mainly in the terminal portions of the proximal convoluted tubule and the initial portions of the proximal straight tubule whereas inorganic Hg caused necrosis in pars recta segments of the proximal tubule.

A recent study from our laboratory has shown that cadmium, a toxic heavy metal, is one of the most effective agents known for inducing hepatocytic transdifferentiation of the rat pancreas. With repeated injections of cadmium, the incidence of rats with pancreatic hepatocytic foci can be as high as 93%. Cadmium is also well known as a very potent inducer of metallothionein, a metal-binding protein that appears to be important in the biologic response to several toxic heavy metals in most tissues, including the pancreas. Therefore, the present study sought to determine if metallothionein was associated with cadmium-induced transdifferentiation of pancreatic cells. Expression of metallothionein was studied immunohistochemically by the peroxidase-antiperoxidase method in tissue sections of the pancreas of rats with pancreatic hepatocytes. High levels of metallothionein were localized primarily within the pancreatic hepatocytes. Surrounding normal pancreatic islet and acinar cells were not immunoreactive. Thus, metallothionein is expressed actively in cells transdifferentiated to hepatocytes by cadmium within the pancreas.

The protective roles of metallothionein (MT) and glutathione (GSH) in acute hepatotoxicity of cadmium (Cd) were investigated in an in vitro system. Liver slices were incubated in a buffer containing cadmium chloride (20-50 ppm) at 37 degrees C for 3 h. Viability of the slices was monitored by measuring intra-cellular potassium (K) content and GSH concentrations. A dose-dependent decrease of intracellular K content of GSH concentrations was observed. Pre-induction of MT (100-fold increase) by injection of zinc sulphate (30 mg Zn/kg body weight) showed protection against decrease in both intracellular K and GSH concentrations in liver slices. Decrease of hepatic GSH (90%) by an injection of buthionine sulfoximine (BSO)(4 mmol/kg body weight) to the rats further enhanced the Cd toxicity in the liver slices. This enhanced toxicity resulting from BSO treatment can be totally overvome by induction of MT by Zn pre-treatment. The cellular uptake of Cd remained unaltered in all experiments. These results demonstrate that hepatic toxicity of Cd may be due to its binding to intracellular sulfhydryl groups and both intracellular GSH and MT levels may provide protection against cytotoxicity of Cd in liver. Moreover, even at low GSH levels, MT could partially protect the hepatic cells from Cd cytotoxicity.

In the present study we examined the effects of zinc pretreatment (to induce the renal synthesis of metallothionein) on the renal accumulation and intrarenal distribution of inorganic mercury in uninephrectomized (NPX) and sham-operated (SO) rats 24 h after the animals were given a 0.75, 1.0 or 1.5 μmol/kg intravenous (i.v.) dose of inorganic mercury. We also examined the effects of zinc pretreatment on the nephropathy induced by the three doses of inorganic mercury. Zinc was administered at a dose of 306 μmol/kg (20 mg/kg) subcutaneously (s.c.) in the form of zinc sulfate once daily for 2 consecutive days prior to the administration of inorganic mercury. Following zinc pretreatment, the renal accumulation of injected inorganic mercury increased in both NPX and SO rats treated with the three doses of inorganic mercury, but the increase was significantly greater in the NPX rats. The enhanced accumulation of mercury was associated with an altered pattern in the intrarenal distribution of mercury, particularly in the NPX rats. The increased renal accumulation of mercury in both the NPX and SO rats was due primarily to its increase in the renal cortex. We have recently found that the synthesis of metallothionein in the renal cortex increases in NPX and SO rats given zinc. Therefore, it appears that there is a relationship between the content of preinduced cellular metallothioein in the cortex and the content of mercury that accumulates in the cortex. Zinc pretreatment also prevented the nephropathy induced by the three doses of inorganic mercury from occurring in both the NPX and SO rats. We propose that some of the protection may be related to the altered intrarenal accumulation and distribution of mercury that occurs after pretreatment with zinc. Hepatic accumulation of mercury also increased in both groups of rats, but increase again was significantly greater in the NPX rats. Our findings show clearly that a significant reduction in renal mass alters the hepatic and renal accumulation of mercury when zinc pretreatment is used to induce the renal and hepatic synthesis of metallothinein. In addition, our findings show that zinc pretreatment protects both normal and remnant kidneys in rats from the nephrotoxic effects of inorganic mercury.

