Nucleophilic Distribution of Metallothionein in Human Tumor Cells

Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 μMCdCl₂did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell.     

Metallothionein redox cycle and function

The biologic function of metallothionein (MT) has been a perplexing topic ever since the discovery of this protein. Many studies have suggested that MT plays a role in the homeostasis of essential metals such as zinc and copper, detoxification of toxic metals such as cadmium, and protection against oxidative stress. However, mechanistic insights into the actions of MT have not been adequately achieved. MT contains high levels of sulfur. The mutual affinity of sulfur and transition metals makes the binding of these metals to MT thermodynamically stable. Under physiologic conditions, zinc-MT is the predominant form of the metal-binding protein. The recognition of the redox regulation of zinc release from or binding to MT provides an alternate perspective on biologic function of MT. Oxidation of the thiolate cluster by a number of mild cellular oxidants causes zinc release and formation of MT-disulfide (or thionin if all metals are released from MT, but this is unlikely to occur in vivo), which have been demonstrated in vivo. Therefore, the thermodynamic stability of zinc binding makes MT an ideal zinc reservoir in vivo, and the redox regulation of zinc mobilization enables MT function in zinc homeostasis. MT-disulfide can be reduced by glutathione in the presence of selenium catalyst, restoring the capacity of the protein to bind zinc. This MT redox cycle may play a crucial role in MT biologic function. It may link to the homeostasis of essential metals, detoxification of toxic metals and protection against oxidative stress.     

Metal binding in metallothioneins: Competition for cadmium and zinc between chelex-100 and metal binding sites in metallothionein

A study of the use of the metal chelation properties of Chelex-100 in metal binding reactions of metallothionein (MT), is described. The stoichiometric ratios of bound metals in MT were determined at several stages during a titration in which the Zn(II) in Zn₇MT was displaced by Cd(II), by using Chelex-100 to sequester the free zinc. The stoichiometric ratios provide convincing supporting evidence that the complicated circular dichroism spectral properties observed during the titration arise because the incoming cadmium is distributed across both domains in the protein. It is shown that Chelex-100 does not sequester zinc or cadmium directly from the metallothionein binding sites. Use of Chelex-100 over the temperature range −20 to 65 °C is demonstrated. The chelation capacity of Chelex-100 (in terms of μ metal ion/mg resin) has been determined for a range of elements important in metal toxicology, including: cadmium (33 μ), zinc (22 μ), copper (19 μ), silver (38 μ), lead (40 μ) and mercury (40 μ).     

Cadmium metabolism by rat liver endothelial and Kupffer cells.

The metabolism of cadmium was investigated in Wistar-rat liver non-parenchymal cells. Kupffer and endothelial cells, the major cell populations lining the sinusoidal tracts, were isolated by collagenase dispersion and purified by centrifugal elutriation. At 20 h after subcutaneous injection of the metal salt (1.5 mg of Cd/kg body weight), endothelial cells accumulated 2-fold higher concentrations of Cd than did Kupffer or parenchymal cells. Most of the Cd in non-parenchymal cells was associated with cytosolic metallothionein (MT), the low-Mr heavy-metal-binding protein(s). When MT was quantified in cytosols from cells isolated from control rats by a 203Hg competitive-binding assay, low levels were found to be present in Kupffer, endothelial and parenchymal cells. Cd injection significantly increased MT levels in all three cell types. The induction of MT synthesis was investigated in vitro by using primary monolayer cultures. The incorporation of [35S]cysteine into MT increased 47% over constitutive levels in endothelial-cell cultures after the addition of 0.8 microM-Cd2+ to the medium for 10 h. MT synthesis in Kupffer cells was not observed. The lack of MT synthesis by monolayer cultures of Kupffer cells in vitro was associated with a decreased capacity of these cells to accumulate heavy metals from the extracellular medium. This apparent decreased ability to transport metals did not reflect a general defect in either cellular function or metabolic activity, since isolated Kupffer cells incorporated [3H]leucine into protein at rates comparable with those shown by liver parenchymal cells and readily phagocytosed particles.     

Signal regulatory protein α regulates the homeostasis of T lymphocytes in the spleen

The molecular basis for formation of lymphoid follicle and its homeostasis in the secondary lymphoid organs remains unclear. Signal regulatory protein α (SIRPα), an Ig superfamily protein that is predominantly expressed in dendritic cells or macrophages, mediates cell-cell signaling by interacting with CD47, another Ig superfamily protein. In this study, we show that the size of the T cell zone as well as the number of CD4(+) T cells were markedly reduced in the spleen of mice bearing a mutant (MT) SIRPα that lacks the cytoplasmic region compared with those of wild-type mice. In addition, the expression of CCL19 and CCL21, as well as of IL-7, which are thought to be important for development or homeostasis of the T cell zone, was markedly decreased in the spleen of SIRPα MT mice. By the use of bone marrow chimera, we found that hematopoietic SIRPα is important for development of the T cell zone as well as the expression of CCL19 and CCL21 in the spleen. The expression of lymphotoxin and its receptor, lymphotoxin β receptor, as well as the in vivo response to lymphotoxin β receptor stimulation were also decreased in the spleen of SIRPα MT mice. CD47-deficient mice also manifested phenotypes similar to SIRPα MT mice. These data suggest that SIRPα as well as its ligand CD47 are thus essential for steady-state homeostasis of T cells in the spleen.     

