Apo-metallothionein emerging as a major player in the cellular activities of metallothionein

Observations of apo-metallothionein (apo-MT) have been made under a variety of physiologic circumstances, including zinc deficiency in cell culture and in rodents, cellular induction of MT by dexamethasone with concurrent Zn deficiency, a variety of tumors under normal Zn conditions, MT induction by Zn and Bi citrate, induction of hepatic MT after tumor cell injection into nude mice, and overexpression of cardiac MT in MT transgenic mice. Experiments demonstrating both the lability of Zn and Cu bound to MT and the cellular stability of apo-MT are described to help rationalize the widespread presence of this metal-depleted species. Finally, comparative in vitro and cellular experiments examined the relative reactivity of Zn- and apo-MT with nitric oxide species, showing that apo-MT is much more reactive chemically and that in cells it may be a principal reactive species within the MT pool.     

Zinc depletion suppresses tumor growth in mice

The hypothesis that in tumor-bearing animals an increase of host hepatic zinc metallothionein (Zn-MT) causes a restriction of zinc in the tumor tissue was studied. Three types of tumors were induced in laboratory mice by cell transplant. Tumor growth appears to be inhibited under zinc-deficient conditions, even in cases where zinc deficiency was started after tumor cell transplant. The survival times of tumor-bearing mice were prolonged by administration of cadmium chloride, which induces the synthesis of a combined zinc-cadmium metallothionein derivative in the host liver, but not in the tumor tissue, leading to an increase of hepatic zinc in the treated animals. The uptake of⁶⁵Zn by the liver of Cd-treated, tumor bearing mice was significantly higher than that of controls whereas uptake of⁶⁵Zn by tumor cells was significantly higher in controls than in the treated animals. These results suggest that restriction of zinc intake suppresses tumor growth.     

Elevation of hepatic levels of metallothionein and zinc in mice bearing experimental tumors.

A tumor growth-dependent elevation in the hepatic levels of Zn and metallothionein (MT), without a change in the level of Cu, was found in mice and rats bearing solid tumors in the inguinal region. The levels of Zn and MT thus elevated gave a significant correlation (r = 0.95) between them. Nevertheless, when tumor-bearing mice and rats were fed a Zn-deficient diet, the hepatic levels of Zn and MT did not increase. In mice in which inflammation was induced at the same region, on the other hand, hepatic levels of Zn and MT increased transiently after the injection of turpentine or carrageenan even when they were fed the Zn-deficient diet. These results suggest that the elevation of MT and Zn levels can be a helpful marker for detecting malignancy.     

Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+

The cadmium-binding properties of rabbit liver Zn7-metallothionein (MT) 2 and apo-MT, rat liver apo-alpha MT and Zn4-alpha MT, and calf liver apo-beta MT, have been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. Both sets of spectra recorded during the titration of Zn7-MT 2 with Cd2+ exhibit a complicated pattern that is quite unexpected. Such behavior is not found at all in sets of spectra recorded during titrations of the apo-species (apo-MT, apo-alpha MT, and apo-beta MT), and is observed to a much lesser extent in the titration of Zn-alpha MT. Comparison between the band centers of the Cd-alpha MT and Cd-beta MT indicates that the CD spectrum of Cd7-MT is dominated by intensity from transitions that originate on Cd-S chromophores in the alpha domain, with little direct contribution from the beta domain. Analysis of the spectra recorded during titrations of Zn7-MT 2 with Cd2+ suggests: (i) that Cd2+ replaces Zn2+ in Zn7-MT isomorphously; (ii) that cadmium binds in a nonspecific, "distributed" manner across both domains; (iii) that cluster formation in the alpha domain only occurs after 4 mol eq of cadmium have been added and is indicated by the presence of a cluster-sensitive, CD spectral feature; (iv) that the characteristic derivative CD spectrum of native Cd4,Zn3-MT is only obtained from "synthetic" Cd4,Zn3-MT following a treatment cycle that allows the redistribution of cadmium into the alpha domain; warming the synthetic "native," Cd4,Zn3-MT, to 65 degrees C results in cadmium being preferentially bound in the alpha domain; and (v) Zn7-MT will bind Cd2+ quite normally at up to 65 degrees C but with greater specificity for the alpha domain compared with titrations carried out at 25 degrees C. These results suggest that the initial presence of zinc in both domains is an important factor in the lack of any domain specificity during cadmium binding to Zn-MT which contrasts the domain specific manner observed for cadmium binding to apo-MT.     