A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1-100 ng for MT-2 and 10-1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1% Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our antiserum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%) for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of zinc (Zn) salts to induce the synthesis of metallothionein (MT) in liver, kidney and pancreas of rats pretreated with cadmium (Cd) salts was investigated. Twenty-four hours after either CdCl2 (2.0 mg Cd/kg, s.c.) or saline pretreatment, rats were injected with saline, CdCl2 (2.0 mg Cd/kg, s.c.) or ZnSO4 (20 mg Zn/kg, s.c.) and the concentrations of MT and MT-1 mRNA in tissues subsequently measured. After a single injection of Cd salts, concentrations of MT and MT-1 mRNA were significantly increased in liver as compared to control. With two injections of Cd, the accumulation of MT in liver was approximately twice the levels of MT following a single injection of Cd. In kidney, MT and MT-1 mRNA expression were significantly increased only after two injections of Cd and in the pancreas, Cd injections did not alter either MT content or MT-1 mRNA expression. Treatment with Zn salts increased MT concentrations in both liver and pancreas. However, the pancreas was the most responsive to injections of Zn salts as compared to the liver in terms of increases in both protein concentration and MT-1 mRNA expression. When Zn injection was preceded by a Cd injection, induction as measured by MT-1 mRNA and MT concentrations were approximately additive in liver. In kidney, although Cd or Zn treatment separately had no effect on MT or MT-1 mRNA content, injection of Cd followed by Zn resulted in significantly increased levels of renal MT and MT-1 mRNA. Fractionation of liver cytosols on a Sephadex G-75 column revealed that in animals receiving two injections of Cd, virtually all the Cd was associated with MT whereas Zn was distributed between both high molecular weight (HMW) proteins and MT. In animals receiving both Cd and Zn injections, cytosolic Cd was still bound predominantly to the MT fraction, while the proportion of cytosolic Zn associated with MT increased. The results of this study suggest that, treatment with Cd salts followed by Zn salt injection can induce further synthesis of MT in liver, kidney and pancreas with subsequent binding of both Zn and Cd to the intracellular MT.

Rats fed diets with cadmium (Cd) concentrations similar to that found in human diets, and nutritionally marginal with respect to iron (Fe), zinc (Zn), and calcium (Ca) retained 10 times more Cd in the duodenum than rats fed adequate mineral diets. In the current study, 2 experiments were performed to determine the role of intestinal metallothionein (MT) in the accumulation of duodenal Cd, and to determine whether endogenous rice grain Cd is as available as Cd exogenously incorporated into the grain. In Expt. 1, wild-type and MT-null mice were fed 40% rice diets containing marginal or adequate amounts of Fe, Zn, and Ca, and 240 mug Cd/kg. Duodenal Cd was 10 times higher in both wild-type and MT-null mice regardless of their mineral status. In Expt. 2, one group of rats was fed 40% rice diets in which Cd was incorporated into the rice during growth and maturation, and another group was fed 40% rice diets in which Cd was incorporated into the rice during cooking. Each group also was fed either marginal or adequate amounts of Zn, Fe, and Ca. After 5 wk, rats were given a single meal labeled with (109)Cd, and the amount of label retained after 7 d was determined by whole-body counting. Rats with marginal mineral status retained 10 times more (109)Cd than those with adequate status; however, there was no difference between rats fed endogenous or exogenous Cd rice. Although duodenal Cd concentration was 8 times higher in the marginally fed rats, MT concentration was unchanged. These 2 experiments indicate that MT induction is not involved in duodenal Cd accumulation in animals with marginal dietary status of Fe, Zn, and Ca. In addition, they support the hypothesis that marginal deficiencies of Fe, Zn, and Ca, commonly found in certain human populations subsisting on rice-based diets, play an important role in increasing the risk of dietary Cd exposure.

The objective of this study was to evaluate the potential role of TNF-alpha in the onset of acute hepatitis in the Long-Evans Cinnamon (LEC) rat, an animal model for inherited copper (Cu) toxicosis. In LEC rats, Cu is accumulated in the liver with age, and clinical signs of acute hepatitis were observed as, icterus, reduced body weight, nasal bleeding, dehydration, and reduced food intake at 12 weeks of age. Cellular changes such as apoptosis in the liver were evident in these rats with increasing age. Positive TNF-alpha and TNFR1 immunostainings were observed in hepatocytes and Kupffer cells in LEC rats. Hepatic levels of caspase-3 activity, TNF-alpha mRNA, and protein were also increased in LEC rats from 6 to 12 weeks of age as compared with control Long-Evans (LE) rats. The neutralization of TNF-alpha by passive immunization or the inhibition of caspase activity can block the apoptotic process initiated by TNF-alpha. In this study, we evaluated the effects of passive immunization of LEC rats with weekly administration of anti-rat TNF-alpha on Cu-induced acute hepatitis. This treatment resulted in a reduction of the percentage of apoptotic cells in the liver, decreased activity of caspase-3, and also in down-regulation of the TNF-alpha gene expression. Thus, these results suggest a major role for TNF-alpha on the pathogenesis of Cu-induced acute hepatitis in LEC rats.

The intestinal uptake and tissue distribution of cadmium (Cd) were studied in control rats and those deficient in zinc (Zn), iron (Fe) or cysteine (SH) using an in situ model where an intestinal loop of 5 cm was incubated with CdCl2 or CdMT (MT, metallothionein) for 30 and 60 min. The intestinal content of Cd after incubation with CdCl2 or Cd MT was not affected by nutritional deficiencies, but the Cd uptake from CdCl2 was always higher than that from Cd MT. However, both Fe and Zn deficiencies had a marked effect on distribution of Cd in liver, kidney and pancreas. After 30 min incubation in situ with CdCl2, Cd was deposited only in liver in control and SH deficient rats, while Cd was detected also in kidney and pancreas of both Fe and Zn deficient rats. After 60 min incubation with CdCl2, the deposition of Cd in the liver, kidney and pancreas of Fe deficient rat was significantly higher than that in the control. The deposition of Cd after CdMT incubation in situ was mainly found in kidney, and nutritional deficiencies increased the tissue deposition of Cd from CdMT. Similarly, the renal deposition of Cd absorbed from CdCl2 was markedly increased in Fe deficient rats. These results suggest that the intestinal uptake mechanisms of Cd from CdCl2 and Cd MT are different and nutritional deficiencies can markedly increase the deposition of Cd in the kidney, the critical organ in chronic cadmium exposure.