Levels of heavy metals and their binding protein metallothionein in type 2 diabetics with kidney disease

Hyperglycemia, a major metabolic disturbance present in diabetes, promotes oxidative stress. Activation of antioxidant defense is an important mechanism to prevent cell damage. Levels of heavy metals and their binding proteins can contribute to oxidative stress. Antiradical capacity and levels of metallothionein (MT), metals (zinc and copper), and selected antioxidants (bilirubin, cysteine, and glutathione) were determined in 70 type 2 diabetes mellitus (T2DM) subjects and 80 healthy subjects of Caucasian origin. Single nucleotide polymorphism (rs28366003) in MT gene was detected. Antiradical capacity, conjugated bilirubin, and copper were significantly increased in diabetics, whereas MT and glutathione were decreased. Genotype AA of rs28366003 was associated with higher zinc levels in the diabetic group. The studied parameters were not influenced by renal function. This is the first study comprehensively investigating differences in MT and metals relevant to oxidative stress in T2DM. Ascertained differences indicate increased oxidative stress in T2DM accompanied by abnormalities in non‐enzymatic antioxidant defense systems.     

Metallothionein response following cadmium exposure in the oligochaete Eisenia fetida

We studied the metallothionein (MT) response in cadmium-exposed worms (Eisenia fetida) both at the protein level by Dot Immunobinding Assay (DIA) with a polyclonal antibody raised against the most immunogenic part of this protein and at the expression level by Northern blotting using a specific probe. MT appeared as two close isoforms. DIA results clearly demonstrated significant differences in MT level of whole worm heat-treated supernatants between E. fetida exposed to Cd concentrations as low as 8 mg Cd kg(-1) of dry soil compared to controls. Northern blotting analysis performed on whole bodies of worms revealed that a single exposure to 8 mg Cd kg(-1) of dry soil for 1 day resulted in the production of MT mRNA. This response was maintained for exposure of at least 1 month. Clear differences of MT gene expression were also observed between worms exposed to different Cd concentrations (8, 80 or 800 mg Cd kg(-1) of dry soil). Immunocytochemistry demonstrated that MT was located in the chloragogenous tissue surrounding the gut where metals are known to be accumulated. This work revealed that E. fetida MT is a sensitive and relevant biomarker of Cd exposure and especially when considering gene expression response. Further experiments have now to prove its usefulness in natural metal-contaminated soil toxicity assessments.     

Analysis of stress-induced gene expression in fish cell lines exposed to heavy meals and heat shock

We have examined the effect of heavy metals on the expression of two major groups of stress-induced proteins in fish cell lines: the 70 kDa heat-shock proteins (hsp70) and metallothioneins (MTs). The rainbow trout hepatoma (RTH) cell line synthesized the hsp70 protein in response to zinc and heat shock, while chinook salmon embryonic (CHSE) cells synthesized this protein in response to these inducers, as well as cadmium. The synthesis of this 70 kDa protein was correlated with the accumulation of hsp70 mRNA as measured by hybridization to a trout hsp70 gene probe. Heavy metals also induced the synthesis of MT in RTH cells. However, heat shock did not result in induction of MT and its mRNA. Unlike RTH cells, CHSE cells did not synthesize MT following exposure to cadmium or zinc. When these cells were treated with 5-azacytidine prior to heavy metal treatment, accumulation of MT mRNA was observed. Northern blot analysis of total RNA from 5-azacytidine treated CHSE cells, using a trout MT (tMT-B) cDNA probe, indicated that the time-course of induction and the maximal level of MT mRNA accumulation in response to cadmium and zinc paralleled that observed in RTH cells. Copper and dexamethasone were ineffective in inducing MT mRNA in 5-azacytidine-treated CHSE cells. These results indicate that MT is specifically induced in response to heavy metal treatment, whereas the synthesis of hsp70 appears to be a general stress response. Furthermore, MT is differentially regulated by heavy metals and dexamethasone in these cell lines and the expression of MT is cell-type-specific.     

