metallothionein and zinc-chloride

Interaction of zinc with mercuric chloride and sodium selenite was studied in the rat at the organ and subcellular levels (liver and kidneys). Zinc chloride was administered subcutaneously at dose of 5 mg Zn/kg, mercury chloride into the tail vein at a dose of 0.5 mg Hg/kg (both metals every other day during 2 weeks) and sodium selenite intragastrically, at doses of 0.1 mg Se/kg, every day. Zinc retention in the rat did not exceed 20% and was unchanged in the presence of mercury. An interaction effect was reflected by an increased whole-body retention of zinc by selenium, mercury, and selenium. In the presence of selenium no peak of metallothionein-like proteins stimulated by zinc or mercury was found in the soluble fraction of the kidneys. The metallothionein level did not differ from that typical for control group animals, too. A significant increase in the level of endogenous copper was found only in the kidneys of rats exposed to zinc in the presence of mercury and selenium.

Topics: Animals; Body Burden; Chlorides; Copper; Drug Interactions; Female; Kidney; Liver; Mercuric Chloride; Mercury; Metallothionein; Rats; Rats, Inbred Strains; Selenious Acid; Selenium; Zinc; Zinc Compounds; Zinc Radioisotopes

This study provides a preliminary characterization of a metallothionein (MT) gene in Septifer virgatus and highlights its potential use in biomonitoring. The full-length SvMT cDNA and the complete sequence of the SvMT gene were identified using reverse transcriptase PCR coupled with the rapid amplification of cDNA ends and the primer walking method. The SvMT cDNA encodes a protein of 72 amino acids having nine classical Cys-X-Cys motifs. Moreover, the deduced amino acids contained the conserved motif (Cys-x-Cys-x(3)-Cys-Thr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys) of MT family 2. Its molecular mass and isoelectric point were estimated to be 7.01 kDa and 7.00, respectively. BLAST-based searching indicated that SvMT shared 81.0% amino acid sequence identity with Mytilus edulis MT-20-II. The SvMT gene has three coding exons and two introns. After exposure to 1 mg/L cadmium chloride, the expression of SvMT increased 15-fold by 3 days (d), with a maximum expression of 27-fold by 5 d compared with the pre-exposure level. After exposure to 2 mg/L zinc chloride, the expression of SvMT increased 2.5-fold by 3 d and 4.7-fold by 5 d compared with the pre-exposure level. A significant increase in the expression level of SvMT mRNA was observed after the exposure of S. virgatus to the combination of 0.003 mg/L cadmium chloride and 0.2 mg/L zinc chloride compared with the pre-exposure level. Our work indicates that the SvMT gene is associated with stress responses and could be a potential biomarker for marine pollution.

Topics: Amino Acid Sequence; Animals; Cadmium Chloride; Chlorides; DNA, Complementary; Environmental Biomarkers; Metallothionein; Mytilidae; Water Pollution, Chemical; Zinc Compounds

1. Cardiotoxicity is an important factor that limits the clinical use of doxorubicin (Dox). Metallothionein (MT) can antagonize the Dox-induced cardiotoxicity. Using a proteomics approach we have detected that major peroxiredoxins (Prxs) may be involved in this process. In the present study, we further investigate the mechanisms of the MT effects against Dox-induced cytotoxicity and the interactions between MT and Prxs. 2. We have established a primary cardiomyocyte culture system from MT-I/II null (MT(-/-)) and corresponding wild type (MT(+/+)) neonatal mice, and pretreated the MT(+/+) cardiomyocytes with ZnCl2 to establish the MT overexpression cardiomyocyte model. 3. Based on the results, in MT(+/+) cardiomyocytes, ZnCl2 pretreatment significantly increased the cardiomyocytes MT levels and inhibited the cardiotoxicity of Dox; it can resist LDH leakage, cardiomyocyte apoptosis, DNA damage, ROS accumulation and inhibit the decrease in activity of antioxidant enzymes induced by Dox. Moreover, ZnCl2 enhanced the expression of Prx-2, -3, -5 and -6, it can inhibit the expression of Prxs decrease in MT(+/+) cardiomyocytes induced by Dox, but had no effect in MT(-/-) cardiomyocytes. 4. Therefore, the present study suggests that ZnCl2 can protect the cardiomyocytes from the Dox-induced oxidative injury and can inhibit the changes in Prxs expression through induced MT overexpression.

Topics: Animals; Cardiotonic Agents; Cardiotoxins; Chlorides; Doxorubicin; Metallothionein; Mice; Myocytes, Cardiac; Peroxiredoxins; Zinc Compounds

We investigated zinc cytotoxicity in mouse neural stem/progenitor cells (NSPCs) and their differentiated progeny (neuronal/glial cells) in correlation with expression of metallothionein (MT) gene. Differentiated cells were less sensitive than NSPCs to ZnCl2 (IC50: 128μM vs. 76μM). Differentiation of immature NSPCs to the differentiated cells led to an increase in expression of MT family genes (Mt1, Mt2, Mt3, and Mt4). Zinc exposure induced a dose-dependent increase in expression level of Mt1 and that of Mt2 in both NSPCs and the differentiated cells. Our results showed that the reduced cytotoxicity of zinc associated with differentiation from NSPCs into their progeny was related to the upregulation of MTs.

Topics: Animals; Biomarkers; Cell Differentiation; Cells, Cultured; Chlorides; Dose-Response Relationship, Drug; Gene Expression Regulation; Metallothionein; Metallothionein 3; Mice; Neural Stem Cells; Neuroglia; Neurons; Up-Regulation; Zinc Compounds

Exposure to environmental toxicants can alter a range of cellular functions involved in the immune response. Increased expression of the stress protein metallothionein 1 (MT1) is one example hereof. Previously, it has been reported that MT1 has several immunosuppressive properties. Furthermore, we earlier showed that functionally tolerogenic dendritic cells (DCs) expressed increased mRNA levels of MT1. Here, we demonstrate that dexamethasone-treated murine DCs are functionally tolerogenic and produce MT1. However, these DCs do not actively transport MT1 to the cell membrane and their regulatory function does not depend on MT1. Alternatively, ZnCl2-treated murine DCs transport MT1 to the cell surface are tolerogenic and promote the expansion of T cells with a regulatory phenotype. Moreover, the membrane-bound MT1 was shown to be essential for ZnCl2-treated DCs to exert their regulatory function. On the basis of this, MT1 can be used as a new marker for functionally tolerogenic DCs. Additionally, we have found a new mechanism for tolerogenic DCs to exert their immune regulatory function.

Topics: Animals; Cell Membrane; Cells, Cultured; Chlorides; Dendritic Cells; Dexamethasone; Female; Forkhead Transcription Factors; Immune Tolerance; Immunosuppressive Agents; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Transgenic; Protein Transport; RNA, Messenger; T-Lymphocytes, Regulatory; Zinc Compounds

Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl(2), a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations.. Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive β-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing "redox sensitive GFP" targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA.. As observed for Mt1a mRNA levels, β-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50-100 µM ZnCl(2) to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late β-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions.. ZnCl(2) reduces mitochondrial oxidative stress and improves rat β-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS.

Topics: Animals; Antioxidants; Apoptosis; Cation Transport Proteins; Cell Culture Techniques; Chelating Agents; Chlorides; Cytoprotection; Dose-Response Relationship, Drug; Gene Expression Regulation; Glucose; Insulin; Insulin Secretion; Insulin-Secreting Cells; Male; Metallothionein; Mitochondria; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Wistar; RNA, Messenger; Sulfhydryl Compounds; Time Factors; Zinc Compounds; Zinc Transporter 8

We developed a suicidal DNA vaccine (pIRF1A-G-pMT-M) for salmonid fish susceptible to Infectious Hematopoietic Necrosis Virus (IHNV). The suicidal vaccine consists of two operons: i) an inducible fish promoter, the interferon regulatory factor 1A promoter (pIRF1A), driving the expression of the IHNV viral glycoprotein (G) gene that induces protection, and ii) a ZnCl(2) inducible fish promoter, the metallothionein promoter (pMT), driving the expression of the IHNV matrix (M) protein that induces apoptosis. The vaccine induces an immune response to the G protein and then induces the cell to undergo apoptosis to eliminate the DNA vaccine-containing cell. Also developed is another suicidal construct (pCMV-luc-pMT-M) for monitoring the persistence of luciferase (luc) expression after induction of apoptosis. In this study, we evaluated the inducibility of the MT promoter with ZnCl(2) and the capacity of cells transfected with the suicidal vector pCMV-luc-pMT-M to undergo apoptosis after ZnCl(2) addition. We also demonstrated the protective immunity elicited by the suicidal DNA vaccine pIRF1A-G-pMT-M, the survival of fish after treatment with ZnCl(2), and the elimination of the suicidal vector in fish after ZnCl(2) treatment.

Topics: Animals; Apoptosis; Cell Line; Chlorides; Fish Diseases; Gene Expression Regulation; Infectious hematopoietic necrosis virus; Luciferases; Metallothionein; Oncorhynchus mykiss; Promoter Regions, Genetic; Rhabdoviridae Infections; Survival Analysis; Transfection; Vaccines, DNA; Zinc Compounds

Previous studies have shown that zinc chloride (ZnCl(2)) can induce metallthionein (MT) in the liver and kidney to protect tissues against toxicants and shows a better corneal wound healing than conventional drugs do. We hypothesized that ZnCl(2) can promote odontogenesis of dental pulp stem cells (DPSCs) via MT. The purpose of this study was to investigate the effects of ZnCl(2) on human DPSCs and the expression of MT.. DPSCs were isolated by flow cytometry with selective surface marker CD146 and STRO-1. After they grew into confluence, DPSCs were induced into odontoblasts with or without ZnCl(2) supplemented in the culture medium for 21 days. The effect of ZnCl(2) on DPSCs differentiation was examined followed by alkaline phosphatase staining/activity and quantitative real-time polymerase chain reaction analysis.. By treating DPSCs with ZnCl(2), the duration of mineralization was shortened and expressions of differentiation markers into odontoblasts were more significant than those without ZnCl(2) stimulation. Besides, the MT gene expression was increased with the increasing expressions of odontoblasts' markers after treated with ZnCl(2).. This was the first report that ZnCl(2) could promote odontoblastic differentiation of DPSCs through the up-regulation of gene MT.

Topics: Adolescent; Adult; Analysis of Variance; Cell Differentiation; Cells, Cultured; Chlorides; Dental Pulp; Female; Humans; Male; Metallothionein; Odontoblasts; Odontogenesis; Reference Values; Root Canal Irrigants; Stem Cells; Up-Regulation; Zinc Compounds

Metal-responsive transcription factor-1 (MTF-1) is essential for the induction of genes encoding metallothionein by metals and hypoxia. Here, we studied the mechanism controlling the activation of MTF-1 by hypoxia. Hypoxia activation of Mt gene transcription is dependent on the presence of metal regulatory elements (MREs) in the promoter of Mt genes. We showed that MREa and MREd are the main elements controlling mouse Mt-1 gene induction by hypoxia. Transfection experiments in Mtf-1-null cells showed that MTF-1 is essential for induction by hypoxia. Chromatin immunoprecipitation analysis showed that MTF-1 DNA-binding activity was strongly enhanced in the presence of zinc but not by hypoxia. Notably, hypoxia inducible factor- (HIF) 1α was recruited to the Mt-1 promoter in response to hypoxia but not to zinc. MTF-1 activation was inhibited by PKC, JNK, and PI3K inhibitors and by the electron transport chain inhibitors rotenone and myxothiazol, but not by the antioxidant N-acetylcysteine. We showed that prolyl-hydroxylase inhibitors can activate MTF-1, but this activation requires the presence of HIF-1α. Finally, HIF-dependent transcription is enhanced in the presence of MTF-1 and induction of an MRE promoter is stimulated by HIF-1α, thus indicating cooperation between these 2 factors. However, coimmunoprecipitation experiments did not suggest direct interaction between MTF-1 and HIF-1α.

Topics: Acetylcysteine; Animals; Blotting, Northern; Cell Hypoxia; Chlorides; Chromosome Mapping; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Genes, Reporter; HEK293 Cells; Hep G2 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoprecipitation; Luciferases; Metallothionein; Methacrylates; Mice; Mutagenesis, Site-Directed; NIH 3T3 Cells; Oxidative Stress; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Plasmids; Promoter Regions, Genetic; Protein Kinase Inhibitors; Protein Stability; Rotenone; Signal Transduction; Thiazoles; Transcription Factor MTF-1; Transcription Factors; Transcriptional Activation; Transfection; Zinc Compounds

Few quantitative analyses of the induction of metallothioneins (MTs) have been conducted, and there are no reports on the contribution of MTs to the protective mechanism against ultraviolet (UV) radiation in the lens. In this study, we quantitatively analyzed the induction of MTs and analyzed the resulting protective effects against both metal- and UV radiation-induced damage in the cultured lens epithelial cell line, alphaTN4-1.. The induction profiles of MTs by ZnCl(2) treatment in alphaTN4-1 cells were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The cells in which MTs were induced were either treated with high concentrations of ZnCl(2) or CdCl(2), or irradiated with UV-C, UV-B, or UV-A radiation, followed by analysis of cell viability. The (3)H-thymidine incorporation rate was used as an indicator of cell viability.. mRNA expression of MT-I and MT-II, the main MT isoform classes, was induced by ZnCl(2) treatment in a dose-dependent manner. MT induction increased the protective effects against both metaland UV-A radiation-induced cell damage.. Our results suggest that MTs play an important role in the protection against damage induced by both toxic metals and UV-A radiation in lens epithelial cells.

Topics: Animals; Cadmium Chloride; Cell Survival; Cells, Cultured; Chlorides; Cytoprotection; DNA Damage; DNA Repair; Dose-Response Relationship, Drug; Epithelial Cells; Lens, Crystalline; Metallothionein; Mice; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Ultraviolet Rays; Zinc Compounds

We recently reported that induction of metallothionein (MT) was critical in limiting nickel (Ni)-induced lung injury in intact mice. Nonetheless, the mechanism by which Ni induces MT expression is unclear. We hypothesized that the ability of Ni to mobilize zinc (Zn) may contribute to such regulation and therefore, we examined the mechanism for Ni-induced MT2A expression in human airway epithelial (BEAS-2B) cells. Ni induced MT2A transcript levels and protein expression by 4 hours. Ni also increased the activity of a metal response element (MRE) promoter luciferase reporter construct, suggesting that Ni induces MRE binding of the metal transcription factor (MTF-1). Exposure to Ni resulted in the nuclear translocation of MTF-1, and Ni failed to induce MT in mouse embryonic fibroblasts lacking MTF-1. As Zn is the only metal known to directly bind MTF-1, we then showed that Ni increased a labile pool of intracellular Zn in cells as revealed by fluorescence-activated cell sorter using the Zn-sensitive fluorophore, FluoZin-3. Ni-induced increases in MT2A mRNA and MRE-luciferase activity were sensitive to the Zn chelator, TPEN, supporting an important role for Zn in mediating the effect of Ni. Although neither the source of labile Zn nor the mechanism by which Ni liberates labile Zn was apparent, it was noteworthy that Ni increased intracellular reactive oxygen species (ROS). Although both N-acetyl cysteine (NAC) and ascorbic acid (AA) decreased Ni-induced increases in ROS, only NAC prevented Ni-induced increases in MT2A mRNA, suggesting a special role for interactions of Ni, thiols, and Zn release.