The protective roles of metallothionein (MT) in streptozotocin (STZ)-induced pancreatic damage were investigated using zinc (Zn) as the inducer of MT synthesis in rats. Diabetes was produced in a group of Sprague Dawley rats by a single injection of STZ. In another group of rats, to induce the synthesis of MT, Zn was injected subcutaneously about 12 h before injection of STZ. Rats were sacrificed at about 30 h, 3 and 6 weeks after administration of STZ. The plasma glucose levels in STZ and Zn+STZ group increased significantly in 30 h, 3 and 6 weeks compared with the control group, but the level of glucose in Zn+ STZ group was lower than that in the STZ group. Both Zn and Mt levels increased in pancreas, liver, and kidney of rats injected with Zn or Zn + STZ. The induction of MT synthesis in pancreas of Zn-injected rats was also demonstrated by Western Blot analysis. Lipid peroxidation induced by STZ was inhibited significantly by pretreatment with Zn. Injection of STZ decreased the activities of superoxide dismutase (SOD) in plasma, liver and pancreas, but injection of Zn had no effect on the activities of SOD. The results support that pretreatment with Zn can partially prevent the development of diabetes induced by STZ injection and it may be related to the effects of MT as a scavenger for the oxygen free radicals.

The specificity of three populations of antibodies to the two metallothionein (MT) isoforms has been characterized using a competitive enzyme-linked immunosorbent assay (ELISA). A polyclonal sheep anti-MT-1 antibody (PAb 1) showed higher affinity to MT-1 than to MT-2 with I50 (concentrations required to achieve 50% inhibition) being 18 and 480 ng for MT-1 and MT-2, respectively. Conversely, a polyclonal rabbit anti-MT-2 antibody (PAb 2) showed higher affinity to MT-2 than to MT-1 (I50 being 250 and 15 ng for MT-1 and MT-2, respectively). A third antibody (PAb 3), isolated by removal of anti-MT-1 antibodies in PAB 2 by immunoaffinity purification, showed no cross-reactivity with MT-1. This result suggests that there are two populations of antibodies which cross-react specifically with the MT isoforms. Rat tissue MT concentrations were quantified by ELISA using the three different antibodies and silver-saturation method. The sum of MT isoform concentrations (ELISA estimation using antibodies PAb 1 and PAb 3) were not significantly different (p < 0.05) from the total MT concentrations obtained by silver-saturation method. Therefore, the ELISA can be used to estimate the relative quantities of the two MT isoforms in rat tissues. The predominant isoform in rat tissues was MT-2 and the proportion of MT-1 increased with induced synthesis of MT by metal injection.

The effects of unilateral nephrectomy and compensatory renal growth on renal metallothionein metabolism were evaluated in the present study. In rats, the renal content of metallothionein increased in proportion to the increase in renal mass after unilateral nephrectomy and compensatory renal growth. However, when zinc was used to induce the synthesis of renal metallothionein, the remnant kidney in uninephrectomized (NPX) rats produced significantly greater amounts of metallothionein on a per gram kidney basis than a normal kidney in sham-operated (SO) rats. In both NPX and SO rats, zinc pretreatment caused metallothionein synthesis to increase primarily in the renal cortex and renal outer stripe of the outer medulla. Zinc pretreatment also changed the pattern for the intrarenal accumulation of inorganic mercury in NPX rats. After pretreatment with zinc, the accumulation of inorganic mercury predominated in the renal cortex rather than in the outer stripe of the outer medulla in the NPX rats. In addition, both NPX and SO rats were afforded complete protection against the nephrotoxic effects of a low, toxic dose of inorganic mercury when they were pretreated with inorganic zinc. The protection is postulated to be related to the alteration in the pattern of renal accumulation of inorganic mercury. In conclusion, the capacity to synthesize metallothionein increases significantly in rats after they have undergone unilateral nephrectomy and compensatory renal growth. The increased capacity of the remnant kidney to synthesize metallothionein may involve adaptive changes both in transcriptional and/or translational controls of metallothionein synthesis.

The effect of two vicinal dithiols, 2,3-dimercaptopropan-1-ol (BAL) and N -(2,3-dimercaptopropyl)phthalamidic acid (DMPA), and a dithiocarbamate, sodium N -(4-methoxybenzyl) d -glucamine dithiocarbamate (MeOBGDTC), on the biliary excretion of cadmium was examined in rats. Tissue cadmium levels were also determined following the measurements of biliary excretion of cadmium. At 30 min after the injection of CdCl 2 · 2.5H 2 O (1 mg/kg, iv) each rat was given 400 μmol/kg ip of one of the compounds, BAL, DMPA or MeOBGDTC. While all the compounds increased the biliary excretion of cadmium, the most effective was MeOBGDTC, whose administration resulted in a 580% increase in biliary cadmium content. The effectiveness of the MeOBGDTC may be due to the presence of both nonpolar and nonionizing polar groups attached to nitrogen. MeOBGDTC was able to mobilize cadmium to the bile even after the occurrence of the synthesis of metallothionein and the incorporation of the cadmium into it. An attempt was also made to determine the chemical nature of the Cd-MeOBGDTC complex present in the bile by comparing a newly synthesized authentic sample of Cd(MeOBGDTC) 2 complex with the hot dioxane extract of the freeze-dried bile samples using thin-layer chromatography and proton NMR. The results suggested that cadmium excreted in the bile in part, is complexed to MeOBGDTC and glutathione.

