metallothionein and cadmium-sulfate

Nanotechnology has gained increasing commercial attention over recent years and its use has raised concerns about its potential release in the environment. The purpose of this study was to determine the size distribution of CdTe in freshwater, bioavailability and potential toxic effects of cadmium telluride quantum dots (CdTe QD) to the freshwater mussel Elliptio complanata. Mussels were exposed to increasing concentrations (0 to 8 mg Cd L(-1)) of CdTe and 0.5 mg/L CdSO4 for 24 h at 15 degrees C to examine the initial uptake and toxic effects of Cd from CdTe QDs and dissolved CdSO4. After the exposure period, Cd bioaccumulation in the gills, digestive gland and gonad tissues and metallothionein (MT) levels were determined. The results revealed that about 80% of Cd was retained by a 450 nm pore filter (aggregates) and that 14% of the Cd was in the dissolved phase (i.e., eluted through a 1 kDa ultrafiltration membrane) which suggested that uncoated CdTe QDs were not stable in freshwater. In mussels, Cd was accumulated principally by the gills and digestive gland and the bioaccumulation factors of Cd from CdTe were similar to that of dissolved Cd. Indeed, tissue-levels of Cd were below the proportion of dissolved Cd from CdTe which suggests that Cd rather comes from the dissociation of Cd from the ingested QDs than from the internalization of the QDs in mussel tissues. The levels of MT were induced in both the digestive gland and gonad but were readily decreased in the gills by both CdTe and CdSO4. The observed decrease in the metallic form of MT might result from the oxidative stress by CdTe and dissolved Cd. In conclusion, uncoated CdTe QD in freshwater leads to aggregates and a dissolved component of Cd where the latter explained the contribution of the observed accumulation pattern in mussel tissues and effects on MT levels in mussels.

Topics: Animals; Biological Availability; Bivalvia; Body Burden; Cadmium Compounds; Digestive System; Dose-Response Relationship, Drug; Fresh Water; Gills; Gonads; Metallothionein; Oxidative Stress; Quantum Dots; Solubility; Sulfates; Tellurium; Up-Regulation; Water Pollutants, Chemical

Metallothioneins (MTs) have an important role in zinc homeostasis and may counteract the impact of oversupply. Both intracellular zinc and MT expression have been implicated in proliferation control and resistance to cellular stress, although the interdependency is unclear. The study addresses the consequences of a steady-state overexpression of MT-1 for intracellular zinc levels, cell cycle progression, and protection from zinc toxicity using a panel of cell lines with differential expression of MT-1. The panel comprised parental Chinese hamster ovary-K1 cells with low endogenous expression of MT and transfectants with enhanced expression of mouse MT-1 on an autonomously replicating expression vector with a noninducible promoter. Cell cycle progression, determined by flow cytometry and time-lapse microscopy, revealed that enhanced cytoplasmic expression of MT-1 does not impact on normal cell cycle operation, suggesting that basal levels of MT-1 expression are not limiting for background levels of oxidative stress. MT-1 overexpression correlated with a steady-state increase in cytoplasmic free Zn(2+), assessed using the fluorescent zinc-sensor Zinquin, particularly at high levels of overexpression, further suggesting that zinc availability is normally not limiting for cell cycle progression. Enhanced MT-1 expression, over a 10-fold range, had a clear impact on resistance to Cd(2+) and Zn(2+) toxicity. In the case of Zn(2+), the degree of protection afforded was less, indicating that MT-1 has a limited range and saturable capacity for effecting resistance. The results have implications for the use of cellular stress responses to exogenously supplied zinc and zinc-based systemic therapies.

Topics: Animals; Cadmium Compounds; Cell Cycle; Cell Proliferation; Cell Survival; CHO Cells; Cricetinae; Cricetulus; Cytoplasm; Cytoprotection; Dose-Response Relationship, Drug; Metallothionein; Mice; Stress, Physiological; Sulfates; Time Factors; Transfection; Up-Regulation; Zinc Sulfate