Topics: Acetylcysteine; Animals; Antioxidants; Ascorbic Acid; Bronchi; Cell Separation; Cells, Cultured; Chelating Agents; Chlorides; DNA-Binding Proteins; Epithelial Cells; Ethylamines; Flow Cytometry; Fluorescent Dyes; Humans; Metallothionein; Mice; Mice, Knockout; Nickel; Polycyclic Compounds; Pyridines; Pyrimidines; Reactive Oxygen Species; RNA, Messenger; Time Factors; Transcription Factor MTF-1; Transcription Factors; Transcriptional Activation; Transfection; Up-Regulation; Zinc Compounds

Zinc (Zn) administration at non-toxic doses protects against the hepatotoxicity produced by many agents, but the underlying mechanisms remain elusive.. To examine the basis of Zn-induced generalised hepatoprotective effects.. Rats and mice were given Zn at known hepatoprotective levels (100 mumol ZnCl2/kg/day, s.c., for 4 days) and molecular responses were assessed.. Zn treatment produced changes in 5% of the genes on custom-designed mouse liver array and Rat Toxicology-II array. Changes in gene expression were further confirmed and extended by real-time reverse transcriptase-polymerase chain reaction. Zn treatment dramatically increased the expression of the metallothionein (Mt), and modestly increased the expression of acute-phase protein genes (ceruloplasmin, Stat3, egr1, Cxc chemokines and heat-shock proteins). For genes encoding for antioxidant enzymes, some were increased (Nrf2 and Nqo1), while others remained unaltered (Cu, Zn SOD and glutathione S-transferases). Expressions of cytokine and pro-inflammatory genes were not affected, while genes related to cell proliferation (cyclin D1) were modestly upregulated. Some metabolic enzyme genes, including cytochrome P450s and UDP-glucuronosyltransferase, were modestly suppressed, perhaps to switch cellular metabolic energy to acute-phase responses. Liver Zn content was increased between 1.6- and 2.1-fold, while hepatic MT protein was increased between 50 and 200-fold. Mice typically showed greater responses than rats.. Such gene expression changes, particularly the dramatic induction of MT and Nrf2 antioxidant pathway, occur in the absence of overt liver injury, and are probably important in the hepatoprotective effects of Zn against toxic insults.

Topics: Animals; Chlorides; Gene Expression; Gene Expression Profiling; Injections, Subcutaneous; Liver; Male; Metallothionein; NF-E2-Related Factor 2; Oligonucleotide Array Sequence Analysis; Protective Agents; Rats; Rats, Sprague-Dawley; Zinc Compounds

Several studies have described mercury toxicity and the role of metallothioneins (MT) in the detoxification and regulation of metal homeostasis. However, little data exist on this topic during the specific post-natal developmental phase in young mammals. This developmental phase is particularly important since young animals are more sensitive to toxicants than adults. The objective of this work was to investigate whether MT participates in the mechanism of protection conferred by zinc pre-treatment on the toxic effects induced by mercury in neonate rats. Pups were exposed to ZnCl(2) (5 doses of 27 mg/kg/day, s.c.) and subsequently to HgCl(2) (5 doses of 5 mg/kg/day, s.c.); metal (Zn and Hg) and MT contents were analyzed in the liver, kidney, and blood. MT was induced in the liver and kidney of pups of both Zn-sal and Zn-Hg groups, although the greatest increase was in neonates exposed to Zn only. A direct relationship exists between MT and metals for both hepatic and renal tissues, which indicates that the increase in metal levels occurs in parallel to the increase in MT content. Although the heat-treated cytosolic fraction is rich in MT and metals, higher Zn and Hg contents were detected in the insoluble fraction of all tissues. These results suggest that MT is, at least in part, responsible for preventing Hg accumulation in the liver and blood and decreasing renal toxicity.

Topics: Animals; Animals, Newborn; Chlorides; Female; Kidney; Liver; Male; Mercuric Chloride; Mercury Poisoning, Nervous System; Metallothionein; Rats; Rats, Wistar; Zinc Compounds

We previously showed that the major Zn-binding protein, metallothionein (MT) is a critical target for nitric oxide (NO) with resultant increases in labile Zn. We now show that NO donors also affected the activity of the metal responsive transcription factor MTF-1 that translocates from the cytosol to the nucleus in response to physiologically relevant increases in intracellular Zn and transactivates MT gene expression. Exposing mouse lung endothelial cells (MLEC) to ZnCl(2) or the NO donor, S-Nitroso-N-acetylpenicillamine (SNAP, 200 microM), caused nuclear translocation of a reporter molecule consisting of enhanced green fluorescent protein (EGFP) fused to MTF-1 (pEGFP-MTF-1). In separate experiments, NO donors induced increases in MT protein levels (Western blot). In contrast, NO did not cause nuclear translocation of EGFP-MTF-1 in MLEC from MT knockouts, demonstrating a central role for MT in mediating this response. These data suggest that S-nitrosation of Zn-thiolate clusters in MT and subsequent alterations in Zn homeostasis are participants in intracellular NO signaling pathways affecting gene expression.

Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Cells, Cultured; Chlorides; DNA-Binding Proteins; Endothelial Cells; Green Fluorescent Proteins; Lung; Metallothionein; Mice; Mice, Knockout; Nitric Oxide; Nitric Oxide Donors; Recombinant Fusion Proteins; S-Nitroso-N-Acetylpenicillamine; Sheep; Transcription Factor MTF-1; Transcription Factors; Transfection; Zinc; Zinc Compounds

Most people generally ingest cadmium in their food. Cadmium that has accumulated in tissues induces the synthesis of metallothioneins (MTs) which are metal-binding proteins that bind tightly to cadmium to inhibit its renal toxicity. Individuals whose ability to induce the synthesis of MTs is low seem likely to be particularly susceptible to the toxic effects of cadmium. In this study, we analyzed the polymorphism of the promoter region of the gene for MT-IIA, the major species of MT in humans, in 119 adult Japanese subjects. We found that about 18% of the subjects had an A --> G single-nucleotide polymorphism in the core region of the promoter near the TATA box. A reporter-gene assay using HEK293 cells showed that replacement of A by G at position -5 reduced the efficiency of the cadmium-induced transcription of the gene for MT-IIA. This single-nucleotide polymorphism inhibited the binding of nuclear proteins to the core promoter region of the gene for MT-IIA. When the promoter region upstream of the TATA box was replaced by a sequence that contained three dioxin-responsive elements, the reporter-gene assay demonstrated that the A --> G single-nucleotide polymorphism resulted in a marked reduction in the rate of dioxin-induced transcription. These results suggest that the A --> G single-nucleotide polymorphism reduces the efficiency of those aspects of the transcription of the gene for MT-IIA that are controlled by general transcription factors.

Topics: Adult; Base Sequence; Cadmium Chloride; Cell Line; Chlorides; Dioxins; DNA; DNA Mutational Analysis; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Gene Frequency; Genotype; Humans; Luciferases; Metallothionein; Oligonucleotides; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Polymorphism, Single-Stranded Conformational; Promoter Regions, Genetic; Protein Binding; TATA Box; Transfection; Zinc Compounds

Metallothionein (MT) is the protein that has been shown to bind heavy metals, scavenge free radicals, protect DNA from radiation damage, and alleviate disease symptoms. However, only very limited success has been achieved in expression and production of active recombinant metallothionein. In this study, human metallothionein 1A (hMT1A) was transformed into yeast Pichia pastoris for expression with secretion of the protein into the medium. The expression system was optimized to obtain the targeted protein in active form at 335 mg per litre culture. hMT1A showed the character of extreme instability in the experiment. High concentration, aeration and heavy metal ions are the main factors affecting hMT1A stability.

Topics: Carbon; Chlorides; Cysteine; Gene Expression; Hemolysis; Histidine; Humans; Metallothionein; Recombinant Proteins; Saccharomyces cerevisiae; Zinc Compounds

Methamphetamine (METH) is a drug of abuse and neurotoxin that induces Parkinson's-like pathology after chronic usage by targeting dopaminergic neurons. Elucidation of the intracellular mechanisms that underlie METH-induced dopaminergic neuron toxicity may help in understanding the mechanism by which neurons die in Parkinson's disease. In the present study, we examined the role of reactive oxygen species (ROS) in the METH-induced death of human dopaminergic SK-N-SH cells and further assessed the neuroprotective effects of zinc and metallothionein (MT) against METH-induced toxicity in culture. METH significantly increased the production of reactive oxygen species, decreased intracellular ATP levels and reduced the cell viability. Pre-treatment with zinc markedly prevented the loss of cell viability caused by METH treatment. Zinc pre-treatment mainly increased the expression of metallothionein and prevented the generation of reactive oxygen species and ATP depletion caused by METH. Chelation of zinc by CaEDTA caused a significant decrease in MT expression and loss of protective effects of MT against METH toxicity. These results suggest that zinc-induced MT expression protects dopaminergic neurons via preventing the accumulation of toxic reactive oxygen species and halting the decrease in ATP levels. Furthermore, MT may prevent the loss of mitochondrial functions caused by neurotoxins. In conclusion, our study suggests that MT, a potent scavenger of free radicals is neuroprotective against dopaminergic toxicity in conditions such as drug of abuse and in Parkinson's disease.

Topics: Adenosine Triphosphate; Cell Line; Cell Survival; Chelating Agents; Chlorides; Dopamine; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Free Radical Scavengers; Humans; Metallothionein; Methamphetamine; Microscopy, Fluorescence; Neurons; Neuroprotective Agents; Reactive Oxygen Species; Zinc Compounds

Nitric oxide (NO) is a potent intercellular mediator of melanogenesis, whereas metallothionein (MT) is an inducible intracellular antioxidant that has been reported to scavenge NO. We investigated the existence and induction of MT in melanocytes, and its inhibitory effect on NO-induced melanogenesis. The expression of MT was detected in melanocytes, however, at a lower level than in keratinocytes, and its induction was possible by the addition of zinc chloride. Further, an NO-stimulated increase of tyrosinase activity in melanocytes was remarkably suppressed, when MT was induced prior to NO stimulation. Melanogenesis was also suppressed, when dexamethasone was used to induce MT. However, an NO-stimulated increase of tyrosinase expression was not suppressed at the gene and protein level, when MT was induced in melanocytes. The same suppressive effect of melanogenesis was also observed, when alpha-melanocyte-stimulating hormone or endothelin-1 was used as a stimulator. Because these results implied a mechanism other than NO scavenging to explain the suppressive effect of MT induction on melanogenesis, the direct inhibition of tyrosinase by MT was examined. Melanosome fractions were prepared from melanocytes, whose melanogenesis was suppressed by the induction of MT. Tyrosinase suppression was observed in the melanosome fractions, which was neutralized by the addition of anti-MT antibody. These results suggest that MT induction may be effective to suppress melanogenesis stimulated by NO as well as other melanogens, and these suppressive effects might be due to a direct inhibition of tyrosinase activity in melanosome and not a scavenging effect of NO.

Topics: alpha-MSH; Blotting, Northern; Blotting, Western; Chlorides; Dexamethasone; Endothelin-1; Fibroblasts; Glucocorticoids; HeLa Cells; Humans; Keratinocytes; Melanocytes; Melanosomes; Metallothionein; Monophenol Monooxygenase; Nitric Oxide; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Zinc Compounds

Mutations in Cu/Zn-superoxide dismutase 1 (SOD1) are responsible for a familial form of amyotrophic lateral sclerosis (FALS). It has been proposed that oxidative stress and abnormal metal homeostasis contribute to death of motor neurons in this disease. Also, inability of motor neurons to upregulate protective proteins under stress may contribute to their preferential vulnerability to toxicity. Metallothioneins (MT) are low molecular weight, metal-binding proteins with established antioxidant capabilities. This study investigated the ability of motor neurons to upregulate MT isoforms in response to expression of mutant SOD1(G93A) or exposure to other neurotoxicants, and the ability of MT-I gene transfer to protect motor neurons from these stresses. MT isoform-I and -II were expressed constitutively in astrocytes and other non-neuronal cells of dissociated spinal cord cultures, but not in motor neurons. MT-I/II was upregulated in astrocytes, but not motor neurons, following treatment with ZnCl(2) or excitotoxic concentrations of glutamate. MT-III expression was restricted to neurons and was unaffected by treatment with ZnCl(2), paraquat, or glutamate. Overexpression of MT-I in motor neurons by gene transfer reduced the toxicity of ZnCl(2) and paraquat, but failed to protect them against glutamate or SOD1(G93A). These data are evidence against metal-catalyzed, oxidative stress being the primary mechanisms of toxicity conferred by disease-causing mutations in SOD1.

Topics: Amyotrophic Lateral Sclerosis; Animals; Blotting, Western; Cell Survival; Cells, Cultured; Chlorides; Genetic Vectors; Herbicides; Immunohistochemistry; Inclusion Bodies; Isoenzymes; Isomerism; Metallothionein; Mice; Motor Neurons; Mutation; Oxidative Stress; Paraquat; Plasmids; Superoxide Dismutase; Zinc Compounds

Metallothionein (MT), a cysteine-rich, metal-binding protein, is involved in homeostatic regulation of essential metals and protection of cells against oxidative injury. It has been shown that oxidative stress is associated with pathogenesis of osteoporosis and is capable of inhibiting osteoblastic differentiation of bone cells by nuclear factor-kappaB (NF-kappaB). In this study, the effect of MT on oxidative stress-induced inhibition of osteoblast differentiation was examined. 50-200 microM hydrogen peroxide-induced oxidative stress suppressed the osteoblastic differentiation process of primary mouse bone marrow stromal cells (BMSCs), manifested by a reduction in the differentiation marker alkaline phosphatase (ALP). The presence of exogenous MT (20-500 microM) or induction of endogenous MT by ZnCl2 (50-200 microM) could protect BMSCs against H2O2-induced inhibition of osteoblastic differentiation, manifested by a resumption of H2O2-inhibited ALP activity and ALP positive cells. Furthermore, adding exogenous MT or inducing endogenous MT expression impaired H2O2-stimulated NF-kappaB signaling. These data indicate the ability of MT to protect BMSCs against oxidative stress-induced inhibition of osteoblastic differentiation.