Gestational zinc deficiency amplifies the regulation of metallothionein induction in adult mice

To determine if prenatal zinc deficiency has a persistent effect on metallothionein (MT) regulation, Swiss-Webster mice were mated and fed a diet containing either control (100 micrograms Zn/g) or low levels of zinc (5 micrograms Zn/g) from Day 7 of gestation to parturition. After birth all mice were given the control diet. Liver zinc and MT levels were 50% lower in newborn pups from dams fed the low zinc diets than in control pups. In control pups, liver zinc and MT concentrations were relatively stable during the first week of postnatal life. In contrast, in pups prenatally deprived of zinc, liver levels of zinc and MT increased such that by Day 3 of postnatal life, the levels were not significantly different from controls. At Day 56, serum IgM concentrations were significantly lower in the low zinc offspring. Liver zinc concentrations in the two groups of mice were similar at Day 70 postnatal, and in both groups liver MT levels were below detection limits. However, when Day 70 mice were given zinc injections to stimulate MT synthesis, the prenatally zinc deprived offspring showed markedly higher liver MT levels than did control mice given similar injections, despite similar liver zinc concentrations in the two groups. These results show that prenatal zinc deficiency has pronounced effects on postnatal MT metabolism which can persist into adulthood.     

Effect of surgically induced cholestasis on the levels of hepatic zinc and metallothionein in rat liver

Early effects of experimental cholestasis on the homeostasis of zinc (Zn) and metallothionein (MT) were studied in rats which had undergone bile duct ligation for 0, 3, 6, 9, 12, 16, 20, and 24 h. Transient increases in hepatic Zn levels were observed at 9 h but returned to control values at 12 h. Serum Zn levels increased at 24 h. Cholestasis was confirmed by increased serum alkaline phosphatase (AP) activity. MT increased at 3 h and reached a maximum level at 12 h and remained elevated even at 24 h after the onset of experimental cholestasis. No hepatocellular damage was detected according to the results of alanine aminotransferase (ALT) activities in serum. These results shown that the increases in Zn observed in liver are related to bile stagnation rather than to a hepatocellular damage and that increased MT occurs concurrently with increased hepatic Zn. These observations suggest that the cellular levels of Zn in cholestasis is regulated by homeostatic mechanisms, of which one could be mediated by MT.     

锌_7与镉_7－金属硫蛋白清除羟自由基的比较

分离及纯化兔肝金属硫蛋白制备去金属金属硫蛋白、锌７与镉７金属硫蛋白．在不同ｐＨ条件下,比较后二者清除羟自由基能力;在ｐＨ６条件下,比较锌７－金属硫蛋白与有关蛋白和无机锌盐清除羟自由基效果．结论是在近生理ｐＨ条件下锌７－金属硫蛋白清除羟自由基能力远强于镉７－金属硫蛋白．金属硫蛋白清除羟自由基的能力主要来源于蛋白中处于还原态的流基．     