Glutathione (GSH) is the precursor of the phytochelatins (PC), which in plants and fungi are involved in heavy metal sequestration. The regulatory enzyme gamma-glutamylcysteine synthetase (gamma-ECS) catalyzes the first step in GSH biosynthesis. For the heavy metal accumulator Brassica juncea L. a partial gamma-ECS cDNA was cloned by RT-PCR. Treatment of suspension-cultured dark grown seedlings with micromolar concentrations of CuSO4 resulted in a strong increase of gamma-ECS mRNA in roots and shoots, concomitant with an increase of GSH and phytochelatins. A significant up-regulation of gamma-ECS mRNA was observed at 25 microM CuSO4 (shoot growth: -11%), whereas maximum up-regulation was obtained at 100 microM CuSO4 (shoot growth: -60%). Unexpectedly, metallothionein 2 (MT2) mRNA was decreased in response to the CuSO4 treatments. CdSO4 at a concentration of 50 microM caused a 72% reduction in shoot growth without affecting the amounts of gamma-ECS- and MT2 mRNAs. ZnSO4 at a concentration of 500 microM did not reduce growth but induced transient increases of gamma-ECS- and MT2 mRNAs. The implications of the results with respect to differential regulation of gamma-ECS and MT2 during heavy metal exposure are discussed.

Topics: Arabidopsis; Base Sequence; Brassica; Cadmium Compounds; Cloning, Molecular; Copper; Copper Sulfate; DNA Primers; Gene Expression Regulation, Plant; Glutamate-Cysteine Ligase; Metallothionein; Molecular Sequence Data; Plant Leaves; Plant Roots; Polymerase Chain Reaction; Recombinant Fusion Proteins; RNA, Messenger; Sulfates; Transcription, Genetic; Zinc Sulfate

The thermolabile glucocorticoid receptor (GR) in fibroblasts from a patient with familial glucocorticoid resistance (FGR) was characterized by solution hybridization, Northern blot analysis and Western immunoblotting using an hGR and cRNA probe and a GR specific monoclonal antibody. Specific DNA binding was measured by binding of cytosolic GR to mouse mammary tumour virus (MMTV) DNA. Northern blot analysis of total cellular RNA isolated from the fibroblasts showed hybridization of the hGR probe to 7.0 and 6.1 kb RNA species. Basal expression of hGR mRNA was 1.8 times higher in fibroblasts derived from the patient compared to control fibroblasts as assayed by solution hybridization. Even though nonsignificant, dexamethasone treatment maximally caused at 60% down-regulation of GR mRNA in normal fibroblasts after 12 h but only a 40% down-regulation in fibroblasts from the patient. In both cases, the initial mRNA values were restored after 72 h. No difference in GR mRNA stability was observed between fibroblasts from the patient and from controls. The induction of the glucocorticoid-regulated gene metallothionein IIA (MTIIA) by dexamethasone and cadmium sulphate was studied at different temperatures using a cRNA probe for human MTIIA. At elevated temperatures, cadmium sulphate but not dexamethasone increased MTIIA mRNA levels approximately three-fold in fibroblasts from the patient, whereas in normal fibroblasts regardless of temperature both cadmium sulphate and dexamethasone increased MTIIA mRNA levels approximately three- and two-fold, respectively. Cytosolic GR from FGR-fibroblasts showed an increased specific binding to MMTV DNA at 4 degrees C. These data support our previous findings of a thermolabile GR, probably due to a defect intrinsic to the GR protein, in this patient with primary cortisol resistance and indicate a compensatory mechanism at the transcriptional level of GR expression. The data also indicate a receptor defect affecting specific DNA binding in vitro.

Topics: Binding Sites; Blotting, Northern; Cadmium; Cadmium Compounds; Cells, Cultured; Dexamethasone; DNA, Viral; Down-Regulation; Drug Resistance; Female; Fibroblasts; Gene Expression Regulation; Humans; Mammary Tumor Virus, Mouse; Metallothionein; Plasmids; Receptors, Glucocorticoid; RNA Probes; RNA, Messenger; Sulfates

A feature of the cascade regulation of herpes simplex virus 1 gene expression in productive infection is that the first genes to be expressed, the alpha genes, are transactivated by a structural component of the virion designated as the alpha transinducing factor (alpha TIF). In this study, we have tested the hypothesis that latent infection of sensory neurons results from the failure of alpha TIF, a tegument protein, to be transported from the nerve endings to the nucleus of the sensory neuron. Two viruses were constructed. The first recombinant virus (R6003) contained a second copy of the alpha TIF gene placed under the control of a metallothionein promoter. The second recombinant virus (R6004) is identical to R6003 except for the presence of a stop codon inserted at amino acid 70 of the second alpha TIF gene. The metallothionein promoter inserted into the viral genome was shown to be expressed, and alpha TIF mRNA was detected by in situ hybridization of sections of trigeminal ganglia of mice infected with R6003, both untreated and those given cadmium injections. In all experiments, there were no significant differences in the recovery of latent virus from mice infected with R6003 or R6004, whether injected with cadmium or not. Cadmium administration at the time of infection and at intervals thereafter did not preclude establishment of latency. In another series of experiments, transgenic mice expressing the metallothionein-driven alpha TIF did not differ from nontransgenic siblings with respect to the incidence of latent virus in trigeminal ganglia. We conclude that the absence of alpha TIF cannot alone account for the establishment of latency.