~Metallothionein contains about 30% cysteine and is a major protein in newborn rat liver. This protein and glutathione constitute two major intracellular cysteine pools in newborn rat liver. When pregnant rats were fed on a soya-bean-protein diet, low in sulphur amino acids, the hepatic glutathione levels of the dams were decreased. However, this did not affect the levels of glutathione or metallothionein in the pups. The activity of the glutathione-degrading enzyme y-glutamyltransferase (EC 2.3.2.2) in the livers of pups was maximum at birth and gradually decreased with age when the hepatic glutathione was transported to the kidney. In the pups born from dams fed on soya-hean-protein diet the decline in the hepatic enzyme activity was delayed, suggesting a continued degradation of glutathione in the liver. These results suggest that even with a maternal nutritional deficiency of sulphur amino acids, the transfer of cysteine to the fetus is not impaired. However, the hepatic intra-organ degradation of glutathione is continued in these pups for a prolonged period after birth compared with pups born from control mothers. The increased degradation of glutathione in the liver may be essential to meet the requirement of cysteine in pups born from dams fed on the soya-bean-protein diet. Glutathione : Metallothionein : Sulphur metabolism : Rat

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.

The metallothionein (MT) content of fetal rat liver was measured daily during the final week of gestation, by both cadmium saturation and polarographic methods. MT levels rise sharply at day 18 of gestation and continue to increase into the neonatal period. In late gestation, MT serves to bind Cu and Zn from the preexisting hepatic pools of these metals, as well as to accumulate additional amounts of both metals The fetal MT is similar to the adult rat protein both in terms of its protein composition and metal-binding properties. Perinatally the Zn/MT ratio remains constant for several days suggesting a carefully regulated process. At birth, most of the hepatic Zn and a significant amount of hepatic Cu are bound to MT. Immunohistochemical localization of MT shows a progressive increase in cytoplasmic MT with the appearance of nuclear MT by day 20 of gestation in fetal rat liver. The results are discussed in terms of a model for regulation of MT synthesis and for the metal storage role of MT in perinatal development.

Metallothionein (MT) of various tissues contains bound zinc (Zn) and any change in Zn status can alter its synthesis and tissue deposition. The changes in MT levels and its inducibility in Zn-injected and Zn-deficient (Zn-D) rats were studied. MT levels in 11 tissues (brain, lung, heart, liver, kidney, stomach, small intestine, pancreas, spleen, testes and muscle) of control and rats injected with different doses of ZnSO 4 (20 mg Zn/kg for 2, 4 or 7 times) were measured by the cadmium-hemolysate (Cd-hem) method. A dose dependent increase in MT levels was observed only in the pancreas, liver, small intestine and kidney after ZnSO 4 injection — the highest level being in the pancreas. A positive correlation was found between Zn and MT concentrations and also the relative inducibility of MT was similar in these 4 tissues (slopes of regression equations were 12.6–15.5). In order to study the effect of Zn-D in MT induction, rats were fed a diet containing 1 ppm Zn for 18 days and CdCl 2 (1 mg Cd/kg) was injected subcutaneously 3 times at 48-h intervals to control and Zn-D rats. Although the tissue distribution of Cd was similar in both the groups, MT concentrations in pancreas and kidney were significantly decreased in Zn-D. The plasma and tissue levels of Zn were also decreased in Zn-D rats injected with CdCl 2 . The decrease in both Zn and MT levels was more prominent in pancreas than other organs of Zn-D rats. The results suggest that of all the organs studied, the induction of pancreatic MT is sensitive to Zn status and Zn may be a primary inducer of MT.

Renal glutathione (GSH) concentrations were reduced approximately 80% at 4 hr after a single injection of buthionine sulfoxime (BSO) (4 mmol/kg body wt) and remained reduced for at least 16 hr in male rats. Following BSO injection, rats were injected with a nephrotoxic dose of cadmium-metallothionein (Cd-MT) (0.3 mg Cd as Cd-MT/kg body wt) and killed 1, 4, or 12 hr later. Damage to the kidney was assessed histologically and by measurement of p-aminohippuric acid (PAH) uptake into renal cortical slices. Although the renal accumulation of Cd following Cd-MT injection was significantly lower in BSO-pretreated rats as compared to nonpretreated rats, the damage to kidney was more severe. At 4 and 12 hr, both Cd-MT-induced inhibition of PAH uptake and morphological damage were significantly increased in BSO-pretreated rats. In certain experiments, the induction of renal intracellular MT synthesis by zinc pretreatment slightly decreased the renal toxicity of Cd-MT in the BSO-treated rats. The results demonstrate that although GSH depletion decreases the renal accumulation of Cd in rats injected with Cd-MT, the nephrotoxicity of Cd-MT is increased. Preinduction of MT in the kidney can only partially overcome this increase in toxicity. Therefore both GSH and intracellular MT levels can influence the renal toxicity of injected Cd-MT.

