M. George Cherian

Metallothioneins and their role in the metabolism and toxicity of metals

Abstract Recent investigations have provided considerable new information regarding the biological role of metallothioneins. The synthesis of this protein is induced in cells by certain metals. It can tightly bind with zinc, copper, cadmium, mercury or silver reducing the availability of diffusible forms of these metals within cells and therefore decreasing their toxic potential. The metallothioneins may also have an important role in regulating the normal absorption and homeostasis of zinc and copper. It is paradoxical, however, in that a protein synthesized within the cell to reduce toxicity, may, in itself, be toxic when excreted or leaked out from the cell to the extracellular space. Further studies are required to elucidate the mechanisms involved in these effects.

Comparison of metallothionein determination by polarographic and cadmium-saturation methods☆

Abstract 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.

The induced synthesis of metallothionein in various tissues of rat in response to metals. I. Effect of repeated injection of cadmium salts.

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.

Metallothionein in radiation exposure : its induction and protective role

Since its discovery about 40 years ago, there has been a wide interdisciplinary research interest in metallothionein (MT) on its physiological and toxicological aspects. Functionally, MT is involved not only in metal detoxification and homeostasis, but also in scavenging free radicals during oxidative damage. Among over 4500 publications which can be retrieved by Medline search, only about 50 reports have been published on the relationship of MT with ionizing and UV radiation. In this review, we have evaluated critically the published data on the induced synthesis of MT by radiation, and the potential functions of MT in radiation induced cell damage. MT mRNA expression or MT synthesis was found to be induced by exposure of cells in vitro or tissues in vivo to ionizing or UV radiation. In most of the studies in animals and tissue cultures, high doses of ionizing radiation were used to induce MT, and, therefore, it is difficult to extrapolate these results to low level of repeated exposures to radiation in humans. Induced synthesis of MT is considered as one of the mechanisms involved in the adaptive response to low dose radiation exposure. The presence of MT in normal cells may provide protective effects from radiation-induced genotoxicity and cytotoxicity. However, in tumor cells, the presence of MT can result in drug and radiation resistance as well. These effects are modulated by other cellular factors, besides MT, such as antioxidants, and by the cell cycle stages in cell proliferation and differentiation.

Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of rat liver and kidney

Abstract 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.

Changes in the intracellular accumulation and distribution of metallothionein in rat liver and kidney during postnatal development

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.

Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of fetal human liver and kidney and its changes during development.

&NA; The distribution of metallothionein (MT) during human development was investigated using both immunohistochemical and biochemical methods. The level of MT in the fetal liver was higher than the adult liver levels. Higher levels of zinc (Zn) and copper (Cu) were also detected in the fetal liver compared to the adult liver. Although cadmium (Cd) was present in detectable levels in the human adult liver, none was detected in the human fetal liver. MT was localized in the nucleus and the cytoplasm of human fetal and neonatal hepatocytes, using a specific rabbit antibody raised to rat liver MT. In the adult human liver cells, MT was localized mainly in the cytoplasm. In the fetal and neonatal human kidney, MT was localized mainly in the nucleus and the cytoplasm of the proximal tubular epithelial cells. In the adult kidney, in addition to nuclear‐cytoplasmic localization of MT, intraluminal localization was also observed.

The induced synthesis of metallothionein in various tissues of rats in response to metals. II. Influence of zinc status and specific effect on pancreatic metallothionein.

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 ZnSO4 (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 ZnSO4 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 CdCl2 (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 CdCl2. 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.

The search for chelate antagonists for chronic cadmium intoxication.

Cadmium is unique among the metals because of its combination of toxicity in low dosages, long biological half-life (of about 30 years in humans), its low rate of excretion from the body and the fact that it is stored predominantly in the soft tissues (liver and kidney). There has been an increase in exposure to cadmium because its presence in fertilizers and sewage sludge and also its increased industrial use in Cd-Ni batteries. Although there are a number of reports on occupational and environmental exposures to cadmium compounds, treatment of cadmium poisoning has been difficult because there is neither a safe practical means of evaluating bioavailable body burden nor is there a recommended therapeutic chelating agent for chronic cadmium intoxication. In this review, the various factors affecting the chelation of cadmium such as its binding to intracellular metallothionein, the structural requirements of compounds for effective removal of cadmium, the excretion pattern of cadmium after its mobilization from intracellular stores and the recent developments in the design and synthesis of new compounds for cadmium chelation are discussed. The importance of protecting sensitive organs such as kidney and brain during cadmium chelation is addressed. The progress made during the last decade on the synthesis of new compounds, especially derivatives of dithiocarbamates, is remarkable. Some of these compounds provide promise for development of a useful and safe therapeutic chelating agent which can be used for the assessment of cadmium body burden and for preventive removal of cadmium as well as for use in overt cadmium poisoning in humans.

Protective roles of metallothionein and glutathione in hepatotoxicity of cadmium

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.