Topics: Alkaline Phosphatase; Animals; Biomarkers; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Chlorides; Hydrogen Peroxide; Metallothionein; Mice; NF-kappa B; Osteoblasts; Osteogenesis; Osteoporosis; Oxidants; Oxidative Stress; Signal Transduction; Stromal Cells; Zinc Compounds

An increased body retention of Cd in rats orally pretreated with Cd or Zn is explained by induction of hepatic and renal metallothionein. Whether intestinal absorption of Cd increases after such treatments is not clear yet. To approach this problem we measured jejunal transfer rates of 109Cd in vitro and in vivo in pretreated rats (0.44 mmol Cd/l or 4.6 mmol Zn/l in the drinking water for 10 days) and compared them with those of untreated controls. Isolated jejunal segments were used for in vitro perfusion. In vivo perfusion was performed in anaesthetized rats with blood collected from mesenteric venules substituting corresponding losses by reinfusion of rat blood. Water and glucose transfer did not differ between controls and pretreated rats. At a luminal concentration of 5 micromol 109CdCl2/l, Cd and Zn pretreatment significantly increased the transfer rate of 109Cd in vitro and in vivo similarly. The 109Cd transfer rates in controls in the final perfusion intervals (80-120 min) were 0.06 (pmol/cm/min) in vivo and 0.05 in vitro; the corresponding rates in Cd or Zn pretreated rats were significantly higher (P<0.05) and amounted to 0.11 and 0.18 or 0.15 and 0.23, respectively. Mucosal concentrations of 109Cd measured at the end of the perfusion period tended to be lower in the pretreated animals than in the controls. This suggests that pretreatment with Cd or Zn reduces the amount of 109Cd bound to the tissue leaving more 109Cd for the transfer step. As compared to a level of mucosal metallothionein of 8 microg/g wet weight in controls, increased amounts of 67 or 52 microg/g wet weight in the Cd or Zn pretreated rats, respectively, thus did not decrease but increased transfer rates of 109Cd. Therefore, increased small intestinal transfer rates of Cd can contribute to increase the body retention of Cd seen after oral pretreatment with Cd or Zn.

Topics: Animals; Cadmium; Cadmium Chloride; Cadmium Radioisotopes; Chlorides; Chromatography, Gel; Female; In Vitro Techniques; Intestinal Absorption; Intestinal Mucosa; Jejunum; Metallothionein; Random Allocation; Rats; Rats, Sprague-Dawley; Zinc Compounds

Metallothioneins (MT) are ubiquitous low-molecular-weight metal-binding intracellular proteins. We used wild type mouse embryo fibroblasts, GKA1, and its MT-null variant, named GKA2, in order to correlate the presence of MT to the response to a number of different antitumor drugs with different mechanisms of action. We studied sensitivity of GKA1 and GKA2 cells to metal-based compounds having alkylating property, or able to generate reactive oxygen species (ROS); as well as to drugs acting with different mechanisms. The absence of MT in GKA2 cells was correlated to higher sensitivity to the metal-based drugs compared to that of GKA1. No marked differences in sensitivity of two cell lines against gemcitabine, taxol, and vinblastine were observed. No significant change in sensitivity of either GKA1 or GKA2 cells to these non-alkylating drugs was seen after heavy metal pretreatments. In GKA1 cells, MT biosynthesis was induced by copper and cadmium but not by zinc treatment under the conditions of these experiments. Induction of MT was directly proportional to decrease in sensitivity of GKA1 cells to the compounds used in this experiment. In contrast to GKA1 cells, the MT-null cells (GKA2) expressed no detectable metallothionein either constitutively or after treatment with zinc, copper, or cadmium. Nonetheless, heavy metal pretreatment of GKA2 cells did not cause any change in their sensitivity.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Cadmium Chloride; Carboplatin; Cell Division; Cell Line; Chlorides; Cisplatin; Copper Sulfate; Deoxycytidine; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Ferrous Compounds; Fibroblasts; Gemcitabine; Inhibitory Concentration 50; Metallothionein; Metals, Heavy; Mice; Mice, Knockout; Paclitaxel; Spectrophotometry, Atomic; Survival Analysis; Survival Rate; Vinblastine; Zinc Compounds

The effects of CdCl2 and ZnCl2 pretreatments on the inhibition of delta-ALA-D (delta-aminolevulinic acid dehydratase) activity and Hg contents in liver and kidneys of suckling rats intoxicated with HgCl2 were investigated. Zn-pretreatment prevented the effects of mercury at a higher magnitude than CdCl2. Hepatic and renal delta-ALA-D activities were significantly inhibited by HgCl2 and prior exposure to CdCl2 partially prevented the renal effect of mercury but not altered the mercury levels in both tissues. Pretreatment with ZnCl2 abolished mercury-induced delta-ALA-D-inhibition in kidneys and liver and induced an increase in renal (about three times) and a decrease in hepatic (to one-third) Hg contents when compared to the group injected only with mercury. In face of zinc effects to prevent Hg-delta-ALA-D inhibition and to alter Hg-deposition levels in kidney and liver, these results suggest that these effects may be partially due to the synthesis of metallothioneins (MT). In fact, liver MT content presented by animals pretreated with zinc was significantly greater than control and Hg-treated groups, but the increase showed by renal tissue (about 60%) was not significant. Although the MT is rich in cysteine (-SH) and consequently can form a great number of MT-Hg complex, other mechanisms should be also involved in zinc protection on mercury toxicity.

Topics: Animals; Animals, Suckling; Cadmium Chloride; Chlorides; Female; Injections, Subcutaneous; Kidney; Liver; Male; Mercuric Chloride; Mercury Poisoning; Metallothionein; Organ Size; Porphobilinogen Synthase; Rats; Rats, Wistar; Tissue Distribution; Zinc Compounds

The metal binding protein metallothionein (MT) confers drug resistance when MT is induced in tumor tissues. Cisplatin is known to induce MT synthesis in tumor tissues, which may lead to drug resistance. We examined whether a difference in the administration schedule of cisplatin affect the efficiency of MT induction.. Balb/c nude mice were inoculated with NMB-1 human bladder tumor tissues and injected with cisplatin (total dose of 64 micromol/kg) in a single injection or fractioned daily injections. Tumor MT concentration was determined 24 hours after the last injection of cisplatin by mercury binding assay. The effect of pretreatment with ZnCl2 on antitumor activity of cisplatin was examined in NMB-1 bearing mice.. Tumor MT levels increased significantly with the increase in the number of cisplatin injections. Pretreatment of NMB-1 bearing mice with ZnCl2 (200 micromol/kg x 2) caused the same level of MT induction (1.6-fold) as that of fractioned injections of cisplatin (4 x 16 micromol/kg). Pretreatment of NMB-1 bearing mice with ZnCl2 (200 micromol/kg x 2) depressed cisplatin antitumor activity by about 50%.. The induction of MT to a moderate extent (1.6-fold) in NMB-1 tumor inoculated in mice conferred cisplatin resistance. This level of MT induction can be achieved by fractioned daily injections of cisplatin but not by a single injection. Therefore, it is preferable to administer cisplatin as a single injection rather than as fractioned injections to achieve effective antitumor activity with minimum MT induction.

Topics: Animals; Antineoplastic Agents; Carcinoma, Transitional Cell; Cell Line, Tumor; Chlorides; Cisplatin; Drug Administration Schedule; Drug Resistance, Neoplasm; Male; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Urinary Bladder Neoplasms; Zinc Compounds

Zinc is an essential oligoelement for cell growth and cell survival and has been demonstrated to protect cells from oxidative stress induced by UVA or from genotoxic stress due to UVB. In a recent work we demonstrated that the antioxidant role of zinc could be related to its ability to induce metallothioneins (MTs). In this study we identified the mechanism of zinc protection against solar-simulated light (SSL) injury. Cultured human keratinocytes (HaCaT) were used to examine MTs expression and localization in response to solar-simulated radiation. We found translocation to the nucleus, with overexpression of MTs in irradiated cells, a novel observation. The genoprotective effect of zinc was dependent on time and protein synthesis. DNA damage was significantly decreased after 48 h of ZnCl(2) (100 microM) treatment and is inhibited by actinomycin D. ZnCl(2) treatment (100 microM) led to an intense induction, redistribution, and accumulation of MT in the nucleus of irradiated cells. MT expression correlated with the time period of ZnCl(2) treatment. CdCl(2), a potent MT inducer, did not show any genoprotection, although the MTs were expressed in the nucleus. Overall our findings demonstrate that MTs could be a good candidate for explaining the genoprotection mediated by zinc on irradiated cells.

Topics: Cadmium; Cell Nucleus; Cells, Cultured; Chlorides; Comet Assay; DNA Damage; Humans; Keratinocytes; Metallothionein; Radiation-Protective Agents; Sunlight; Time Factors; Zinc; Zinc Compounds

Intracellular safeguarding functions of metallothioneins (MTs) include sequestering transition and heavy metals, scavenging free radicals and protecting against electrophiles. We report that MT protection against Cu-induced cytotoxicity can be reversed and pro-oxidant and pro-apoptotic effects can be induced in HL-60 cells exposed to NO. We demonstrate that in ZnCl(2)-pretreated HL-60 cells loaded with copper nitrilotriacetate (Cu-NTA), exposure to an NO donor, S-nitroso-N-acetyl penicillamine, resulted in S-nitrosylation and oxidation of MT cysteines. This disruption of MT Cu-binding thiolate clusters caused loosening and release of redox-active Cu, enhanced redox-cycling activity of Cu and increased peroxidation of major classes of membrane phospholipids. We also found that Cu-induced oxidative stress in ZnCl(2)-pretreated/Cu-NTA-loaded HL-60 cells was accompanied by apoptosis documented by characteristic changes of nuclear morphology, internucleosomal DNA cleavage, externalization of phosphatidylserine, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. We conclude that in Cu-challenged cells, NO can reverse the protective role of MTs and convert them into pro-oxidant, pro-apoptotic implements.

Topics: Annexin A5; Apoptosis; Carcinogens; Caspase 3; Caspases; Chlorides; Chromatography, High Pressure Liquid; Cytochrome c Group; DNA Fragmentation; Electron Spin Resonance Spectroscopy; Enzyme Activation; HL-60 Cells; Humans; Lipid Peroxidation; Metallothionein; Nitric Oxide; Nitric Oxide Donors; Nitrilotriacetic Acid; Organometallic Compounds; Oxidants; Oxidation-Reduction; Penicillamine; Phospholipids; Zinc Compounds

The hepatic and the renal subcellular distribution of zinc, cadmium or mercury and induction of tissue metallothionein (MT) at 24, 48 and 72 h following an oral equimolar dose (15 micro;mol metal/kg) of zinc (II) chloride, cadmium (II) chloride or mercury (II) chloride in male albino mice were investigated. There was a moderate increase in hepatic and renal zinc levels mainly in their nuclear mitochondrial fraction (NMF) 24 h post zinc chloride administration. Subsequently, the hepatic zinc increased and the renal zinc declined with time. The zinc-induced hepatic MT level was maximum at 48 h, which decreased slightly thereafter, while there was no marked increase in renal MT level at any time interval. The cadmium was equally distributed in liver and kidney more in their supernatant cytosol fraction (SCF) than in their NMF at 24 h after a dose of cadmium chloride. The cadmium levels showed a decreasing trend in hepatic fractions and an increasing trend in renal fractions with time. The cadmium-induced hepatic and renal MT were substantial at 24 h post cadmium administration, the former decreased thereafter while the latter enhanced at 48 h before declining. The accumulation of mercury in kidney was 1.5 times that in liver, which was localised more in their SCF than in their NMF at 24 h in response to a dose of mercuric chloride. The mercury levels of hepatic and renal subcellular fractions started declining after 24 h and at 72 h they were significantly lower. The induction of hepatic and renal MT was maximum at 24 h after mercuric chloride administration, which declined thereafter concomitant with the decrease in their mercury levels. However, the MT levels in both the organs remained considerably higher than in normal animals at 72 h post exposure. The results show that the accumulation of metal in liver and kidney follows the order: Hg > Cd > Zn and the induction of MT follows Hg > Cd > Zn in liver and Cd > Hg > Zn in kidney. The alterations in zinc and copper homeostasis were more marked in liver than in kidney and follows the order: Hg > Cd > Zn.

Topics: Animals; Cadmium; Cadmium Chloride; Chlorides; Enzyme Induction; Homeostasis; Kidney; Liver; Male; Mercury; Metabolic Clearance Rate; Metallothionein; Mice; Subcellular Fractions; Time Factors; Zinc; Zinc Compounds

Metallothioneins (MTs) are the major low molecular weight, zinc-binding proteins in mammalian cells. It has been hypothesized that they play a role in the function of zinc-dependent signal transduction proteins and transcription factors. We investigated the capacity of zinc and other metal ions and conditions to increase both Zn-associated MT levels and the receptiveness of cells to transcriptional activation mediated by the zinc-dependent glucocorticoid receptor (GR). We studied, in a GR-responsive mouse mammary-tumor cell line, the ability of dexamethasone (DEX) to stimulate transcription of a chloramphenicol acetyltransferase (CAT) gene controlled by a mouse mammary-tumor virus promoter. In cells pretreated with 20 to 100 microM ZnCl(2), DEX-induced CAT activity correlated with zinc-induced MT levels. However, 0.05 to 0.5 microM CdCl(2) had no effect on CAT activity, despite an increase in Cd-associated MT. Copper-associated MT was detected in cells treated with 20 microM CuCl(2,) but there was no change in the level of Zn-MT, nor was CAT activity altered in cells exposed to 5 to 20 microM CuCl(2). These results may reflect a functional difference between zinc-associated MT, and MT associated with other metals. Significantly more CAT activity was observed in both heat-shocked cells and in cells exposed to 40 or 50 nM HgCl(2). Although absolute amounts of MT were unchanged by these two treatments, a higher percentage of total cellular zinc was associated with the MT protein fractions after treatment. Changes in GR levels could not account for variations in CAT activity. These data indicate that hormonal signalling can be altered by exposure to metal salts and heat shock, and the effect is correlated with the level of Zn-MT.