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

Metallothionein (Mt) is a low molecular weight metalloprotein and its synthesis is induced by various divalent metals (Cd, Zn, Hg and Cu). The Intracellular distribution of Mt in hepatic and renal cells from control and CdCl2 injected rats was investigated by immunohistochemical methods. Antiserum to purified rat-liver Mt was prepared in rabbits after partial polymerization of the protein. The unlabelled peroxidase-antiperoxidase method using the specific rabbit anti-rat liver Mt provided a sensitive technique to localize Mt in tissue sections. In control rats, Mt or thionein (metal-free protein) was mainly localized in the cytoplasm of hepatocytes, renal collecting duct epithelium and distal convoluted tubular epithelium. In rats, injected intraperitoneally (i.p.) with CdCl2 (0.6 mg/kg) for 2 weeks, Mt was present mainly in the nuclei which were largely negative in control rats. Repeated injection with CdCl2 for 4–8 weeks resulted in the appearance of Mt in both the nucleus and cytoplasm. Intraluminal staining was also noted in proximal convoluted tubules along with marked vacuolation in the cytoplasm at 6 and 8 weeks. High intensity staining was observed in proximal convoluted tubules and collecting duct epithelium of the kidneys in CdCl2 injected rats. The bile duct epithelium in liver samples, renal glomerular mesangial cells, glomerular visceral epithelial cells and vascular smooth muscle cells showed weak to moderately intense staining. No staining was seen in vascular endothelial cells, fibroblasts or leukocytes. The staining for Mt in this technique was abolished when the antibody was absorbed with rat-liver Mt in vitro or by substitution of the antiserum with normal rabbit serum, demonstrating the specificity of the staining reaction for Mt. The results showed the presence of small amounts of Mt predominantly in the cytoplasm of control rat hepatocytes and renal tubular epithelium and its appearance in both the nucleus and cytoplasm after its synthesis induced by CdCl2 injections.

Cells were isolated from adult rat kidney by mild trypsin digestion and maintained in Eagles MEM-d-valine medium for 3 days to form a monolayer primary culture of epithelial cells. When these cells in monolayer were exposed to a medium containing lead nitrate (4.5 × 10−4m) complexed with glutamic acid (5 × 10−4m), inclusion bodies with characteristic ultrastructure were detected morphologically in the cytoplasm within 4 hr. At 24 hr after lead exposure the inclusion bodies were localized mainly in the nucleus. Addition of actinomycin D (0.05 and 5 μg/ml) or cycloheximide (5 and 10 μg/ml) directly to cultures prior to lead exposure resulted in the absence of inclusion bodies at high concentrations and in fewer inclusions at low concentrations. These inhibitors did not affect the uptake of lead by the cell; but they markedly inhibited incorporation of [3H]leucine into cellular proteins. The initial appearance of inclusion bodies in cytoplasm after lead exposure, its subsequent relocalization in the nucleus and the effect of inhibitors in these processes were also confirmed by quantitation of inclusion bodies ultrastructurally in randomly selected cells in each experiment. These results indicate that active protein synthesis is required for the formation of lead-induced inclusion bodies.

Metallothionein (MT) bound to zinc and copper was detected in high concentration in fetal and newborn rat livers by a cadmium saturation method. The levels of both hepatic zinc and MT remained high for the first 14 days after birth and decreased to adult levels by 24 days of age. There was a direct linear relationship between hepatic metallothionein and zinc concentrations during the first 31 days after birth. The ratio of MT to zinc levels also decreased with age suggesting a rapid degradation of MT during postnatal development. Immunohistochemical localization of MT by peroxidase-antiperoxidase technique, using a specific antibody to MT, showed intense intranuclear staining for MT in fetal and newborn rat liver which persisted until Day 9. The nuclear MT staining decreased with age; at 11 days it was equal both in nucleus and cytoplasm and at 14 days, MT was localized mainly in the cytoplasm, similar to adult rat liver pattern. The intranuclear localization of MT in neonates could be considered as a typical fetal-neonatal morphological pattern and its subsequent presence in the cytoplasm, an adult pattern.

The objectives of this investigation were to study the role of zinc-thionein in the metabolism of excessive amounts of zinc and the re-utilization of zinc bound to this protein. Rats were injected ip with 5 mg Zn/kg body weight as zinc chloride for 5 days to induce the synthesis of zinc-thionein. Proteins with molecular weight of 10,000 with high affinity for zinc were synthesized in rat liver and intestine. These proteins were heat stable. Large scale isolation of this protein from rat liver was achieved by a simple method consisting of a heating step and sephadex gel fractionation. Intravenous injection of isolated 65Zn-thionein to rats resulted in similar organ distribution of zinc as that after 65ZnCL2 injection. Gel filtration experiments with liver supernatant from rats injected with 65Zn-thionein showed that a major portion of 65Zn was transfered to other protein fractions and was re-ultilized similar to inorganic salts of zinc. Morphological studies showed no toxic effects after injection of zinc-thionein. These results show that zinc-thionein is metabolically active and non-toxic.