Topics: Animals; Biological Transport; Cadmium; Cadmium Compounds; Cells, Cultured; Gene Expression Regulation, Viral; Genes, Viral; Herpes Simplex; Male; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Transgenic; Neurons; Promoter Regions, Genetic; RNA, Messenger; Simplexvirus; Sulfates; Trans-Activators; Trigeminal Ganglion; Virus Activation

A Cd-binding protein in the Cd2+-resistant strain 301N of Saccharomyces cerevisiae was induced by administration to 0.5 mM CdSO4. The protein was purified by a gel-permeation and subsequent ion-exchange column chromatographies. The purified Cd-binding protein had the characteristics of metallothioneins: (1) low molecular weight (9.0 kDa), (2) high Cd content (63 micrograms/mg protein), (3) amino-acid composition rich in cysteine (18%), basic and acidic amino acids and free from aromatic amino acids, and (4) an absorption shoulder at near 250 nm. Acid pH or EDTA treatments abolished 250 nm absorption of the Cd-binding protein, and the formed apoprotein was capable of binding Cd2+, Cu2+ and Zn2+, respectively. Heat treatment (75 degrees C) little affected the ultraviolet absorption or sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of Cd-binding protein. These results suggest that metallothionein generally found in animals also occurs in Cd-adapted yeast cells and thus has a role in its Cd-resistance.

Topics: Amino Acids; Cadmium; Cadmium Compounds; Chromatography, Gel; Chromatography, Ion Exchange; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Metallothionein; Molecular Weight; Saccharomyces cerevisiae; Spectrophotometry, Ultraviolet; Sulfates

We have constructed a recombinant plasmid pCPS12 containing the hepatitis B viral surface antigen (HBsAg) gene linked to the mouse metallothionein promoter on a BPV-pML2 vector. Two stable clones S12-8 and S12-2, obtained by transfection of the mouse C127 cells with pCPS12 propagated in dam+ dcm+ and dam- dcm- Escherichia coli respectively, exhibited different types of response to 5-azacytidine (5-aza-CR) and cadmium (Cd) induction. In S12-8, the productivity of HBsAg was enhanced by 5-aza-CR or 5-aza-CR plus Cd, but not by Cd alone. In S12-2, the expression of HBsAg was not affected by 5-aza-CR but was induced by Cd in the presence or absence of 5-aza-CR. This suggests that methylation may be important in controlling the HBsAg expression and the inducibility of Cd in the transfectants.

Topics: Animals; Azacitidine; Cadmium; Cadmium Compounds; Cell Line; DNA, Recombinant; Genetic Vectors; Hepatitis B Surface Antigens; Metallothionein; Methylation; Mice; Papillomaviridae; Sulfates; Transfection

Cadmium (Cd) exposure in mice induces transcription of metallothionein (MT) mRNA and protein accumulation in both liver and kidney. Resistance to hepatotoxicity through chronic exposure to heavy metals is the result of this induction. However, the same chronic exposure results in damage to kidney. We report here that acute exposure of mice to Cd as cadmium sulfate (CdSO4), which resulted in preferential accumulation of metal in liver, or Cd-metallothionein (CdMT), which resulted in preferential metal accumulation in kidney, induced MT mRNA accumulation in both liver and kidney. However, MT mRNA accumulated to a level twofold higher in liver than in kidney in response to CdSO4. Equivalent doses of CdMT induced MT mRNA accumulation to an equal degree in kidney and liver. While MT mRNA accumulation in kidney was directly proportional to the amount of cadmium in the organ, this was not the case in liver. There, liver MT mRNA was elevated in the absence of elevated tissue cadmium levels. Interestingly, CdMT induced alphafetal protein (AFP) mRNA accumulation in kidney, but not liver. It appears that (a) maximal MT mRNA accumulation in kidney is less than in liver, and (b) liver is capable of accumulating MT mRNA in response to even very low cadmium exposure that may not result in elevated tissue cadmium.