Hepatic responses to dietary stress in zinc- and metallothionein-deficient mice

Metallothionein (MT) and zinc are both reported to be protective against oxidative and inflammatory stress and may also influence energy metabolism. The role of MT in regulating intracellular labile zinc, thus influencing zinc (Zn)-modulated protein activity, may be a key factor in the response to stress and other metabolic challenges. The objective of this study was to investigate the influence of dietary zinc intake and MT on hepatic responses to a pro-oxidant stress and energy challenge in the form of a high dietary intake of linoleic acid, an omega-6 polyunsaturated fatty acid. Male MT-null (KO) and wild-type (WT) mice, aged 16 weeks, were given semisynthetic diets containing 16% fat and either 5 (marginally zinc-deficient [ZD]) or 35 (zinc-adequate [ZA]) mg Zn/kg. For comparison, separate groups of KO and WT mice were given a rodent chow diet containing 3.36% fat and 86.6 mg Zn/kg. After 4 months on these diets, the body weights of all mice were equal, but liver size, weight, and lipid content were much greater in the animals that consumed semisynthetic diets compared to the chow diet. The increase in liver size was significantly lower in ZA but not ZD KO mice, compared with WT mice. Principally, MT appears to affect the diet-induced increase in liver tissue but it also influences the concentration of hepatic lipid. Plasma levels of C-reactive protein (CRP), a marker of inflammation, were increased by zinc deficiency in WT mice, suggesting that marginal zinc deficiency is proinflammatory. CRP was unaffected by zinc deficiency in KO mice, indicating a role for MT in modulating the influence of zinc. Neither zinc nor MT deficiency affects the level of soluble liver proteins, as determined using two-dimensional (2D) gel proteomics. This study highlights the close association between zinc and MT in the manifestation of stress responses.     

Induction of metallothionein by zinc in lethal milk mutant mice

Adult lethal milk (lm/lm) mutant mice display increased induction of hepatic metallothionein synthesis compared to wild-type mice following the subcutaneous injection of 40 µmol ZnCl₂/kg mouse. At this zinc dose the rate of incorporation of |³⁵S| cysteine into hepatic metallothionein in adult (100-to 230-day-old) lm/lm mice was approximately 2.4-fold greater than the rate of incorporation of isotope in wild-type animals. At a higher zinc dose (160 µmol ZnCl₂/kg) the incorporation of |³⁵S| cysteine into hepatic metallothionein was similar in lm/lm and wild-type mice. The altered dose-response to zinc administration was not due to a change in hepatic zinc, copper, or manganese levels, to a difference in ⁶⁵Zn uptake, or to an alteration in ⁶⁵Zn bound to differential centrifugation fractions of adult lm/lm liver. ⁶⁵Zn bound to hepatic metallothionein was, however, increased in aging lm/lm mice with symptomatic skin lesions.     

Induction of Metallothionein in Rat Liver by Zinc Exposure: A Dose and Time Dependent Study

Metallothioneins (MTs) are low molecular weight ubiquitous metalloproteins with high cysteine (thiol) content. The intracellular concentration of zinc (Zn) is tightly regulated and MT plays a crucial role in it. The present study investigates the relationship between the Zn status (as a function of Zn concentration and time) in the rat liver and the occurrence of hepatic MT. For dose dependent study, four experimental groups, one control and three receiving different levels of metal supplementation, were chosen [Group 1 control and Group 2, Group 3, Group 4 receiving subcutaneous dose of 10, 50 and 100 mg of Zn/kg body weight (in the form of ZnSO₄·7H₂O), respectively]. For the time dependent expression of MT, again four experimental groups, i.e. Group 5 control and Group 6, Group 7, Group 8 receiving 50 mg of Zn/kg body weight (in the form of ZnSO₄·7H₂O) subcutaneously and sacrificed at different time intervals after last injection i.e. 6, 18, 48 h, respectively were chosen. Isolation of MT was done by using combination of gel filtration and ion exchange chromatography while characterization of MT fraction was carried in the wavelength range 200–400 nm. Expression of MT was studied by using Western blot analysis. The results revealed that the MT expression increases with increasing the dose of Zn administered and maximum at 18 h after last Zn injection. Accumulation of MT with increase dose would help in maintaining the intracellular Zn concentration by its sequestration which further reduces the possibility of undesirable binding of Zn to other proteins significantly and maintains Zn homeostasis. The maximum expression of MT at 18 h is indicative of its half life.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

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.     

Zinc binding ligands and cellular zinc trafficking: apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn(2+) binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial "thermodynamic sink". Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn(2+) chelating agents including N,N,N',N'-(2-pyridylethyl)ethylenediammine (TPEN), EDTA, and [(2,2'-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2-3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK(1) cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn(3)-Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn(3)-Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn(3)-Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK(1) cells to TPEN for 24h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Zn-proteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn(2+).     