The binding of Ag+ to metallothionein (MT) was investigated, and a Ag-saturation assay was developed for the measurement of MT in tissues. When samples of purified hepatic Zn-MT or Cd-MT were titrated with Ag+ followed by hemolysate-heat treatment, it was found that saturation of MT occurred at 17 to 18 g-atoms Ag+/mol protein. The rank order of potencies of metals to displace Ag+ from 110mAg-labeled Ag-MT was Ag+ greater than Cu2+ greater than Cd2+ greater than Hg2+ greater than Zn2+ at pH 8.5 in 0.5 M glycine buffer. When a Ag-saturation (Ag-hem) assay was compared with a Cd-saturation (Cd-hem) technique, excellent correspondence was obtained in the measurement of MT from various sources including purified Zn-MT (II), MT in human kidney and liver tissue samples, hepatic MT in Cd-injected adult rats, and renal MT in Hg-injected adult rats. However, in cases where significant amounts of Cu-MT were present, such as in the livers of Cu-injected rats or in the kidneys of Cd-injected rats, the Cd-hem assay consistently underestimated the MT concentrations compared to the Ag-hem method. This is attributed to the inability of Cd to displace Cu effectively from MT at pH 8.5. Thus the Cd-hem assay is not recommended for the measurement of metallothioneins containing a high Cu content. Under such conditions, the Ag-hem method seems superior.

The factors which determine the deposition of cadmium (Cd) were investigated. While intravenously administered Cd2+ salts result in Cd2+ binding loosely to plasma proteins, and subsequently being deposited in the liver, Cd complexed with metallothionein remains tightly bound during glomerular filtration, and most of the dose of Cd is deposited in the kidney. 2 Different chemically erosslinked polymers of MT have been prepared. Cadmuim bound to these polymers remains in circulation longer than any other known form of the metal, and the reticuloendothelial system plays a part in its clearance. Using these species, mechanisms of distribution and metabolism of Cd-protein complexes are discussed.

The effects of maternal Zn, Cu, or Fe deficiencies during late gestation on hepatic levels of metals and metallothionein (MT) and the binding of Zn and Cu to protein fractions were investigated in newborn rats. Timed pregnant rats were fed one of the following diets: Zn deficient (Zn-D), Cu-D, Fe-D, or control from day 12 of gestation until birth. The specific nutritional deficiency status of the dams was confirmed by low plasma levels of the deficient metal. Livers from pups were analyzed for MT, metal content, and metal–protein binding. Maternal Zn-D resulted in a greater than 50% reduction of hepatic MT levels in pups, whereas Cu-D and Fe-D had no significant effects. Pups in each deficient group showed a significant decrease in the hepatic levels of the respective metals. Fractionation of hepatic cytosols from the pups by Sephadex G-75 gel filtration showed that in both Fe-D and Cu-D pups the respective metals were depleted from the high molecular weight protein fraction, whereas in Zn-D pups the Zn was depleted mainly from the MT fraction (Ve/V0 ~ 2). Incorporation of [35S]cysteine into MT fractions was significantly lower in Zn-D pups as compared with control pups. These results indicate that there is a specific effect of the maternal Zn-D on the hepatic storage of Zn as MT in newborn rats.

A nonhistone protein component (NHPIns) firmly bound to DNA of rat kidney nuclei has been isolated and partially characterized. In vivo studies show that this protein specifically incorporates 35 to 45 times more 203Hg than any other nuclear protein fractions. No difference in the ratio of NHPIns to DNA between normal and mercury-poisoned rat kidney nuclei was observed. NHPIns protein gives a single major band by sodium dodecyl sulfate gel electrophoresis. Electrophoretic pattern as well as amino acid composition of this protein isolated from both normal and mercurypoisoned rats are also found to be similar. Cysteine content is 1.3 to 1.4 mole per cent.

Metallothionein is a low-molecular-weight protein with a large cysteine content and a high affinity for metals. These properties were utilized in the determination of metallothionein by a polarographic method and by a cadmium saturation method. In this study, these two methods were compared for the estimation of metallothionein from rat liver and kidney after injection with CdCl 2 and ZnSO 4 . Rats were injected ip with CdCl 2 (0.6 mg Cd/kg) or ZnSO 4 (10 mg Zn/kg); liver and kidneys were removed after 16 or 32 hr for metallothionein estimation by both methods. Comparison of the results showed good agreement in liver and kidney (correlation coefficient, r = 0.92 and r = 0.95, respectively). The values obtained by the polarographic method were slightly higher than those by the Cd-saturation method. A dose-dependent increase in metallothionein levels was also observed by both methods when the rats were injected repeatedly (1, 3, 5, or 10 times) with CdCl 2 (1 mg Cd/kg). There was relatively good agreement between these two methods on the determination of both hepatic and renal metallothioneins containing zinc and cadmium.

Summary Metallothionein was induced in rats in response to cadmium and labeled in vivo with either 109Cd, 14C or 35S. The labeled proteins were isolated from liver tissue and their metabolism was investigated after i.v. injection in rats. Tissue distribution and urinary excretion of cadmium from 109Cd-metallothionein and 109CdCl2 were different. The deposition of cadmium in kidney was higher than in liver and a significant urinary excretion of the metal was observed when administered in the form of metallothionein. In kidney and in urine 109Cd remained bound to metallothionein. The distribution and excretion of the protein moiety was very similar to that of the metal. However, gel filtration of kidney soluble fraction and urine indicated some degradation of the protein.