Topics: Animals; Cadmium Chloride; Cell Fractionation; Cell Nucleus; Chlorides; Copper; Dexamethasone; Gene Expression Regulation; Genes, Reporter; Glucocorticoids; Hot Temperature; Mercuric Chloride; Metallothionein; Mice; Promoter Regions, Genetic; Receptors, Glucocorticoid; Transcription, Genetic; Tumor Cells, Cultured; Zinc; Zinc Compounds

Based on previous findings that liver zinc and metallothionein (MT) levels increase after tumor transplantation, zinc metabolism in tumor-bearing mice was studied to clarify the role of zinc-MT in host defense systems. Zinc in the hepatic cytosolic MT fraction did not increase in tumor-bearing mice fed a zinc-deficient diet, suggesting that dietary zinc is necessary for apo-MT induction in the liver after tumor transplantation and is then incorporated into the apo-MT. When (65)ZnCl(2) was intravenously injected, liver (65)Zn levels in the tumor-bearing mice were higher than those in control mice for 72 h after the injection. Pancreatic and blood (65)Zn levels in tumor-bearing mice were lower than those in controls for 24 h (pancreas) and 6 h (blood) after the injection. These findings indicate that the hepatic zinc response via MT induction influences zinc metabolism in the body after tumor transplantation. Moreover, (65)Zn uptake in the liver of MT-deficient tumor-bearing mice was lower than that in control tumor-bearing mice 1 h after injection. (65)Zn uptake in the tumor and blood (65)Zn levels in the MT-deficient tumor-bearing mice were higher than those in the control tumor-bearing mice. Tumor weight increased more in MT-deficient mice than in control mice. The formation of zinc-MT in the liver of tumor-bearing mice might decrease blood zinc availability for tumors and other tissues, such as the pancreas.

Topics: Animals; Carcinoma, Ehrlich Tumor; Chlorides; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; Mice; Mice, Inbred Strains; Mice, Knockout; Pancreas; Rats; Rats, Inbred Strains; Zinc; Zinc Compounds; Zinc Radioisotopes

Antioxidant activity is believed to be an important intracellular function of metallothioneins (MT), yet the specific mechanisms of their antioxidant action are not known. Under conditions when cells are challenged with elevated concentrations of free copper as a result of metabolic disturbances or environmental and occupational exposures, MTs may be ideally suited for antioxidant function as effective copper chelators. In the study presented here, we tested this hypothesis using a recently established model of copper nitrilotriacetate-induced oxidative stress in HL-60 cells. Since copper-induced oxidative stress triggers apoptosis, we further investigated antiapoptotic function of MTs in HL-60 cells. Using a Sephadex G-75 chromatographic partial purification of MTs from cell homogenates with subsequent immuno-dot-blot assay, we showed that zinc pretreatment yielded a pronounced induction of MTs in HL-60 cells. We report that zinc-induced MTs were able to (i) completely bind intracellular copper, (ii) completely quench redox-cycling activity of copper, (iii) significantly inhibit copper-dependent oxidative stress in membrane phospholipids, and (iv) prevent copper-dependent apoptosis and its characteristic biochemical features (cytochrome c release from mitochondria into cytosol, caspase-3 activation, and externalization of phosphatidylserine in plasma membranes). In separate experiments, we used lung fibroblasts derived from MT1, MT2 knockout mice (MT(-)(/)(-)) and MT wild-type (MT(+/+)) mice. ZnCl(2) pretreatment resulted in a more than 10-fold induction of MTs in MT(+/+) cells, whereas the MT content in MT(-)(/)(-) cells remained low, at levels approximately 100-fold lower than in their MT wild-type counterparts. MT(-)(/)(-) cells were very sensitive to Cu-NTA and, most importantly, showed no response to ZnCl(2) pretreatment. In contrast, MT(+/+) cells were relatively more resistant to Cu-NTA, and this resistance was remarkably enhanced by ZnCl(2) pretreatment. Combined, our results demonstrate that metallothioneins function as effective antioxidants and an antiapoptotic mechanism in copper-challenged HL-60 cells.

Topics: Animals; Antioxidants; Apoptosis; Camptothecin; Caspase 3; Caspases; Cell Nucleus; Chlorides; Fibroblasts; HL-60 Cells; Humans; Lipid Peroxidation; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrilotriacetic Acid; Organometallic Compounds; Oxidative Stress; Zinc Compounds

To assess whether metallothionein (Met) could improve the nitroglycerin tolerance in vivo.. Nitrate tolerance was induced by 2-d treatment of nitroglycerin (Nit) patch (0.05 mg.h-1). Endogenous Met production was induced by pretreatment of ZnCl2 and coadministration of intravenous Met (15 mg.kg-1.d-1) 2 d with Nit in tolerant rats. The induction of Met production was confirmed by the assay of liver and plasma Met levels.. ZnCl2 induced large amount of endogenous Met production in liver and plasma in Nit + ZnCl2 group than that in control group, (89 +/- 4) micrograms/g tissue vs (11.0 +/- 2.4) micrograms/g tissue in liver, P < 0.01, and in plasma (85 +/- 6) micrograms.L-1 vs (71 +/- 6) micrograms.L-1, P < 0.01. There was no significant difference in plasma Met levels in Nit and control groups [(75 +/- 6) micrograms.L-1 vs (71 +/- 6) micrograms.L-1, P > 0.05]. The endogenous Met production enhanced the hypotensive response in Nit + ZnCl2 group [(15.7 +/- 0.8) kPa vs (11.5 +/- 0.6) kPa, n = 6, P < 0.05]. The maximal vessel relaxation induced by sodium nitroprusside (SNP) was the same in 4 different groups but the highest EC50 (concentration which produces 50% of the maximal response to SNP) was found in tolerant group [(42 +/- 9) nmol.L-1, P < 0.01].. Exogenous Met or induction of endogenous Met production antagonize the development of Nit tolerance.

Topics: Animals; Blood Pressure; Chlorides; Drug Tolerance; Liver; Male; Metallothionein; Nitroglycerin; Rats; Rats, Wistar; Vasodilation; Vasodilator Agents; Zinc Compounds

The induction of metallothionein (MT) by physical and chemical stress was assessed using the fresh-water fish, crucian carp (Carassius cuvieri Temminck et Schlegel). The fish exposed to violent air-pumping stress for 6 days revealed time-dependent induction of MT-like metal-binding proteins in both their livers and kidneys. Their hepatic contents after exposure to stress were elevated to twice the basal level with 24 h, resulting in more than a 3-fold increase at 144 h, whereas their renal contents gradually increased after 24 h and reached the same level as that in the liver around 96 h. Two major inducible proteins were purified from livers of fish exposed to stress and were shown to be MT based upon their chromatographic behavior, UV absorption spectra and their molecular weights. Consequently, they were termed ccMT-1 and ccMT-2, according to their elution sequence upon anion-exchange chromatography. Both proteins mainly bound zinc in their endogenous forms and showed different immunogenicity to rat and rabbit MTs. Dexamethasone, a potent inducer for MT synthesis in mammals, induced the production of both isoforms in crucian carp, whereas cadmium and zinc ions prominently induced the synthesis of ccMT-2. These results indicate that crucian carp have the ability to produce MTs in response to various kinds of environmental stress and that violent air-pumping stress in crucian carp may induce MT synthesis, in part, via the release of endogenous factor(s), such as glucocorticoids.

Topics: Air; Animals; Antibody Specificity; Cadmium Chloride; Carps; Chlorides; Dexamethasone; Glucocorticoids; Kidney; Liver; Metallothionein; Metals; Stress, Physiological; Zinc Compounds

The neurotoxicity of diethyldithiocarbamate (DDC) has been documented for decades. In particular, cytotoxic effects against rat astrocytes have been noted by a decrease in cell viability and numerous ultrastructural defects. This study indicates an in vitro protective effect by zinc administration prior to DDC insult. Cell groups pre-treated with 50 microM ZnCl2 prior to the addition of 35 microg/ml DDC showed significant protection when compared with cells treated with DDC alone. Zinc reduced the DDC-mediated toxicity to astrocytes as indicated by an increase in cell adherence and viability. Morphological evaluation indicated a significant decrease in ultrastructural alterations. Metallothionein (MT), a metal regulatory protein known to be induced by zinc was studied to determine its role in this mechanism of protection. Immunocytochemistry and immunoblots showed increased presence of MT in all zinc treated groups. This suggests a protective effect against DDC cytotoxicity on rat astrocytes in vitro may be associated with an increase in MT concentration.

Topics: Animals; Animals, Newborn; Astrocytes; Blotting, Western; Cell Adhesion; Cell Division; Cell Survival; Cells, Cultured; Chelating Agents; Chlorides; Ditiocarb; Electrophoresis, Polyacrylamide Gel; Metallothionein; Microscopy, Electron; Rats; Rats, Sprague-Dawley; Zinc; Zinc Compounds

We examined the carcinogenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in transgenic mice deficient in the metallothionein (MT) I and II genes and in control (129/Sv) mice. Both strains of mice were given BBN for 8 weeks with or without Zn treatment. All mice were killed at 12 weeks after the cessation of BBN administration. BBN induced bladder tumors in 75% of MT null mice and in 43% of 129/Sv mice. The average number of bladder tumors per mouse was significantly higher in MT null mice (1. 18 +/- 0.27) than in 129/Sv mice (0.43 +/- 0.20). Zn treatment suppressed the carcinogenicity of BBN in 129/Sv mice but not in MT null mice. Histopathological examination of the tumors revealed that the malignant potential of bladder tumors in 129/Sv mice was greater than that in MT null mice. These results indicate that MT is an important modulator of carcinogenicity of BBN in the bladder of mice.

Topics: Animals; Butylhydroxybutylnitrosamine; Carcinogenicity Tests; Carcinogens; Carcinoma, Transitional Cell; Chlorides; Metallothionein; Mice; Mice, Transgenic; Urinary Bladder Neoplasms; Zinc Compounds

Metallothioneins (MT) are low-molecular-weight, cysteine-rich proteins that are induced in response to a variety of chemical stresses and therefore can be used to assess human exposure to environmental agents. In the current study, flow cytometry was used to characterize the basal and cadmium-induced expression of MT in the three major leukocyte populations of human peripheral blood. In the analysis, monocytes were the most sensitive leukocytes to this toxic metal, with significant increases in cellular MT levels being detected at concentrations of cadmium as low as 0.1 microM (24 h). The lymphocyte population also exhibited pronounced treatment-associated elevations in cellular MT, while the granulocyte population was found to be nonresponsive. Although both CdCl2 (3 microM) and ZnCl2 (50 microM) induced MT expression in monocytes to a similar degree and did not affect the expression of this protein in granulocytes, cadmium but not zinc treatment induced dramatic increases in MT levels of lymphocytes. Our results indicate that cellular MT protein levels, as determined by this flow cytometric method, may be used to characterize the differential responsiveness of the major human leukocyte subpopulations to transitional metals. It is evident from the current work that the responsiveness of all peripheral blood leukocyte populations should be analyzed in exposure assessment studies.

Topics: Adult; Cadmium Chloride; Cell Survival; Chlorides; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; In Vitro Techniques; Leukocytes; Lymphocytes; Male; Metallothionein; Monocytes; Phenotype; Zinc Compounds

The kidney, in particular the proximal convoluted tubule, is a major target site for the toxic effects of various metals. However, little is known about the early effects of these metals after acute exposure in man. In the present study we have evaluated the toxicity of several inorganic metal compounds (CdCl2, HgCl2, ZnCl2, and Bi(NO3)3) and the induction of metallothionein by these compounds in cultured human proximal tubular (HPT) cells for up to 4 days. The results showed that bismuth was not toxic even at the highest dose (100 microM) used, while zinc, cadmium and mercury exhibited varying degrees of toxicity, zinc being the least toxic and mercury the most potent. A significant degree of interindividual variation between the different isolates used in these experiments was also observed. All metals used in the present study induced MT, as revealed by immunocytochemistry. All metals showed maximal induction between 1 and 3 days after treatment. Although a certain amount of constitutive MT was present in the cultures, the intensity of the staining varied with time in culture and between the different isolates studied. No correlation could be made between the intensity of the staining in control cultures (indicating total amount of constitutive MT) and the susceptibility of a given isolate to metal toxicity. Furthermore, no correlation could be made between metal-induced MT and the susceptibility of a given isolate to that particular metal.

Topics: Bismuth; Cadmium Chloride; Cells, Cultured; Chlorides; Dose-Response Relationship, Drug; Humans; Immunohistochemistry; Kidney Tubules, Proximal; Mercuric Chloride; Metallothionein; Nitrates; Zinc Compounds

Scavenging of hydroxyl radicals (.OH) by the zinc form of metallothionein (ZnMT) was studied in HL-60 cells and in nuclei from such cells previously treated with ZnCl2 (ZnMT cells). Cells were grown for 48 h to label DNA for alkaline elusion experiments. During the last 24 h 0.1 mM ZnMT was included to induce ZnMT. Generation of DNA single-strand breaks (SSBs) by H2O2 in cells (5 x 10(5)/ml) treated at 4 degrees was increased by approximately 70% in Zn-treated cells by comparison with control cells. These cells had grown from an initial concentration of 5 x 10(5)/ml to a concentration at harvest of 16 x 10(5)/ml. Cells started at 6 x 10(5)/ml and growing to a final concentration of 20 x 10(5)/ml did not exhibit a similar increase in SSBs. This elevation in SSBs was traced to an increase in cell Fe content which exhibited a sharp dependence upon concentrations of cells and of ZnCl2 at the time of addition. The diffusion distance (d) from Fe to DNA of ZnMT cells treated with H2O2 was found to be 3.4 nm. This compares with a distance of 6.1 nm in control cells. SSB generation by hydroxyl radicals formed by 137Cs-gamma rays in Zn-treated cells decreased by 12%, accompanied by a decrease in d from 4.8 nm to 2.9 nm. Thus, ZnMT preferentially reacts with OH formed at some distance from DNA. In nuclei isolated from ZnMT cells started at 5 x 10(5)/ml, SSB generation by H2O2 increased by 60%. The d in these nuclei was 4.9 nm, similar to the distance in control nuclei reported previously. These data suggest that, in addition to altering the scavenging environment, treatment of cells with Zn leads to an increase in reactive Fe in cells and in isolated nuclei which can generate DNA damage through reaction with H2O2.