Parenteral zinc and tissue metallothionein in normal and diabetic rats

The effect of parenteral zinc on tissue metallothionein (MT) was studied in normal and streptozotocin-induced diabetic rats. The accumulation of Zn-MT in liver and pancreas of normal and diabetic rats following the administration (ip) of various amounts of zinc was not different. Renal Zn-MT was higher in the diabetic group, and this was not changed by zinc injection. Although diabetic rats, relative to normal, possessed a markedly higher concentration of Cu-MT in kidney initially, this difference decreased considerably after zinc injection. The ratio of Cu-MT to cytosolic Cu in kidney was not affected by parenteral zinc and was highest in diabetic rats. Zinc injection markedly reduced food intake, water consumption, and urine output in both normal and diabetic rats. Blood glucose of diabetic rats also decreased 24 h after zinc administration. Our results indicate that relative to normal, MT and zinc metabolism are different in kidney, and to some extent liver, but not different in the pancreas of the chemically induced diabetic rat.     

Displacement of zinc and copper from copper-induced metallothionein by cadmium and by mercury: in vivo and ex vivo studies

The in vitro affinity of metals for metallothionein (MT) is Zn less than Cd less than Cu less than Hg. In a previous study Cd(II) and Hg(II) displaced Zn(II) from rat hepatic Zn7-MT in vivo and ex vivo (Day et al., 1984, Chem. Biol. Interact. 50, 159-174). The ability of Cd(II) or Hg(II) to displace Zn(II) and/or Cu(II) from metallothionein in copper-preinduced rat liver (Zn, Cu-MT) was assessed. Cd(II) and Hg(II) can displace zinc from (Zn, Cu)-MT both in vivo and ex vivo. The in vitro displacement of copper from MT by Hg(II) was not confirmed in vivo and ex vivo. Cd(II) treatment did not alter copper levels in (Zn, Cu)-MT, as expected. Hg(II) treatment, however, did not decrease copper levels in MT, but rather increased them. The sum of the copper increase and mercury incorporation into MT matched the zinc decrease under in vivo conditions and actually exceeded the zinc decrease under ex vivo conditions. Short-term exposure of rat liver to exogenous metals can result in incorporation of these metals into MT by displacement of zinc from pre-existing MT. Displacement of copper from pre-existing MT by mercury, as predicted by in vitro experiments, was not confirmed under the conditions of our in vivo and ex vivo experiments. This result is explainable based on the differing affinities and/or preferences of the two metal clusters in MT.     

Effect and possible role of Zn treatment in LEC rats,an animal model of Wilson's disease

The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long-Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.     

Elimination of zinc-65 from the brain under kainate-induced seizures

On the basis of the previous evidence that 65Zn concentrations in the brain of EL (epilepsy) mice was affected by induction of seizures, 65Zn movement in the brain was quantitatively evaluated in ddY mice treated with kainate. Six days after intravenous injection of 65ZnCl2, mice were intraperitoneally injected with kainate (10 mg/kg x 6 times in 2 weeks). Myoclonic jerks were observed during treatment with kainate. Twenty days after 65Zn injection, 65Zn distribution in the brain was compared between the kainite-treated and control mice. 65Zn distribution in the brain of the kainate-treated mice was overall lower than in the control mice. 65Zn concentration was significantly decreased in the frontal cortex, hippocampal CA1, thalamus and hypothalamus by treatment with kainate. These results demonstrate that kainate-induced seizures are linked to decreased zinc concentrations in the brain.     