Although the injection of cadmium salts increases the metallothionein (MT) concentration in a number of organs, including pancreas, the induced synthesis of pancreatic metallothionein was specifically decreased in zinc deficiency. The fate of Cd-induced metallothionein and the change in tissue distribution of Zn and Cd were studied with the onset of Zn deficiency. Rats were repeatedly injected with CdCl2 to induce MT synthesis, and were subsequently made Zn deficient. The lability of the pancreatic Zn pool, as compared with that of liver and kidney, was demonstrated. During Zn deficiency, there was little change in Cd concentration or MT level in either liver or kidney. When Zn deficiency led to a decrease in pre-existing pancreatic MT, Cd was not eliminated from the pancreas, but was transferred to a high molecular weight fraction. On the basis of these results we suggest that the presence of Cd-Zn-MT has little effect on the eventual pancreatic Zn and MT contents, during subsequent Zn deficiency.

Polymers of cadmium metallothionein I and II were prepared and used for distribution studies. Mice were intravenously injected with comparable doses of cadmium as chloride, metallothionein and metallothionein-polymer and were killed at 3 h and 24 h after injection. A considerably higher concentration of cadmium was found in the plasma and spleen of animals exposed to metallothionein-polymer than other groups, while the cadmium concentration in the kidney was less than, but in the liver was greater than those injected with cadmium-metallothionein. Gel filtration of liver and kidney cytosols showed that metallothionein is the most stable form of cadmium in the tissues. The injected polymer was degraded into the monomer within 24 h in tissues. The high concentration of polymer in plasma indicates a slow clearance of cadmium in this form. The increased antigenicity of metallothionein-polymer may be related to its slow clearance from blood and increased deposition in spleen.

Cadmium-thionein (Cd-Th) was isolated from livers of rats repeatedly treated with small doses of cadmium chloride. A major portion (55 per cent) of intraperitoneally injected Cd-Th was deposited in the kidney followed by renal damage. Subcellular fractionation of kidney homogenate at 1, 4, 24 and 48 hr after Cd-Th injection revealed that only less than 7 per cent of the cadmium war, accumulated in the lysosomal fraction and more than 50 per cent of cadmium was recovered from the cytosol. Injection of [ 35 S]cysteine into rats pre-injected with sublethal doses of Cd-Th resulted in the incorporation of radioactivity into newly synthesized thionein in the kidney between 4 and 24 hr. A major portion of the renal cadmium was bound to metallothionein at all time intervals studied. These studies demonstrated that the Cd-Th complex in the processes of entering the renal cells intact produces cell injury. The thionein moiety of the injected Cd-Th is degraded and resynthesized in the kidney, while nearly all of the metal ( > 90 per cent) is always bound to thionein. The role of the lysosomal system in the renal toxicity of Cd-Th is minimal.

The effects of cadmium-metallothionein (Cd-MT) on organic ion uptake in renal cortical slices and lipid peroxidation in the kidney were studied in rats. For in vitro studies, slices were prepared from kidneys of control animals and incubated in buffer containing either cadmium chloride (CdCl2) or Cd-MT in equimolar Cd concentrations ranging from 5 x 10(-6) to 2 x 10(-4) M. Uptake into the slices of the organic anion p-aminohippuric acid (PAH) was found to be inhibited by both forms of Cd in a dose-dependent manner. Although this inhibition was slightly greater in the presence of Cd-MT, accumulation of Cd into the slices was approximately 12 times greater with CdCl2 than Cd-MT. Tetraethylammonium (TEA) uptake was less sensitive to the inhibitory effects of both CdCl2 and Cd-MT, although a dose-dependent inhibition did occur with higher Cd concentrations. To study the in vivo effects of Cd-MT on transport function and lipid peroxidation in the kidney, rats were injected with Cd-MT (0.3 mg Cd per kilogram body weight [bw]) and sacrificed at specific time intervals. Similar to the in vitro studies, PAH uptake into the renal cortical slices was markedly inhibited within 12 hours after Cd-MT injection whereas inhibition of TEA uptake was less and not observed until 48 hours after injection. Only a small increase (1.4-fold) in lipid peroxidation, as measured by generation of malondialdehyde (MDA), in the kidney was detected at four hours postinjection, and no further increase was observed at later time periods. The results suggest that Cd-MT affects the transport of organic anions and cations during its renal uptake but that lipid peroxidation may play only a minor role in Cd-MT-induced renal toxicity.

Body retention and tissue distribution of Cd from oral 109 CdCl 2 and 109 Cd‐metallothionein were studied in C57BL/6J mice given identical doses (60 μg Cd and 2 μCi 109 Cd). Measurement of 109 Cd radioactivity in mice showed identical absorption but differences in tissue distribution of Cd from the two forms. A significantly greater deposition of Cd was observed in the kidney of mice fed Cd‐metallothionein than in the CdCl 2 group where a major portion of Cd was deposited in the liver. Most of the radioactive Cd in the renal and hepatic tissues and in the duodenum was bound to metaltothionein 24 h after oral Cd‐metallothionein, whereas only 30 and 38% of the cadmium in liver and kidney supernatants, respectively, were bound to metallothionein after oral CdCI 2 . These results suggest that the dietary form of Cd may influence the renal deposition of Cd.