Topics: Antioxidants; Cell Nucleus; Cell Survival; Cesium Radioisotopes; Chlorides; DNA Damage; DNA, Single-Stranded; Free Radical Scavengers; Gamma Rays; HL-60 Cells; Humans; Hydrogen Peroxide; Hydroxyl Radical; Iron; Metallothionein; Zinc Compounds

Differential pulse polarography (DPP) was applied to measure metallothionein (MT) content in the RH-35 rat hepatoma cell. The method was sensitive in measuring MT at nanogram concentrations. The MT measurement was not interfered by small molecular weight proteins. Cellular MT content increased upon incubated in medium containing different concentrations of CdCl2 or ZnCl2. The increase was concentration-dependent. In the case of CdCl2, significant increase in cellular MT content was observed at a level as low as 0.18 mg/l. The change in MT occurred when there was no significant change in cell viability or cellular protein content. The concentration of ZnCl2 needed to induce the same amount of MT as that with 0.18 mg/l CdCl2 was 12 mg/l. At high concentrations (> 12 mg/l), ZnCl2 also caused an increase in cellular protein content. Therefore, when calculation was based on cellular protein content, ZnCl2 did not caused further increase in MT. L-ascorbic caused significant increase in cellular MT content when it was presented at levels above 100 mg/l but not at 50 mg/l. The effect of L-ascorbic acid on MT induction was not dose-dependent. At 150 mg/l, the amount of MT induced was not different from that with 100 mg/l. Thus, L-ascorbic might not directly influence cellular MT content. Cysteine at 4 and 40 mg/l did not increase cellular MT content. The results of this study showed that cellular MT content could be measured by DPP. Our finding that CdCl2 at sub-lethal concentrations can effectively induce MT suggests that MT may serve as an early warning signal prior to serve as an early warning signal prior to the occurrence of cell death.

Topics: Animals; Ascorbic Acid; Cadmium Chloride; Cell Survival; Chlorides; Cysteine; Dose-Response Relationship, Drug; Liver Neoplasms, Experimental; Metallothionein; Polarography; Rats; Reproducibility of Results; Tumor Cells, Cultured; Zinc Compounds

The functions of metallothioneins (MTs) have been debated for at least a decade. Because it seems unlikely that they evolved only to protect cells against exogenous heavy metals, it has been suggested that MTs have roles in scavenging reactive intermediates, controlling zinc and copper homeostasis, and controlling transfer of zinc to transcription factors and other proteins. Previously, we demonstrated that Chinese hamster G12 cells which overexpress MT have greatly reduced spontaneous mutation rates, suggesting that MT evolved to prevent spontaneous mutagenesis induced by free nuclear zinc ions. We have now isolated G12 transfectants which express antisense RNA to MT. Immunofluorescent staining reveals MT protein in both the nucleus and the cytoplasm in parental cells. A clone expressing high levels of antisense RNA (AMT30) shows reduced basal and induced levels of MT protein. AMT30 cells are hypersensitive to cadmium, zinc, copper and mercury chlorides as well as to menadione. Glutathione levels in AMT30 and G12 cells do not differ. AMT30 cells are spontaneous mutators, showing a spontaneous mutation rate 5-10 times that of G12 cells or G12 cells transfected with vector alone. Only transfectants which show a high level of MT antisense expression (i.e., AMT30) had greatly elevated spontaneous mutation rates. These results support our hypothesis that a major role of MT is to act as an endogenous antimutagen probably via scavenging of reactive intermediates in the nucleus. AMT30 cells should be useful in delineating the sources of spontaneous mutagenesis.

Topics: Animals; Cadmium Chloride; Cell Nucleus; Cells, Cultured; Chlorides; Cloning, Molecular; Copper; Cricetinae; Cricetulus; Cytoplasm; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Genetic Vectors; Mercuric Chloride; Metallothionein; Mutagenesis; Ribonucleases; RNA, Antisense; Transcription, Genetic; Transfection; Vitamin K; Zinc Compounds

Selenoprotein P-like protein, similar to selenoprotein P, uses multiple TGAs for incorporation of selenocysteines but not as stop codons. It is also characterized by having a His-Pro-rich domain and a regionally differential expression pattern. Hence, in addition to selenium metabolism, this protein is considered to have a developmental function. In the present study, the structure of the selenoprotein P-like protein gene was analyzed. The gene consisted of five exons, and the 5'-flanking region contained a TATA box, TCF-1-CS, bHLH-CS, gamma-IRE-CS, c-Myb-CS, C/EBP-CS, HNF-5-CS, MRE2-CS, etc. The presence of motifs like TCF-1-CS, c-Myb-CS, etc. supports the suggestion that this protein is involved in cellular maturation. Since the presence of MRE2-CS suggests that this protein is related to the antidote effect of selenium against heavy metal intoxication, the availability of this motif was examined using bovine kidney cell lines, CKT-1 and MDBK. Metallothionein mRNA markedly increased 6 h after administration of 10(-6) M CdCl2 and ZnCl2 in both cell lines. No significant alteration was observed in selenoprotein P-like protein mRNA, whereas its basal expression was high, indicating that this protein is constitutively expressed. Thus, it is still possible that this protein acts as an antidote, even though it is not inducible by heavy metals.

Topics: Animals; Base Sequence; Cadmium Chloride; Cattle; Cell Line; Chlorides; DNA, Complementary; Gene Expression Regulation; Kidney; Mercuric Chloride; Metallothionein; Metals, Heavy; Molecular Sequence Data; Organ Specificity; Promoter Regions, Genetic; Proteins; Restriction Mapping; RNA, Messenger; Selenoprotein P; Selenoproteins; Sequence Analysis, DNA; Zinc Compounds

Changes in the concentration of free Zn2+ were monitored in isolated rat hepatocytes using the fluorescent indicator zinquin (ethyl[2-methyl-8-p-toluenesulphonamido-6-quinolyloxy]acetat e). The concentration of Zn2+ in freshly isolated hepatocytes was 1.3 x 10(-6) M (range 0.61-2.7 x 10[-6] M). This value decreased by about 10%-15% during incubation in the absence of zinc and increased in a time- and concentration-dependent manner in the presence of exogenous zinc (Km approximately 10 microM). IIb group metal ions led to a concentration-dependent increase in zinquin fluorescence. The rank of efficacy was Hg approximately Cd > Pb (IVa) >> Cu (Ib) >>> Ni (VIII). This rank resembles their ability to mobilize zinc from metallothioneins. 8-Br-3',5'-cAMP (10[-4]M) caused a rapid decrease in Zn2+ epifluorescence which was apparent within 10 min and was sustained throughout the experiment. This effect was gradually obliterated in the presence of external ZnCl2. The effect was specific for cAMP (or cAMP generating hormones) as the calcium-dependent hormone [arg8]vasopressin (5 x 10[-8] M) did not affect intracellular Zn2+. An integrated role of zinc as a possible mediator in signal transduction is discussed.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Arginine Vasopressin; Cadmium; Chlorides; Copper; Dose-Response Relationship, Drug; Fluorescent Dyes; Hemostatics; Lead; Liver; Mercury; Metallothionein; Nickel; Quinolones; Rats; Tosyl Compounds; Zinc; Zinc Compounds

The mouse metallothionein (MT) gene family consists of four known members (MT-I through IV) clustered on chromosome 8. Studies reported herein examine the expression and regulation of the MT-III and MT-IV genes in specific cell types in the maternal reproductive tract, developing embryo, and fetus known to express the MT-I and -II genes. MT-III and MT-IV mRNAs were absent from the visceral yolk sac, placenta, and fetal liver, tissues with high levels of MT-I and MT-II mRNAs. In contrast, MT-III and MT-IV mRNAs were both abundant in the maternal deciduum, and in experimentally induced deciduoma on 7 and 8 days postcoitum (1 dpc = vaginal plug), as are MT-I and -II mRNAs. The abundance of each of these MT mRNAs increased coordinately during development of the deciduum (6-8 dpc), and in situ hybridization localized MT-I, MT-III, and MT-IV mRNAs to the secondary decidual zone of the antimesometrial region on 8 dpc, where in some regions all of the cells were apparently positive. Thus, all of the known mouse MT genes are co-expressed in at least some of the cells in the secondary decidual zone. Electrophoretic analysis of decidual MT suggested that the MT-I, -II, and -III isoforms are abundant proteins in the secondary deciduum. Bacterial endotoxin-lipopolysaccharide (LPS) and Zn are powerful inducers of MT-I and MT-II gene expression in many adult organs, whereas these agents apparently have little effect on MT-III and MT-IV gene expression. Neither of these agents significantly effected levels of decidual MT-III or MT-IV mRNAs in vivo or in primary cultures of decidual cells in vitro, and only modest effects of Zn on MT-I mRNA levels were noted. During 2 days of in vitro culture, decidual cell MT-I and MT-III mRNA levels remained elevated while MT-IV mRNA levels decreased. Thus, expression of the mouse MT gene locus in the deciduum appears to be developmentally regulated, and in this tissue, the MT genes are refractory to induction by Zn or inflammation.

Topics: Animals; Base Sequence; Chlorides; Cloning, Molecular; Decidua; DNA Primers; Embryonic and Fetal Development; Female; Gene Expression Regulation; Gestational Age; Liver; Metallothionein; Mice; Mice, Inbred Strains; Molecular Sequence Data; Multigene Family; Placenta; Polymerase Chain Reaction; Pregnancy; Pseudopregnancy; RNA, Messenger; Uterus; Yolk Sac; Zinc Compounds

The metal-responsive smt operator/promoter region of Synechococcus PCC7942 was fused to the luxCDABE genes of Vibrio fischeri. Plasmid DNA (pJLE23) carrying this fusion conferred metal ion-inducible luminescence to transformed cyanobacteria. Synechococcus PCC7942 (pJLE23) was sensitive to ZnCl2 concentrations within a range of 0.5-4 microM as demonstrated by induction of luminescence. Trace levels of CuSO24 and CdCl2 were also detected.

Topics: Bacterial Proteins; Biosensing Techniques; Cadmium Chloride; Chlorides; Cloning, Molecular; Copper Sulfate; Cyanobacteria; Luminescent Measurements; Metallothionein; Plasmids; Repressor Proteins; Trans-Activators; Transcription, Genetic; Transformation, Genetic; Zinc Compounds

The localization of metallothionein in control and Zn-exposed embryos of Xenopus laevis was studied by whole-mount immunohistochemical staining. The embryos were grown according to the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) protocol from N/F stage 8 to stage 47, with or without addition of ZnCl2 (300 microM) to the medium. At stages 27, 38, 42, 45 and 47, control and Zn-exposed embryos were fixed in buffered formalin, and whole mounts were stained by an immunoperoxidase technique, using monoclonal murine antibody to equine metallothionein. Staining of metallothionein was evident in myotomal cell nuclei of developing somites by stage 27, stomatodeum, oropharynx, and gills by stage 38, developing kidneys (mesonephros) by stage 45, and liver by stage 47. The staining of metallothionein at these sites was more intense in Zn-exposed embryos than controls. The central nervous system (especially the spinal cord) and the yolk mass were faintly stained for metallothionein in controls and Zn-exposed embryos. Staining of metallothionein in myotomal cell nuclei was most prominent at stage 38, diminished at stages 42 and 45, and practically disappeared by stage 47. This is the first report that metallothionein is expressed in myotomal cell nuclei of Xenopus embryos during normal somitogenesis and becomes increased when the embryos are exposed to teratogenic levels of Zn2+.

Topics: Animals; Cell Nucleus; Chlorides; Cleavage Stage, Ovum; Culture Media; Metallothionein; Xenopus laevis; Zinc; Zinc Compounds

Metallothionein (MT) induction was studied in mineralizing cultures of chicken growth plate chondrocytes and quantitated using a Cd-saturation assay. In serum free media, MT induction was observed for Cd concentrations of 0.1 microM and greater and at Zn concentrations of 100 microM and greater. Supplementation of culture media with cysteine and/or methionine resulted in higher levels of MT induction and reduced toxicity during Cd exposure. Maximum MT induction appeared to coincide with the earliest culture stages during which important enzymes and matrix components are being synthesized. Of non-metal MT inducers tested, sodium butyrate caused a low level induction of MT while interleukin-1 had no effect on basal MT levels. 1,25-dihydroxyvitamin D increased MT induction. The steroid hormone dexamethasone caused a reduction in basal and induced MT levels. These findings suggest that MT regulation in growth plate chondrocytes differs significantly from what is known in other cell types and that this difference may be related to the mineralization of this tissue.

Topics: Animals; Butyrates; Butyric Acid; Cadmium Chloride; Cells, Cultured; Chickens; Chlorides; Culture Media; Cysteine; Dexamethasone; Dihydroxycholecalciferols; Growth Plate; Interleukin-1; Metallothionein; Methionine; Zinc Compounds

The effect of pretreatment with metallothionein (MT) inducers (bismuth nitrate or zinc chloride) on clastogenicity of anticancer drugs was investigated. Bismuth nitrate (50 mumol/kg) or zinc chloride (400 mumol/kg) was administered s.c. to mice once a day for two days prior to treatment with 3.3 mumol/kg of cis-diamminedichloroplatinum(II) (cis-DDP), 3.4 mumol/kg of adriamycin (ADR), 72 mumol/kg of cyclophosphamide (CPA) or 0.41 mumol/kg of L-phenylalanine mustard (L-PAM). The frequency of occurrence of erythrocytes with micronuclei in bone marrow was increased by each anticancer drug at 24 h after treatment. Micronucleus formation was significantly prevented by pretreatment with either bismuth nitrate or zinc chloride. MT concentration in bone marrow cells of mice at the time of treatment with anticancer drugs increased to 2- and 3.5-fold by pretreatment with bismuth nitrate and zinc chloride, respectively. These results indicate that MT induction in bone marrow cells effectively prevents micronucleus induction of anticancer drugs.

Topics: Animals; Antineoplastic Agents; Bismuth; Bone Marrow; Chlorides; Cisplatin; Cyclophosphamide; Doxorubicin; Male; Melphalan; Metallothionein; Mice; Mice, Inbred ICR; Micronucleus Tests; Mutagens; Nitrates; Zinc Compounds

The uptake of 65Zn, determined by gamma-counting and also by autoradiography, significantly increased with the increasing level of metallothionein in liver 4 weeks after the start of feeding a diet containing 3'-methyl-dimethylaminoazobenzene (3'-Me-DAB), at which time the serum gamma-glutamyl transpeptidase activity was not yet significantly elevated. At this time, furthermore, hepatic uptake of 67Ga-citrate did not increase and that of 99mTc-Sn-colloid decreased in 3'-Me-DAB-fed rats. These results suggest that a short-life gamma-emitting isotope such as 69mZn may be useful for the detection at the early stage of hepatic carcinogenesis.