Changes in plasma zinc,copper, iron,and hepatic metallothionein in adjuvant-induced arthritis treated with cyclosporin

The early changes in hepatic metallothionein (MT) and plasma zinc (Zn), copper (Cu), and iron (Fe) were investigated during the induction of adjuvant (AJ) arthritis in rats in conjunction with cyclosporin (CSA) treatment. Plasma Zn decreased after AJ injection (60% of control values at 8 h), and this was associated with a 4.5-fold increase in hepatic MT at 8 h. Plasma Zn was lowest at 16 h (40% of control), whereas hepatic MT concentrations increased to a maximum of 20-fold at 16 h. Changes in plasma Fe paralleled those of Zn, whereas plasma Cu levels were increased. Plasma metal and hepatic MT concentrations returned toward normal from d 1–7. At d 14, when marked paw swelling was apparent, hepatic MT and plasma Cu were again increased and plasma Zn decreased. Administration of CsA decreased MT induction in rats injected with AJ and also caused a marked recovery in plasma Zn and Fe levels. These changes were small but significant even in the early stages (up to 24 h) after AJ injection and were followed by a sustained improvement in all parameters, corresponding to the nonappearance of clinical arthropathy in CsA-treated rats. TNF-α and IL-6 production by peritoneal macrophages isolated from AJ-injected rats was significantly decreased by CsA treatment at d 7 and 14. The inhibition of hepatic MT induction during acute and chronic inflammation by cyclosporin emphasizes the role of the immune system in altered metal homeostasis in inflammation.     

Determination of metallothionein-1/metallothionein-2 ratios in the mouse liver and pancreas by capillary zone electrophoresis using a polyacrylamide-coated capillary at neutral pH

Metallothionein (MT) isoforms, MT-1 and MT-2, in biological specimens are clearly separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated capillary. The effectiveness of CZE analysis in the study of MT isoforms in biological specimens is discussed. We did two experiments to determine the MT-1/MT-2 ratio in biological specimens. The ratio of MT-1/MT-2 can be determined by CZE under a neutral pH without any detergents. One of these studies is time-dependent changes of the MT-1/MT-2 ratio in the cytosol of the pancreas and liver in mice after Zn or Cd injection. In the pancreas, both isoforms were detected in the control mice and the ratio of MT-1/MT-2 was below 1.0. When Zn was injected, the maximum peak areas of both isoforms were obtained at 24 h, and the ratios increased over a value of 1.0 at 3 h and peaked at 10 h. However, in the Cd-injected mice, the peak areas of both isoforms increased up to 72 h, and the ratios were below 1.0 up to 72 h. On the contrary, neither isoform was detected in the livers of control mice. The ratios of Zn-injected mice liver were near the value 1.0 between 6 and 72 h, although the areas of both isoforms showed peaks at 48 h. The ratios of Cd-injected mice livers were detected to be over 1.0 from 10 h, but there were no significant difference between 10 and 72 h, and the areas of both isoforms showed peaks at 24 h. The other experiment investigated the ratio in each fraction of cell fractionation. Cell fractionation was done in the livers of Zn-treated mice. Twenty-four hours after the injection, the ratio of MT-1/MT-2 was 0.80+/-0.12 and 1.19+/-0.21 (mean+/-SD) in nuclear and cytosol fractions, respectively. Neither isoform was detected in mitochondrial or microsomal fraction. From the present results, CZE analysis is a suitable method for observation of the ratio of MT-1/MT-2 in biological specimens, and dynamic changes in both isoforms can be detected.     

Effect and possible role of Zn treatment in LEC rats,an animal model of Wilson's disease

The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long–Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.     

Early effects of surgery on zinc and metallothionein levels in female rats

Time-response effects of experimental surgery on zinc (Zn) and metallothionein (MT) homeostasis were investigated in female rats up to 24 h. Hepatic Zn content increased at 20 and 24 h postsurgery, whereas serum Zn levels decreased. Hepatic MT increased significantly by 9 h postsurgery and peaked at up to twofold of control at 12 h after surgery. Following the peak at 12 h, hepatic MT content decreased with time but did not reach control levels at the end of this study. When MT isoforms were evaluated, MT-II levels were elevated to the highest extent by 12 h after surgery, whereas MT-I levels started to decrease after 3 h postsurgery but then increased by 20 h. The early increases in MT content are probably mediated by nonmetallic mediators released during the postsurgical inflammatory process, favoring the plasma/tissue mobilization of Zn. This process might be part of the overall mechanisms occurring in the inflammation.