The liver of 7-day-old rats contains high levels of metallothionein (MT) and zinc, which can be reduced markedly following treatment of neonates with dexamethasone (1 mg/kg, s.c.) twice daily on days 3, 4, 5 and 6 postpartum. Prior treatment with trace amounts of 65Zn and 109Cd did neither alter the basal MT and zinc concentrations nor their response to dexamethasone. The turnover of 65Zn or 109Cd in newborn rats was studied by measuring the whole body retention of the radioisotopes. Between days 3 and 7 postpartum, the normal turnover of 65Zn was much faster than that of 109Cd, and dexamethasone treatment resulted in further decrease of 65Zn retention without significantly altering that of 109Cd. The data indicate that dexamethasone enhanced the excretion of 65Zn without any effect on 109Cd excretion. The dexamethasone treatment resulted in marked reduction in both hepatic Zn and MT levels in newborn rats. Gel filtration on Sephadex G-75 columns of hepatic cytosols isolated from 7-day-old rats showed significant amounts of total Zn and 65Zn bound to MT fraction. Moreover, dexamethasone treatment markedly reduced their binding to the MT fraction suggesting that most of the Zn lost from the liver of dexamethasone-treated animals was from MT. About 95% of 109Cd in the liver cytosol was present in the MT fraction of 109Cd-injected rats and dexamethasone had little effect on the binding of 109Cd to MT fraction despite a marked reduction in the binding of total Zn. These data suggest that unlike in adult rats, injection of dexamethasone results in marked decrease in hepatic levels of Zn and MT in neonatal rats.

Even though the induce synthesis of metallothionein (MT) after exposure to metals has been known for some time, there is little data on the quantitation of MT in various tissues. In this study we have measured MT levels in eleven different tissues of rat after injection of CdCl2 and also compared the relative MT inducibility of these tissues. Over a period of 16 days, rats were injected subcutaneously once every second day with either saline or 3 different doses - 0.8, 1.5 or 3 mg (7.2, 13.4 or 26.8 mu mol) Cd/kg of CdCl2 and sacrificed 48 h after the last injection. Cadmium and MT were determined in brain, lung, heart, liver, kidney, stomach, small intestine, pancreas, spleen, testes and muscle. A dose dependent increase in MT accumulation was observed in a number of tissues after CdCl2 injection, the highest amount being in liver, kidney and pancreas. Further analysis of the data showed a positive correlation between Cd and MT concentrations in 8 tissues. The relative MT inducibility in liver, pancreas, heart and stomach in response to Cd injection was identical.

Single cells were isolated from rat (100-150 g) kidney cortex slices by mild trypsinization. They were plated on plastic culture dishes and were maintained in a selective Eagle's D-Valine Minimum Essential Media (MEM) to form an epithelial cell monolayer culture. The fibroblast growth was retarded by D-Valine. The cells in monolayer culture accumulated cadmium when they were incubated with a 109CdCl2 (10(-5) M) containing medium. The synthesis of metallothionein by these cells was demonstrated by incorporation of [35S]cysteine and 109CdCl2 in a heat stable protein (mol. wt 10,000) within 8 h of exposure and also by immunoprecipitation using a specific antibody to rat liver metallothionein. The cytotoxic effects of Zn2+, Cu2+, Cd2+ and Hg2+ were studied in culture after addition of various concentrations of metal salts (10(-5)-10(-2) M). Disrupted cellular colonies with severe cell damage were observed after addition of 10(-3) M Cd2+ or Hg2+ as CdCl2 or HgCl2 while similar toxicity was observed only after addition of 10(-2) M Zn2+ or Cu2+ as ZnSO4 or CuSO4 to confluent cell cultures. The cellular damage due to Cd2+ was protected when the cells were pretreated with Cd2+ (10(-5) M) for 24 h and these cells could tolerate much higher concentrations of cadmium (10(-2) M). These results indicate a direct protective role of intracellular metallothionein in the cellular toxicity of cadmium.

Cadmium and metallothionein (MT) levels were measured in lungs derived from Lewis rats exposed 0, 5, 10, 15, 20, 24, or 30 times to a Cd acetate aerosol (1.6 mg/m3). Cd burden rose from 2.21 micrograms in control lungs to 42.55 micrograms in lungs of animals exposed 30 times to the Cd aerosol. Pulmonary MT, expressed as nmoles Cd-thionein, paralleled Cd retention. Levels increased from 7.5 nmol in control animals to a value of 340.5 nmol in animals exposed 30 times to Cd. Energy dispersive X-ray microanalysis and the laser micro-probe analyzer were used to localize Cd within the lung. Lung tissue was prepared by a variety of chemical and physical fixation procedures. Cd localization proved unsuccessful by these techniques, presumably because the cellular Cd levels were below the limits of detectability. Using a immunohistochemical procedure which employed an antibody to MT, pulmonary MT was found in alveolar epithelial cells, fibroblasts, and lymphocytes. The MT was restricted to the nuclei of these cells.