Metal-dependent protein folding: metallation of metallothionein

Metallothionein (MT), a low molecular mass, cysteine-rich protein, is a model system for metal ion-directed folding due to its diverse metal binding properties. In this minireview, the current status of theoretical and experimental studies that have focused primarily on the initial metallation steps involving the metal-free, or apo, MT and divalent metals, Zn²⁺ and Cd²⁺ is described. Apo-MT has recently been reported to be present in the cell in quantities equal to that of the metallated protein, which might indicate a potential role for the protein in the absence of metals. Molecular mechanics-molecular dynamics (MM3/MD) calculations carried out on the demetallation of cadmium-coordinated MT isoform 1a indicate structural stability of the metal-free protein with significant retention of the backbone conformation imposed by the metal-thiolate clusters present in the metallated holo-protein. Significantly, the cysteinyl sulfurs were found inverted to the outside of a quite compact sphere. In contrast, MM3/MD calculations of apo-MT starting from a linear strand did not possess any structural stability and can be described as a random coil conformation. Evidence for the sequence of metallation is discussed, together with current experimental data to support either a cooperative or sequential binding mechanisms.     

Influence of maternal mineral deficiency on the hepatic metallothionein and zinc in newborn rats

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 approximately 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.     

Various responses to copper and manganese exposure of Carassius auratus gibelio from two populations

The aim of this study was to compare the effect of pro-oxidants copper (Cu(2+), 0.005 and 0.050mg L(-1)) or manganese (Mn(2+), 0.17 and 1.7mg L(-1)) on Carassius auratus gibelio from polluted (B) and unpolluted (Z) sites after exposure for fourteen days. Fish from site B showed high levels of lipid peroxidation (TBARS concentration), lower levels of metallothionein (MT)-related metal, total glutathione (GSH), its redox index, superoxide dismutase (Cu,Zn- and Mn-SOD), glutathione-S-transferase (GST) and cholinesterase (ChE) activities and also higher MT-related thiol concentration in the liver and gills. A common effect of exposure was related to genotoxicity, a decrease in GSH and an increase in microsomal ethoxyresorufin O-deethylase activity in the liver. However, the systems of oxidative stress and biotransformation were more efficient in fish from the polluted site, while the responsivity of MTs in this fish was impaired. Principle Component Analysis separated the subgroups from the unpolluted site and fish loaded by lesser concentrations of metals on the one side, and fish from the polluted site jointly with fish exposed to higher concentrations of metals on the other side. The main distinguishing indices of sites and exposures selected by classification and regression tree (CART) analysis were MT characteristics and genotoxicity.     

Tolerance and stress response of sclerotiogenic <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> G15 to copper and lead

Aspergillus oryzae G15 was cultured on Czapek yeast extract agar medium containing different concentrations of copper and lead to investigate the mechanisms sustaining metal tolerance. The effects of heavy metals on biomass, metal accumulation, metallothionein (MT), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were evaluated. Cu and Pb treatment remarkably delayed sclerotial maturation and inhibited mycelial growth, indicating the toxic effects of the metals. Cu decreased sclerotial biomass, whereas Pb led to an increase in sclerotial biomass. G15 bioadsorbed most Cu and Pb ions on the cell surface, revealing the involvement of the extracellular mechanism. Cu treatment significantly elevated MT level in mycelia, and Pb treatment at concentrations of 50–100 mg/L also caused an increase in MT content in mycelia. Both metals significantly increased MDA level in sclerotia. The variations in MT and MDA levels revealed the appearance of heavy metal-induced oxidative stress. The activities of SOD, CAT, and POD varied with heavy metal concentrations, which demonstrated that tolerance of G15 to Cu and Pb was associated with an efficient antioxidant defense system. In sum, the santioxidative detoxification system allowed the strain to survive in high concentrations of Cu and Pb. G15 depended mostly on sclerotial differentiation to defend against Pb stress.     

Androgens stimulate sex change in protogynous grouper,Epinephelus coioides: spawning performance in sex-changed males

We examined the efficacy of androgens (1.0 mg/kg body mass), testosterone (T), 11-ketotestosterone (11-KT), 17alpha-methyltestosterone (MT), testosterone propionate (TP) or androgen mixture (T, MT and TP in an equal ratio), for induction of sex change in protogynous orange-spotted grouper, Epinephelus coioides. The spawning performance in sex-changed males was also investigated. MT and androgen mixture at a dose of 1.0 mg/kg BW induced a sex transition and completion of spermatogenesis up to the functional male phase. The androgen mixture was most effective. Significantly, higher plasma T levels were found in MT and androgen mixture groups compared to control and other androgen implantation (T, TP or 11-KT) groups. We found that plasma levels of estradiol-17beta (E2) or 11-KT were not different among treated groups. Sex-changed males could successfully fertilize mature eggs. Fertilization and hatching rates were of 23.5-70.4% and 8.4-44.6%, respectively. The data demonstrated that induction of sex change by exogenous androgens in groups could apply to the aquaculture field for seed production.     

Fish and molluscan metallothioneins

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.