Topics: alpha-Fetoproteins; Animals; Cadmium; Cadmium Compounds; Kidney; Liver; Male; Metallothionein; Mice; RNA, Messenger; Sulfates

1. A simple method for preparation of metallothionein (Mt) I and II has been developed for the purpose of making standards for use in various biochemical systems and in antibody production. 2. The theoretical content of SH groups in a Mt protein; assuming the mol. wt to be 10,000 and each molecule to contain 20 SH groups was found to be 7.1 and 7.7 times higher than for our purified Mt I and II, respectively. 3. In our native polyacrylamide gel system Mt I ran ahead of Mt II, while the two Mt forms were not separated in the Laemmli SDS system in which it behaved as a protein with mol. wt 10,000. In both gel systems, however, Mt I stained as a very faint band in comparison to Mt II, despite equal absorbance at 254 nm and Cd binding capacity. 4. Compared to staining of polyacrylamide gels with Coomassie Brilliant Blue less than 1/50 parts (1 ng) of the protein could be easily seen after silver staining. 5. It was found that Mt may undergo spontaneous modification, polymerization and loss of metal binding properties. 6. Spontaneous modification and polymerization reduced the antigenic properties of our purified Mt. Only Mt II appeared to be immunologically active.

Topics: Animals; Autoradiography; Cadmium; Cadmium Compounds; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Liver; Metallothionein; Molecular Weight; Rabbits; Rats; Rats, Inbred Strains; Reference Standards; Sulfates

A variety of genes have been shown to change copy number during development, including rRNA genes in amphibians and chorion proteins in insects. Dihydrofolate reductase and metallothionein-1 (MT-1) genes are present in high copy number in cultured mammalian cells subjected to low levels of agents that will select for cells with amplified copies of specific genes. Recent studies have shown that the metallothionein-1 gene in mouse liver is regulated at the transcriptional level by treatment with heavy metals. We report here that, at cadmium concentrations 5 to 10-fold higher than that required to induce maximal transcription of the MT-1 gene, there is a 2 to 3-fold increase in MT-1 gene concentration in liver nuclear DNA by 6 hours after induction, and extra copies persist up to 3 weeks in the absence of further heavy metal treatment. The extra MT-1 gene copies that appear 6 hours after cadmium treatment are in a conformation that renders them relatively nuclease insensitive.

Topics: alpha-Fetoproteins; Animals; Cadmium; Cadmium Compounds; Cell Nucleus; DNA; DNA Replication; Gene Amplification; Kinetics; Liver; Male; Metallothionein; Mice; Micrococcal Nuclease; Nucleic Acid Hybridization; Sulfates; Transcription, Genetic

A mouse hepatocyte cell line selected for growth in 80 microM CdSO4 (Cdr80 cells) was used to test the role of metallothioneins in heavy metal detoxification. The cadmium-resistant (Cdr80) cells have double minute chromosomes carrying amplified copies of the metallothionein-I gene and accumulate ca. 20-fold more metallothionein-I mRNA than unselected cadmium-sensitive (Cds) cells after optimal Cd stimulation. As a consequence, the amount of Cd which inhibits DNA synthesis by 50% is ca. 7.5-fold higher in Cdr80 cells than in Cds cells. Cds and Cdr80 cells were compared in terms of their resistance to other heavy metals. The results indicate that although Zn, Cu, Hg, Ag, Co, Ni, and Bi induce metallothionein-I mRNA accumulation in both Cdr80 and Cds cells, the Cdr80 cells show increased resistance to only a subset of these metals (Zn, Cu, Hg, and Bi). This suggests that not all metals which induce metallothionein mRNA are detoxified by metallothionein and argues against autoregulation of metallothionein genes. Metallothionein-I mRNA is also induced by iodoacetate, suggesting that the regulatory molecule has sensitive sulfhydryl groups.

Topics: Animals; Cadmium; Cadmium Compounds; DNA Replication; Gene Amplification; Genes; Iodoacetates; Iodoacetic Acid; Kinetics; Liver Neoplasms, Experimental; Metallothionein; Metals; Mice; RNA, Messenger; Sulfates; Transcription, Genetic