Topics: Administration, Oral; Animals; Autoradiography; Chlorides; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; p-Dimethylaminoazobenzene; Rats; Rats, Wistar; Tissue Distribution; Zinc Compounds; Zinc Radioisotopes

Metallothioneins (MTs) are sulfhydryl-rich proteins. MT-I and MT-II are found in all tissues of the body, while MT-III exists only in brain. Regulation of MT-I and MT-III mRNA was studied in brain and liver of control C57BL/6J mice and mice given chemicals known to increase MT-I, namely, lipopolysaccharide (LPS), zinc chloride (Zn), cadmium chloride (Cd), dexamethasone (Dex), ethanol, and kainic acid (KA). Northern blot analysis revealed that MT-I mRNA levels in liver were induced dramatically (12-27-fold over basal levels) by all of the chemicals, while in brain only LPS produced an increase in MT-I mRNA (2-fold). Interestingly, the MT-I inducers, Cd, Dex, ethanol, and KA, down-regulated brain MT-III mRNA levels by approx. 30%. Because brain is such a heterogenous tissue, in situ hybridization was used to localize MT-I and MT-III mRNA in control and treated mice. MT-I mRNA signal, which was most abundant in the glial cells of the Purkinje cell layer of the cerebellum in control mice, appeared to be enhanced in mice given the MT-I inducers (LPS, Zn, Cd, Dex, ethanol, and KA). MT-I mRNA hybridization signal was also enhanced in the olfactory bulbs from LPS- and Cd-treated mice, while this signal was present but weak in control brains. MT-III mRNA hybridization signals were localized in hippocampus and co-localized with MT-I message in the glial cells of the Purkinje cell layer of the cerebellum. In addition, diffuse MT-III mRNA signals were visible in areas of the cerebral cortex, and in the molecular layer of the cerebellum. Signals for MT-III in hippocampus appeared to be reduced by KA, Dex and LPS treatment, while in the cortical region, MT-III mRNA signals appeared to be enhanced by KA, Cd, and ethanol treatment. In conclusion, both MT-I and MT-III expression in brain appears to be modulated by exogenous treatment, however, the changes are small in relation to those observed in liver. Chemical-induced alterations of MT mRNA are non-uniform throughout the brain, and thus best studied in a region-specific manner.

Topics: Animals; Base Sequence; Blotting, Northern; Brain; Cadmium; Cadmium Chloride; Chlorides; Dexamethasone; Ethanol; Gene Expression Regulation; Kainic Acid; Lipopolysaccharides; Liver; Male; Metallothionein; Mice; Mice, Inbred C57BL; Molecular Sequence Data; RNA, Messenger; Zinc Compounds

Heme oxygenase-1, an essential enzyme in heme catabolism, and metallothionein IIA, a small metal-binding protein with clusters of cysteins, are remarkably induced in HeLa cells following the treatment with cadmium or zinc. Both proteins are considered to be involved in the defense system against metal toxicity. Here we showed by transient expression assays that the cadmium-responsive element (CdRE) of the human heme oxygenase-1 gene is not responsive to zinc, whereas a metal-regulatory element (MRE) of the human metallothionein IIA gene is able to respond to either cadmium or zinc. The CdRE is recognized by a certain nuclear protein(s) which is however unable to bind to an MRE of the metallothionein IIA gene. These results suggest that the metal-selective activation of each gene promoter is mediated by a separate mechanism.

Topics: Base Sequence; Cadmium; Cadmium Chloride; Chlorides; Consensus Sequence; Enzyme Induction; Gene Expression Regulation; HeLa Cells; Heme Oxygenase (Decyclizing); Humans; Metallothionein; Molecular Sequence Data; Plasmids; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Zinc Compounds

One mechanism by which chemicals cause cellular injury is the formation of reactive oxygen species. In vitro studies have shown that metallothionein (MT), a small metal-binding, sulfhydryl-rich, readily inducible protein, can scavenge reactive oxygen species, especially hydroxyl radicals. Nevertheless, whether or not MT protects against oxidative stress in the intact animal is not known. Experimental induction of MT could help to clarify this question, however, it is unclear whether agents that induce MT also influence known antioxidant systems. Therefore, the present study was designed to determine whether the well-known MT inducers are specific for induction of MT or whether they might also influence other hepatic systems that protect against oxidative stress. Male rats were administered cadmium chloride (Cd; 30 mumol/kg, s.c.), zinc chloride (Zn; 1000 mumol/kg, s.c.), alpha-hederin (alpha-H, 30 mumol/kg, s.c.) or lipopolysaccharide (LPS; 1 mg/kg, s.c.) 24 h prior to measurement of antioxidant systems. Zn and alpha-H increased hepatic GSH concentration 20% and 55%, respectively. Cd significantly increased, whereas LPS reduced, the activities of selenium-dependent glutathione peroxidase and glutathione reductase. Glutathione S-transferases were not altered by any of the inducers. Cd also increased DT-diaphorase activity. Cd, Zn and alpha-H all decreased catalase activity 20-35%, while the activity of superoxide dismutase was unaffected by the inducers. The amount of total cytochrome P450 enzymes and cytochrome b5 were decreased by LPS, Cd and alpha-H, while Zn appeared to have no effect. The activities of P450 enzymes towards testosterone oxidation were also decreased by LPS, Cd and alpha-H. In conclusion, all four MT inducers examined affect systems known to protect cells against oxidative stress. Therefore, using these chemicals to determine the in vivo role of MT in protecting against oxidative stress poses difficulties.

Topics: Analysis of Variance; Animals; Cadmium; Cadmium Chloride; Carcinogens; Catalase; Chlorides; Cytochrome P-450 Enzyme System; Cytochromes b5; Cytosol; Dihydrolipoamide Dehydrogenase; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Lipopolysaccharides; Liver; Male; Metallothionein; Oleanolic Acid; Oxidative Stress; Rats; Rats, Sprague-Dawley; Saponins; Selenium; Superoxide Dismutase; Testosterone; Zinc Compounds

Transgenic mice which expressed human IGF-binding protein-3 (hIGFBP-3) were generated by pronuclear injection of an hIGFBP-3 cDNA driven by the mouse metallothionein 1 promoter. Two of the seven founder mice had measurable levels of hIGFBP-3 in the circulation. The serum levels of hIGFBP-3 increased as the mice were bred to homozygosity and were further induced by supplementing the drinking water with 25 mM ZnCl2. While the birth weight, litter size and body weight of transgenic mice were not significantly different from non-transgenic litter mates or wild-type mice derived from the same genetic background, the transgenic mice demonstrated selective organomegaly. The spleen, liver and heart of mice derived from both founders were significantly heavier compared with organs from non-transgenic mice (P < 0.05, P < 0.005 and P < 0.01 respectively). The weights of the brain and kidney were similar in transgenic and non-transgenic mice. Expression of the transgene was detected in the kidney, small intestine and colon by Northern blot analysis. Western ligand blotting of serum from transgenic mice did not demonstrate any change in the abundance of the IGFBPs detected by this method. When serum from transgenic mice was incubated with 125I-labeled IGF-I and analyzed by Sephacryl S-200 chromatography under neutral conditions a significantly (P < 0.05) increased amount of the radioactivity was found in the 140 kDa ternary complex compared with serum from wild-type mice. Immunoreactive hIGFBP-3 was detected in the 140 kDa ternary complex but the majority of immunoreactive hIGFBP-3 present in transgenic mouse serum eluted in later fractions indicating that it was not associated with the acid-labile subunit. These data demonstrate that modest constitutive expression of hIGFBP-3 has a selective effect on organ growth and development. The establishment of these IGFBP-3 transgenic mouse strains may provide useful models to investigate further the physiological role of IGFBP-3.

Topics: Animals; Cardiomegaly; Chlorides; DNA, Complementary; Female; Gene Expression Regulation, Developmental; Hepatomegaly; Humans; Insulin-Like Growth Factor Binding Protein 3; Male; Metallothionein; Mice; Mice, Inbred Strains; Mice, Transgenic; Organ Size; Promoter Regions, Genetic; Recombinant Fusion Proteins; Splenomegaly; Transgenes; Zinc Compounds

The effect of Glutathione (GSH) depletion on the induction of metallothionein (MT) synthesis by paraquat (PQ) was examined in ICR mice. An increase in hepatic MT level in mice was observed after a single PQ administration. Pretreatment of mice with L-buthionine-SR-sulfoximine (BSO), an inhibitor of GSH synthesis, enhanced the induction of hepatic and renal MT synthesis by PQ depending on the decreased tissue GSH level. A similar result was obtained by pretreatment with diethylmaleate, a GSH depleting agent. The ratio of hepatic MT-I to MT-II induced by PQ was not changed by BSO pretreatment. An increase in the hepatic MT level in GSH depleted mice was observed from 3 hr to 24 hr after PQ administration. An increase in the hepatic MT-I mRNA level after treatment with PQ was observed prior to hepatic MT induction in BSO pretreated mice. Pretreatment with actinomycin D, an inhibitor of mRNA synthesis, inhibits the PQ-induced increase in hepatic MT and MT-I mRNA levels in BSO pretreated mice. Pretreatment with BSO did not affect the induction of MT synthesis by zinc, cadmium or dexamethasone. Pretreatment with dexamethasone, an anti-inflammatory agent, enhanced the hepatic MT induction by PQ treatment in GSH depleted mice, while dexamethasone reduced the MT induction by turpentine oil, which is known to induce inflammation and hepatic MT synthesis. These findings suggest that GSH depletion enhances the induction of MT synthesis by PQ because of an increase in the transcription rate, and this enhancement of MT synthesis is not due to an inflammatory response caused by PQ.

Topics: Animals; Antimetabolites; Blotting, Northern; Buthionine Sulfoximine; Cadmium; Chlorides; Chromatography, Gel; Chromatography, Ion Exchange; Dactinomycin; Dexamethasone; Glucocorticoids; Glutathione; Herbicides; Kidney; Liver; Male; Maleates; Metallothionein; Mice; Mice, Inbred ICR; Paraquat; Protein Synthesis Inhibitors; Zinc Compounds

Synthesis of metallothionein-I (MT-I) and heme oxygenase mRNAs is rapidly and transiently induced by H2O2 in mouse hepatoma cells (Hepa) and this effect is blocked by catalase. Menadione, which generates free radicals, also induces these mRNAs. Deletion mutagenesis revealed that a region between -42 and -153 in the mouse MT-I promoter was essential for induction of a CAT reporter gene. A multimer of a 16 bp sequence (-101 to -86) that includes an antioxidant response element and overlapping adenovirus major late transcription factor binding site elevated basal expression and allowed induction by H2O2 when inserted upstream of a minimal promoter. However, deletion of this region (-100 to -89) from the intact MT-I promoter (-153) did not completely eliminate response. Multiple copies of a metal response element also permitted response to H2O2. These results suggest that induction of MT-I gene transcription by H2O2 is mediated by at least two different elements within the proximal MT-I gene promoter and suggest a previously undescribed function of the MRE. Induction of MT gene transcription by ROS and the subsequent scavenging of ROS by the MT peptide is reminiscent of the metal regulatory loop and is consistent with the hypothesized protective functions of MT.

Topics: Animals; Antioxidants; Base Sequence; Carcinoma, Hepatocellular; Chlorides; Consensus Sequence; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Heme Oxygenase (Decyclizing); Hydrogen Peroxide; Metallothionein; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA, Messenger; Sequence Deletion; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured; Viral Proteins; Vitamin K; Zinc Compounds

G12, a transgenic Chinese hamster V79 cell derivative which contains a single copy of the Escherichia coli gpt gene as a target for mutagenesis, has little constitutive metallothionein (MT) expression. It was transfected with a vector containing the mouse MT-I gene, and MT-I-overproducing lines were isolated. MT-I transfectants had lower spontaneous mutation frequencies compared with the G12 parental cell line. Mutagenesis by alkylating agents was unchanged. MT expression in G12 and MT transfectants could be modulated by exposure to Zn(II) or Cd(II). The spontaneous mutation frequencies in Zn(II)- and Cd(II)-treated cells was inversely related to MT expression. In G12 cells grown in concentrations of Zn(II) up to 12 microM, a significant dose-dependent increase in spontaneous mutagenesis was observed. At higher (but subtoxic) concentrations in which endogenous MT was induced, a dramatic decrease in spontaneous mutagenesis was observed. In contrast, MT-I transfectants exhibited much lower spontaneous mutagenesis after growth in all concentrations of Zn(II). These data demonstrate a possible role for MT in modulating spontaneous mutagenesis and point to a role for Zn(II) in contributing to spontaneous mutagenesis. Because there is variability in human MT expression, low MT expression might be a risk factor for cancer.

Topics: Animals; Cadmium; Cell Line; Chlorides; Cricetinae; Cricetulus; Drug Resistance; Ethyl Methanesulfonate; Metallothionein; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutation; Transfection; Zinc Compounds

The effect of the stress-induced, cysteine-rich protein, metallothionein I (MT), on oxygen consumption by rat liver mitochondria was studied. Using a Clark-type oxygen electrode we found that electron transport from succinate to oxygen was enhanced by MT whereas ADP-initiated oxygen consumption was inhibited by MT. The MT effect was concentration dependent. No evidence was found for the provision by MT of an alternate pathway to oxygen.

Topics: Adenosine Diphosphate; Animals; Cadmium; Cadmium Chloride; Chlorides; Dose-Response Relationship, Drug; Electron Transport; Male; Metallothionein; Mitochondria, Liver; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Succinates; Succinic Acid; Zinc Compounds

Metallothioneins (MTs) are low-molecular-mass cysteine-rich proteins implicated in metal homoeostasis and resistance to toxicity induced by heavy metals and alkylating agents. We report high hepatic MT protein accumulation (greater than 100-fold compared with wild-type mice) in toxic milk (tx) mice, along with markedly higher cytosol copper and zinc levels. Increased MT-gene transcription alone could not account for the high constitutive MT protein levels, since MT mRNA levels were not increased in tx mouse livers. However, hepatic MT was significantly more stable in adult tx mice: MT half-life (t1/2) was 79 or 77% greater than in wild-type mice before and after Cd induction respectively. Cd or Zn treatment increased MT mRNA, but not MT protein, accumulation in tx mouse livers: Cd displaced MT-bound Zn and Cu in preexisting MT. Thus tx mice appear to accumulate hepatic MT as a result of decreased protein degradation. These animals may provide a useful model to study the physiological role of MT, and human diseases (such as Wilson's disease) with abnormal copper metabolism.

Topics: Animals; Cadmium; Cadmium Chloride; Chlorides; Copper; Cytosol; Female; Glutathione; Half-Life; Kidney; Kinetics; Liver; Male; Metallothionein; Mice; Mice, Mutant Strains; Organ Specificity; Reference Values; RNA, Messenger; Zinc; Zinc Compounds

We examined the efficacy of metallothionein induction in the prevention of the carcinogenic action of cis-platinum and melphalan administered repeatedly to mice over a relatively long period. The increased pulmonary metallothionein induced by bismuth or zinc compounds during the period of chemotherapy with cis-platinum or melphalan protected the mice from carcinogenesis of these drugs in the lung. These results suggested the efficacy of metallothionein inducers in suppression of carcinogenicity considered as a secondary effect of anticancer agents in cancer chemotherapy.

Topics: Animals; Anticarcinogenic Agents; Bismuth; Carcinogens; Chlorides; Cisplatin; Female; Lung; Lung Neoplasms; Melphalan; Metallothionein; Mice; Mice, Inbred A; Nitrates; Zinc Compounds

The effects of cytokines and heavy metals on the expression and localization of metallothioneins (MTs) within U373MG astrocytoma cells were analyzed by using indirect immunofluorescence using a monoclonal anti-MT antibody (MT45). IL-1, CdCl2 (50 microM) or ZnCl2 (500 microM) remarkably augmented intracellular MT levels, whereas IL-6 or 10 microM of ZnCl2 showed no inducing activity. From 24 to 48 h after the addition of CdCl2 or IL-1, immunoreactive MTs were found in the cytoplasm and the nucleus. After 72 h, immunoreactive MTs accumulated in a granular form near the cell surfaces in the presence of CdCl2 (50 microM) or IL-1 plus ZnCl2 (10 microM). However, this accumulation was not observed when only IL-1 was added. Thus, Zn2+ facilitated the appearance of the granular form of immunoreactive MTs at a concentration where they do not induce MTs by themselves.

Topics: Astrocytoma; Cadmium; Cadmium Chloride; Chlorides; Humans; Interleukin-1; Metallothionein; Metals; RNA, Messenger; Tumor Cells, Cultured; Zinc; Zinc Compounds

Properties of the reactions of cis-dichlorodiammine Pt(II) and related complexes with zinc metallothionein or apometallothionein have been investigated. During these reactions, platinum binds stoichiometrically to protein sulfhydryl groups and zinc, if present, is displaced. The ammine ligands are also lost in the process, suggesting that Pt(II) has tetrathiolate coordination in metallothionein. This conclusion is supported by extended x-ray absorption-fine structure studies which indicate that there are 4 sulfurs in the first coordination sphere of the platinum centers. The product contains 10 +/- 2 Pt(II) per mol of protein and migrates over Sephadex G-75 as a structure of similar size to zinc metallothionein. The kinetics of reaction are biphasic as monitored by the formation of Pt-thiolate bonds or by the release of zinc from the protein. Both methods yield identical rate laws for the reaction. The first step is independent of Pt(II) concentration but involves the binding of as many as four platinum atoms to the protein with little Pt-sulfhydryl bond formation and without much loss of zinc. The second rate process is first order in both zinc or sulfhydryl binding sites and Pt(II). Neither kinetic step is sensitive to the chloride ion concentration over the range 0-0.5 M. However, the reaction is sensitive to pH between 5.5 and 8.0. trans-Dichlorodiammineplatinum(II) reacts with zinc metallothionein with similar kinetics.

Topics: Animals; Chlorides; Cisplatin; Hydrogen-Ion Concentration; Kinetics; Liver; Metallothionein; Protein Binding; Rats; Rats, Inbred Strains; Spectrophotometry; Zinc; Zinc Compounds

Topics: Animals; Cadmium; Cadmium Chloride; Chlorides; Horses; Humans; Liver; Male; Metallothionein; Rabbits; Rats; Rats, Inbred Strains; Species Specificity; Zinc; Zinc Compounds

Topics: Amino Acids; Animals; Apoproteins; Cadmium; Chlorides; Dithionitrobenzoic Acid; Endopeptidases; Kinetics; Metallothionein; Peptide Mapping; Rabbits; Rats; Zinc; Zinc Compounds

Compared to non-metal toxicants (ammonia, 1.56 ppm; and phenol, 10 ppm), the metals (CdCl2, 30 ppm; HgCl2, 16.7 ppb; and ZnCl2, 6 ppm) significantly induced hepatic metallothionein (MT) concentrations in C. punctatus, exposed independently to non-lethal doses of these toxicants for 28 days. It is suggested that hepatic MT serves as a metal-sequestering protein and is involved in the detoxication of metals only and ensures protection from toxic chemicals in ambience.

Topics: Ammonium Hydroxide; Animals; Chlorides; Fishes; Gene Expression Regulation; Hydroxides; Liver; Mercuric Chloride; Metallothionein; Phenol; Phenols; Stimulation, Chemical; Zinc; Zinc Compounds

Topics: Administration, Cutaneous; Animals; Chlorides; Dose-Response Relationship, Drug; Guinea Pigs; Kidney; Liver; Metallothionein; Rats; Skin; Skin Absorption; Zinc; Zinc Compounds

DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.

Topics: Animals; Blotting, Southern; Cell Division; Cell Line; Chlorides; DNA Polymerase I; Gene Amplification; Gene Expression Regulation, Enzymologic; Genetic Vectors; Genomic Library; Metallothionein; Mice; Promoter Regions, Genetic; RNA; RNA, Antisense; Transfection; Zinc; Zinc Compounds

The MER5 cDNA was cloned from RNA preferentially synthesized in murine erythroleukemia (MEL) cells during the early period of MEL cell differentiation. To understand the role of the MER5 gene in the differentiation, we have transferred the MER5 cDNA into MEL cells in both sense and antisense orientations under control of the promoter of the human metallothionein gene. Only in the transformants with the antisense MER5 cDNA, did their elevated expression inhibit differentiation. The result suggests that the MER5 gene product may promote early events in the differentiation of MEL cells.

Topics: Amino Acid Sequence; Animals; Blotting, Western; Cell Differentiation; Chlorides; Cloning, Molecular; Erythrocytes; Genes; Kinetics; Leukemia, Erythroblastic, Acute; Metallothionein; Mice; Molecular Sequence Data; Promoter Regions, Genetic; RNA, Antisense; Transfection; Transformation, Genetic; Tumor Cells, Cultured; Zinc; Zinc Compounds

Only one metallothionein appears in domesticated duck upon zinc induction. The complete amino acid sequence has been elucidated. This metallothionein has the same sequence as the chicken metallothionein, as determined by chemical sequencing of overlapping peptides produced by selective proteinase digestion and confirmed by mass spectrometry. The observation that animals of divergent origins share a common gene product presents an example of extreme conservation of a stress-inducible protein.

Topics: Amino Acid Sequence; Animals; Chlorides; Chromatography, High Pressure Liquid; Ducks; Metallothionein; Molecular Sequence Data; Peptide Fragments; Zinc; Zinc Compounds

Low dietary concentrations of methionine and cysteine are known to decrease hepatic glutathione content. However, it is not known if restricting the dietary content of these sulfur containing amino acids also affects hepatic levels of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the cofactor for sulfation, or metallothionein, a protein rich in sulfhydryl groups. Rats were fed diets lacking cysteine and containing various concentrations of methionine (0.15, 0.3, or 0.6%) for 8 days. Control diet contained 0.3% each of methionine and cysteine. Hepatic glutathione levels were decreased approximately 75% in rats fed diets containing 0.15 or 0.3% methionine. In contrast, PAPS and hepatic metallothionein concentrations were not decreased by the low sulfhydryl diets. Additionally, rats on the various diets were challenged by the administration of ZnCl2 (3 mmol/kg. sc). In both control rats and rats maintained on sulfhydryl-deficient diets, ZnCl2 increased hepatic metallothionein to the same level. However, significantly lower levels of PAPS were observed after ZnCl2 in rats receiving sulfhydryl-deficient diets than in controls. In summary, restriction of dietary sulfhydryl markedly decreases the hepatic content of glutathione and has a minor effect on PAPS concentration, but does not decrease the basal hepatic concentration of metallothionein or its induction by ZnCl2.

Topics: Adenine Nucleotides; Animals; Chlorides; Cysteine; Cystine; Diet; Glutathione; Liver; Male; Metallothionein; Methionine; Phosphoadenosine Phosphosulfate; Rats; Rats, Inbred Strains; Zinc; Zinc Compounds

Exposure of a trout gonadal fibroblast (RTG-2) cell line to ZnCl2, CdCl2 and CuCl2 resulted in differential levels of accumulation of metallothionein (MT) mRNA. ZnCl2 being the most effective agent induced MT mRNA in 3 h, with 172-fold induction after 48 h and continued accumulation up to 144 h. Following CdCl2 treatment, mRNA could be detected after 24 h, reaching peak levels at 72 h. Furthermore, trout MT mRNA could be detected up to 8 days after withdrawal of extraneous ZnCl2. Using a novel technique of primer extension and DNA sequencing with total RNA as template, specificity of the trout MTa and MTb gene-specific primers was established. Primer extension studies revealed a higher response of MTa to ZnCl2 and CdCl2 compared to MTb. Insensitivity of MT mRNA induction to cycloheximide suggested that induction by the metals was independent of de novo protein synthesis. However, simultaneous exposure of cells to actinomycin D and metals completely inhibited MT mRNA synthesis implying control at the transcriptional level.

Topics: Animals; Base Sequence; Blotting, Northern; Cadmium; Cadmium Chloride; Cell Line; Chlorides; Cycloheximide; Dactinomycin; Fibroblasts; Gene Expression Regulation; Kinetics; Metallothionein; Molecular Sequence Data; RNA, Messenger; Transcription, Genetic; Trout; Zinc; Zinc Compounds

During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-induced differentiation of these transfectants was inhibited in proportion to the level of exogenous c-myb mRNA expression. By adding or removing ZnCl2 at different times during the induction process, it was possible to show that up-regulation of exogenous c-myb limited to the first 2 days of induction had little or no effect on differentiation. In contrast, continuous expression of exogenous c-myb beginning at any time during the period of induction blocked further differentiation. These results suggest that during HMBA induction of MEL cells, the early down-regulation of c-myb mRNA is not necessary for terminal differentiation, whereas the down-regulation of c-myb at a later time is necessary.

Topics: Animals; Cell Differentiation; Cell Line; Chlorides; Gene Expression Regulation, Neoplastic; Kinetics; Leukemia, Erythroblastic, Acute; Metallothionein; Mice; Plasmids; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Proto-Oncogenes; Restriction Mapping; RNA, Neoplasm; Transfection; Zinc; Zinc Compounds

Gold sodium thiomalate was incubated with one cadmium-sensitive cell line and two cadmium-resistant variants. The resistant lines have been reported to synthesize metallothionein (MT) in response to both cadmium and zinc, whereas the sensitive line does not. All cell lines showed a dose-dependent inhibition of growth as a result of gold sodium thiomalate treatment. However, daily comparisons of cell numbers indicate that the cadmium-resistant lines actually increase in number at the highest gold concentrations, whereas numbers of cells in the nonresistant line decrease. MT biosynthesis was measured by monitoring the incorporation of [35S )cysteine into low molecular weight protein. None of the cells synthesized MT in response to gold. When incubated with both zinc and gold, MT was synthesized by both of the cadmium resistant lines; however, the amount of MT synthesized was reduced in the presence of gold which appears to inhibit the uptake of [35S]cysteine by all the cell lines. Although MT is synthesized in the presence of zinc and gold sodium thiomalate, the MT does not have a significant effect on the ability of these cells to withstand high concentrations of gold.

Topics: Animals; Cadmium; Cell Division; Cell Line; Chlorides; Chromatography; Contraceptives, Oral, Combined; Cricetinae; Cysteine; Drug Resistance; Female; Gold Sodium Thiomalate; Mesocricetus; Metallothionein; Ovary; Zinc; Zinc Compounds

Immunocytochemical and electrophoretic techniques were used to investigate the presence of metallothionein, a metal-binding protein, in the dorsolateral and ventral lobes of the developing rat prostate. Male rats aged 7 and 14 days were injected subcutaneously with 6 and 20 mg/kg body weight of cadmium and zinc, respectively, or with saline for controls, 24 h prior to tissue sampling. Immunohistochemical localization of metallothionein was observed in the epithelial tissues of the dorsolateral prostate from 7 and 14 day-old animals and in 1 day-old untreated rats. This staining pattern did not appear to be significantly affected by cadmium or zinc treatment. In contrast, metallothionein localization in the ventral prostate decreased with age but demonstrated a slight response to metal-ion treatment in the 7 day-old animals. Electrophoretic and immunoblot analysis confirmed the presence of metallothionein in the control and metal-induced prostate samples from neonatal rats. Lobe-specific differences in localization suggest a functional significance for metallothionein, independent of inducible protein.

Topics: Animals; Animals, Newborn; Blotting, Western; Cadmium; Cadmium Chloride; Chlorides; Electrophoresis, Polyacrylamide Gel; Immunoenzyme Techniques; Male; Metallothionein; Prostate; Rats; Rats, Inbred Strains; Zinc; Zinc Compounds

In the intact animal, inorganic Cd distributes mainly to the liver and produces hepatotoxicity, while Cd-metallothionein (CdMT) distributes primarily to the kidney and produces nephrotoxicity. CdMT has also been demonstrated to be more toxic than Cd in cultured kidney cells, but it is not known if CdMT is more toxic to all cultured cells or if there is a good correlation between in vitro and in vivo toxicity. Therefore, hepatocytes, which were isolated and grown in monolayer culture for 24 h, were incubated with CdCl2 (1-100 microM) or CdMT (3-100 microM Cd). The intracellular K+ content was quantitated 24 h later as an index of toxicity. The K+ concentration of the hepatocytes was decreased 50% by 4 microM CdCl2, whereas 25 microM CdMT was required to produce similar injury. In the intact animal, zinc induces the synthesis of MT and decreases the hepatotoxicity of Cd. ZnCl2 added to the media (100 microM) for 24 h before exposure to Cd or CdMT increased the intracellular MT concentration 700%. This elevation in MT reduced the toxicity of CdCl2 approximately 80% but did not alter the toxicity of CdMT. In summary, CdCl2 is more toxic to cultured hepatocytes than Cd-MT, and MT induction decreases the toxicity of CdCl2 in hepatocytes, as has been observed in the intact animal. This indicates that cultured hepatocytes appear to be an excellent model for examining the hepatotoxicity of Cd.

Topics: Animals; Cadmium; Cadmium Chloride; Cells, Cultured; Chlorides; Liver; Male; Metallothionein; Potassium; Rats; Zinc; Zinc Compounds

The induction of the mouse metallothionein-I (MT-I) transcriptional regulatory region by heavy metals in cultured avian cells was studied in a transient-expression assay system for a growth hormone. The MT-I promoter was shown to be inducible by 10 microM to 90 microM ZnCl2 in a dose-dependent manner.

Topics: Animals; Cells, Cultured; Chick Embryo; Chlorides; Cloning, Molecular; Gene Expression Regulation; Genetic Vectors; Growth Hormone; Metallothionein; Plasmids; Precipitin Tests; Promoter Regions, Genetic; Transfection; Zinc; Zinc Compounds

The effect of dietary zinc status on biliary excretion of zinc, cadmium and mercury administered as a bolus of metal chloride (1 mg metal/kg body weight i.v.) was studied. Female rats were fed a purified diet containing either 9 micrograms/g (low), 45 micrograms/g (adequate) or 1150 micrograms/g (high) zinc for 8 d. Hepatic metallothionein (MT) was similar in low- and adequate-zinc groups, but was 18-fold higher in the high-zinc group than in the other two groups. Liver zinc content varied in relation to dietary zinc level. Biliary excretion of all metals studied was significantly lower in the high-zinc group than in the low-zinc group. The cumulative excretion of zinc, cadmium and mercury over 2.5 h in rats fed these two diets was 6.2 +/- 1.4 vs. 33.5 +/- 7.7, 0.006 +/- 0.02 vs. 22.8 +/- 8.4 and 1.6 +/- 0.6 vs. 14.9 +/- 5.3 micrograms/kg body weight, respectively. A relationship was found between the disposition of metal in liver and the extent of biliary metal excretion. Biliary metal excretion was highly correlated with liver cytosolic non-MT-bound metal; r = 0.999, 0.998 and 0.993 for endogenous + exogenous zinc, cadmium and mercury, respectively.

Topics: Animals; Bile; Cadmium; Cadmium Chloride; Chlorides; Cytosol; Diet; Female; Liver; Mercuric Chloride; Metallothionein; Rats; Rats, Inbred Strains; Spectrophotometry, Atomic; Zinc; Zinc Compounds

Metallothionein (MT) genes contain multiple metal regulatory elements (MREs) that are responsible for metal induction. A protein blotting procedure and a synthetic oligonucleotide have been used to identify nuclear factors interacting with a MRE (MREd) of the mouse MT-1 gene. We report the specific binding of the probe to a protein of apparent Mr 108,000 (p108). The specificity of the interaction was demonstrated by mutation analysis and competition experiments. Furthermore, the probe contains the Sp1 consensus binding sequence 5'CCGCCC3', in addition to the MRE consensus sequence, 5'TGCAC3', and we show that a Simian Virus 40 DNA fragment which contains six Sp1 binding sites did not bind p108 nor did it compete for the protein(s) interacting with MREd in a DNA footprinting assay. These results show that a metal regulatory element of the mouse MT-1 gene interacts specifically with a nuclear protein of Mr 108,000 and that this protein is distinct from the transcription factor Sp1.

Topics: Animals; Base Sequence; Cadmium; Cadmium Chloride; Cell Nucleus; Chlorides; DNA; Genes; Genes, Regulator; L Cells; Metallothionein; Mice; Molecular Sequence Data; Nuclear Proteins; Oligonucleotide Probes; Zinc; Zinc Compounds

Deletions in chromosome 7 of the mouse affect the expression of the metallothionein gene Mt-1, which maps on chromosome 8, and steady-state levels of Mt-1 mRNA are reduced to 15-40% of normal in livers of newborn mice homozygous for either the c3H or c14CoS deletion. Glucocorticoids fail to induce hepatic Mt-1 mRNA levels in deletion homozygotes in contrast to normal littermates. However, zinc chloride is effective in inducing Mt-1 mRNA levels in livers of deletion homozygotes as well as of their normal littermates. Other tissues (e.g., kidney and intestine) of deletion homozygotes express basal levels of Mt-1 mRNA higher than those of normal littermates. In the intestine these are furthermore inducible by both hormonal and metal agents. Thus, loss of inducibility of the Mt-1 gene in deletion homozygotes concerns glucocorticoids only and is furthermore restricted to specific cell types (i.e., hepatocytes). The trans-acting factor(s) normally encoded in the deleted region of chromosome 7 appears to be instrumental in conferring on the metallothionein gene in hepatocytes the essential competence to respond to hormonal inducing signals.

Topics: Albinism; Animals; Chlorides; Chromosome Deletion; Chromosome Mapping; Dexamethasone; Gene Expression Regulation; Homozygote; Intestinal Mucosa; Kidney; Liver; Metallothionein; Mice; Mice, Mutant Strains; RNA, Messenger; Zinc; Zinc Compounds

We have studied DNA repair in the Chinese Hamster Ovary (CHO) metallothionein (MT) gene after UV-light induced damage. The repair was examined comparatively with or without transcriptional activation of the gene by incubation in the presence of the heavy metal ZnCl2. Whereas the repair efficiency was very low in the uninduced state, it increased significantly after induction of the gene. The presence of ZnCl2 did not appear to change other repair parameters in the cells. The overall genome DNA repair efficiency after UV irradiation was similar whether or not the gene was induced and the preferential DNA repair pattern in the essential dihydrofolate reductase (DHFR) gene which we have previously described was unaffected by the presence of ZnCl2. Based upon repair analysis in two different restriction fragments containing the MT I gene, we conclude that the region of efficient repair after induction is considerably larger than the 1 kb size of the gene. The results suggest that the accessibility of a genomic region to DNA repair enzymes may be regulated by the local chromatin structure in a dynamic manner.

Topics: Animals; Cell Line; Chlorides; Cricetinae; Cricetulus; DNA Repair; DNA Restriction Enzymes; Gene Expression Regulation; Metallothionein; Tetrahydrofolate Dehydrogenase; Transcription, Genetic; Zinc; Zinc Compounds

Metallothioneins (MT) contain a high concentration of cysteine which bind heavy metals. Exposure of liver cells to metals induces the synthesis of MT and thus causes the cells to draw upon their sulfhydryl (SH) pools. The utilization of methionine as compared with that of cysteine as a source of SH for the synthesis of MT has not been shown. Therefore, studies were designed to determine whether methionine, in addition to cysteine, serves as an SH donor for Zn-induced synthesis of MT in rat hepatocyte cultures. Hepatocytes were able to synthesize only low levels of MT when the concentration of both amino acids was extremely low; however, when either of the amino acids was present at a high concentration, production of MT was independent of the other amino acid concentration. Subsequently, induction of MT was compared in four media: complete (0.5 mM methionine, 0.5 mM cysteine), Met (0.5 mM methionine), Cys (0.5 mM cysteine), and SH free (-SH). Somewhat higher concentrations of MT were produced by the hepatocytes in the Met than in the Cys media and no differences were observed between the Met and the complete media. By contrast, GSH synthesis was much more dependent on methionine than on cysteine for its synthesis. Incorporation studies with 35S-labeled cysteine and methionine indicated that lower concentrations of MT found in hepatocytes in the Cys media may be due to less accumulation of cysteine by the hepatocytes. Cellular accumulation of cysteine was initially rapid and then reached a plateau, whereas the rate for methionine accumulation was more constant and eventually obtained higher cellular levels. To provide additional evidence for the role of methionine in MT production, a known inhibitor of the cystathionine pathway, DL-propargylglycine (PPG), was added to each of the four media. Reductions in MT levels were not observed in the cells cultured in the complete and Cys media; however, a 95% reduction was observed in the cells cultured in the Met media. In summary, the present results suggest that both cysteine and methionine can serve as a SH source for MT synthesis, and that the availability of SH in most culture mediums would not limit the synthesis of MT. Whereas methionine is a much better SH source than cysteine for GSH synthesis in hepatocyte cultures, it is only slightly better for MT synthesis.

Topics: Animals; Chlorides; Cysteine; Glutathione; Liver; Male; Metallothionein; Methionine; Rats; Rats, Inbred Strains; Sulfates; Time Factors; Zinc; Zinc Compounds

During the second half of gestation in the mouse there is a rise in both fetal (4-fold) and maternal (10-fold) metallothionein-I (MT-I) mRNA in the liver (but not in the kidney). There is a large increase in plasma corticosterone (the predominant murine glucocorticoid hormone), as well as an increase in hepatic zinc, which is coincident with the induction of MT-I mRNA. Considering that both of these compounds are known to be effective inducers of MT-I mRNA, we set out to determine whether either one or both were involved in the developmental regulation of MT-I genes. Several lines of evidence suggest that corticosterone is the principal inducer of fetal MT-I mRNA: The induction of MT-I mRNA in the liver, but not in the kidney, mimics glucocorticoid regulation but not metal regulation. Reduction of maternal corticosterone levels by treating mice with metyrapone lowered MT-I mRNA levels but had no effect on zinc levels. A line of transgenic mice carrying a metallothionein-growth hormone fusion gene that is responsive to metals but unresponsive to glucocorticoids was not developmentally regulated. Based on these observations, we propose that corticosterone is responsible for the induction of MT-I mRNA and that the resulting MT sequesters zinc and copper which may be used later in development.

Topics: Animals; Chlorides; Corticosterone; Dexamethasone; Female; Gastric Mucosa; Genes; Glucocorticoids; Intestinal Mucosa; Intestines; Kidney; Liver; Metallothionein; Mice; Mice, Inbred BALB C; Pregnancy; Promoter Regions, Genetic; RNA, Messenger; Stomach; Tissue Distribution; Zinc; Zinc Compounds

A comparison of the Hg radioassay (i.e. "Piotrowski" assay), the Cd radioassay (i.e. "cadmium-hemolysate" assay), and the radioimmunoassay (RIA) for metallothionein (MT) was performed for livers from control, Cd-, and Zn-pretreated rats. Results indicate that the Cd and Hg radioassays gave similar results in all cases. The RIA values compared very favorably to those obtained by the metal saturation assays for livers from control rats. The values obtained for livers from Cd- or Zn-treated animals were generally lower in the RIA than in the Hg or Cd radioassay, ranging from 36 to 75% of the metal saturation assays. The source of this discrepancy cannot be discerned from the present data. However, the various assay systems employed did yield MT concentrations that followed similar intraassay patterns, being lowest in control, elevated to an intermediate level with Cd treatment, and highest following Zn treatment.

Topics: Animals; Cadmium; Cadmium Chloride; Chlorides; Liver; Male; Mercury; Metallothionein; Radioimmunoassay; Radioligand Assay; Rats; Rats, Inbred Strains; Zinc; Zinc Compounds

The copper complex of 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazone) or CuKTS is reduced and dissociated upon reaction with Ehrlich cells. Titration of the cells with the complex leads to the specific binding of copper to metallothionein with 1 to 1 displacement of its complement of zinc. Under conditions of complete titration of metallothionein, 1.25-2.5 nmol CuKTS/10(7) cells, cellular DNA synthesis is rapidly inhibited but no long term effects on cell proliferation are observed. The kinetics of redistribution of Cu and Zn in Ehrlich cells in culture and in animals were studied after pulse reaction of CuKTS with cells. After exposure of cells to the noncytotoxic concentration of 2.5 nmol of CuKTS/10(7) cells, nonmetallothionein bound copper is lost rapidly from the cells, after which copper in metallothionein decays. New zinc metallothionein is made as soon as exposed cells are placed in culture. New synthesis stops when the level of zinc in metallothionein reaches control levels. A second pulse treatment of cells with CuKTS to displace zinc from metallothionein again stimulates new synthesis of the protein to restore its normal concentration. The kinetics of metal metabolism in Ehrlich cells exposed to 5.5 nmol of CuKTS/10(7) cells, which inhibits cell proliferation, are qualitatively similar except there is a pronounced lag before new zinc metallothionein is synthesized. The Ehrlich ascites tumor in mice responds to CuKTS similarly to cells in culture. It is also shown that cultured Ehrlich cells do not make extra zinc metallothionein in the presence of high levels of ZnCl2, and fail to accumulate copper in the presence of large concentrations of CuCl2.

Topics: Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Cell Division; Cells, Cultured; Chlorides; Copper; Female; Kinetics; Metallothionein; Mice; Mice, Inbred ICR; Organometallic Compounds; Thiosemicarbazones; Time Factors; Zinc; Zinc Compounds

Synchronized Chinese hamster cells were induced with ZnCl2 at multiple stages of the cell cycle and labeled with [35S]cysteine, and the 35S-labeled proteins were isolated and separated into metallothionein and nonmetallothionein fractions. Metallothionein was found to be inducible in all stages of the cell cycle and in G1-arrested cells.

Topics: Animals; Cell Cycle; Chlorides; Cricetinae; Cricetulus; Cysteine; Metallothionein; Zinc; Zinc Compounds

A number of toxic chemical and physical agents elicit the induction of a series of protein species, some of which react with the agents and render them nontoxic. A few of the induced species (such as metallothionein) are rich in thiol groups that might be expected to react with alkylating agents and render them nontoxic. If a safe means could be found for selectively enhancing the synthesis of alkylating-agent-reactive species in normal but not tumor cells, such a procedure would have ramifications in the area of cancer chemotherapy. In this report, we have utilized a variety of trace elements (Zn, Se, Cu, As) as inducers of synthesis of protective species in line CHO Chinese hamster cells and in a number of derived variants to determine whether this type of approach can be utilized to increase resistance to alkylating-agent toxicity. Our results indicate that Zn, Se and Cu elicit a protective response (increased survival, monitored by colony-forming ability) against the toxic effects of iodoacetate or melphalan, and, at least in the case of zinc, at levels that are physiologically reasonable. Arsenite appears to be a marginally effective inducer in the CHO cell and an ineffective inducer in the Cdr20F4 variant cell. The increased survival is not attributable to metallothionein inducibility, decreased availability of the alkylating agent in the medium, or decreased uptake of the drug into the trace-element-pretreated cells. The protective responses induced by zinc or selenite alone are additive in cells receiving both trace elements prior to exposure to alkylating agent, which suggests that different domains of response are elicited by the two metals. In view of reported differences in inducibility of protective proteins between normal and tumor cells, a possibility is raised for a novel approach to alkylating-agent chemotherapy that is somewhat analogous to the protocol utilized in high-dose methotrexate therapy.

Topics: Animals; Biotransformation; Cell Line; Cell Nucleus; Cell Survival; Chlorides; Cricetinae; Cricetulus; Cytoplasm; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Female; Iodoacetates; Melphalan; Metalloproteins; Metallothionein; Ovary; Trace Elements; Zinc; Zinc Compounds

Topics: Animals; Apoproteins; Cadmium; Cadmium Chloride; Cell Nucleus; Chlorides; Copper; Lead; Liver; Male; Metallothionein; Metals; Mice; RNA; Zinc; Zinc Compounds

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Carbon Tetrachloride Poisoning; Chlorides; Glutathione; Liver; Metalloproteins; Metallothionein; Microsomes, Liver; Rats; Zinc; Zinc Compounds