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METALLOTHIONEIN

Metallothionein is a cysteine-based protein that transports metals such as copper, zinc, and cadmium in the body.

Structure and function of metal chelators produced by plants: the case for organic acids, amino acids, phytin, and metallothioneins.

Rauser WE

Department of Botany, University of Guelph, ON, Canada. wrauser@uoguelph.ca

Plants produce a range of ligands for cadmium (Cd), copper (Cu), nickel (Ni), and zinc (Zn). Cd- and Zn-citrate complexes are prevalent in leaves, even though malate is more abundant. In the xylem sap moving from roots to leaves, citrate and histidine are the principal ligands for Cu, Ni, and Zn. Phosphorus-rich globular bodies in young roots are probably Zn-phytate. Metallothioneins (MTs) are cysteine (Cys)-rich ligands. Plants produce class II MTs (MT-IIs) which differ from the archetypal mammalian MT-I in the location and number of Cys. The Ec protein from wheat embryos has Cys in three domains, binds Zn, and disappears with seedling development. The first 59 amino acids have been sequenced for the protein. Fifty-eight genes for MT-IIs, from a range of plants and tissues, predict proteins with Cys in two domains. Most of the predicted proteins have not been isolated, and their metal binding is poorly documented. Three protein bands, corresponding to six MT genes, have been isolated from Arabidopsis, and the amino acids sequenced for nine fragments. The MT-IIIs are atypical, nontranslationally synthesized polypeptides with variously repeating gamma-glutamylcysteine units. Of the five families known, those with carboxy-terminal glycine are the most widespread among plants, algae, and certain yeasts. A heterogeneous grouping of these molecules form Cd-binding complexes with tetrahedral coordination and a Cd-sulfur interatomic distance of 2.52 A. One complex is cytosolic, the dominant one is vacuolar. Together, they can bind a large proportion of cellular Cd; other ligands may also function. Little is known about the counterpart situation for Cu and Zn.

Toxicol Appl Pharmacol 1999 Oct 1;160(1):76-85

Cadmium uptake and transepithelial transport in control and long-term exposed Caco-2 cells: the role of metallothionein.

Blais A, Lecoeur S, Milhaud G, Tome D, Kolf-Clauw M

Laboratoire de Toxicologie, Ecole Nationale Veterinaire d'Alfort, Maisons Alfort, France.

Exposure of humans to cadmium, a common environmental pollutant, is mainly through food intake. However, the mechanisms of intestinal absorption have not been clearly elucidated for this toxic metal ion. In order to investigate the effects of long-term exposure to this metal and the role of metallothioneins in cadmium absorption, we used human-derived Caco-2 cells cultured on porous membrane filters. We first validated this model by quantifying metal uptake and transepithelial transport on control cells and cells adapted to grow for 2 to 5 weeks in the presence of low doses of cadmium in the culture medium. The nontoxic doses of cadmium (0.1, 1.0, and 5 microM), in which Caco-2 cells could be cultured for many passages without deleterious effects, were determined by evaluating transepithelial resistance of the cells and lactate dehydrogenase leakage. After 24 h of 1 microM Cd exposure, intracellular cadmium levels were 3- and 6-fold higher for cells exposed for extended periods to 1 and 5 microM cadmium, respectively, compared to control cells. In control and long-term exposed cells, this accumulation was inhibited by zinc, copper, and pCMBS, but not by verapamil or ouabain. Intracellular metallothionein content was increased 1.5-, 5-, and 12-fold for the cells grown in the presence of 0.1, 1.0, and 5 microM cadmium, respectively, in the culture medium. The amount of metallothionein synthesized and released by the cells was highly correlated with cadmium accumulation and transport. Our results suggest that Caco-2 cell monolayers are a good predictive model for the study of cadmium intestinal absorption following exposure to repeated low doses of cadmium, and confirm the essential role of metallothionein in the regulation of cadmium intestinal absorption.

 

Placenta 1992 Jul-Aug;13(4):349-55

Cellular localization of metallothionein in human term placenta.

Goyer RA, Haust MD, Cherian MG

Department of Pathology, University of Western Ontario, London, Canada.

Cellular localization of metallothionein (MT) in placenta may provide information on its function as a metal binding protein. Rabbit antibodies to rat liver MT cross-reacted with human MT and were used to localize MT in human term placenta by avidin-biotin peroxidase technique. Serial sections (5 microns) were cut from paraffin-embedded placentae obtained at term from five normal women and incubated with rabbit antibodies to MT. Normal rabbit serum was used as a negative control. The slides were incubated with biotinylated swine anti-rabbit IgG (linking antibody) then with avidin-biotin horseradish peroxidase complex and developed with diaminobenzidine in hydrogen peroxide (0.03 per cent) substrate. The optimum staining of MT was obtained at a 1:800 antibody dilution. MT was identified in fetal amniotic cells, syncytial trophoblasts and villous interstitial cells, and in maternal decidual cells. The presence of MT at specific cellular sites suggests that it may regulate the transplacental transport of metals such as zinc, copper and cadmium. Since the level of cadmium is lower and that of zinc and copper higher in fetal than in maternal blood, this may suggest that placental MT may restrict cadmium while enhancing zinc and copper transport.

Exp Gerontol 1993 Jan-Feb;28(1):17-38

Metallothionein expression and stress responses in aging human diploid fibroblasts.

Luce MC, Schyberg JP, Bunn CL

Department of Biology, University of South Carolina, Columbia 29208.

Metallothioneins (MTs) are low molecular weight proteins with a high cysteine content that are inducible by heavy metals and by other conditions of environmental stress. This laboratory was investigated in human diploid fibroblasts the induction of MTs by cadmium and by dexamethasone, and the induction of heat shock proteins, as models for age-related changes in gene expression that reflect the ability of old cells to respond to environmental stress. Old cells were more sensitive to the toxic effects of CdCl2 in the concentration range 100-175 microM. Analysis of 35S-cysteine-labelled cell extracts by polyacrylamide gel electrophoresis and fluorography showed that in the absence of any inducer, old cells have a 3.7-fold increase over young cells in the basal level of MT. The rate and extent of induction of MT by CdCl2 was reduced in old cells: Exposure of old cells to 100 microM CdCl2 for 18 h resulted in MT levels about 33% of the amount in young cells. Northern blot analysis showed that the changes in MT protein levels occurred in parallel with changes in mRNA levels, which implicates transcriptional control as the origin of these aging changes. These young/old differences in MT synthesis were maintained in density-arrested cultures, indicating that the aging changes were not due to differences in the cell cycle status of these cell populations. The rate and extent of induction of a 68-kDa heat shock protein were also reduced in old cells, which showed an increase in basal, uninduced level of this protein similar to MT. In contrast, old cells retained the ability to synthesize MTs in response to dexamethasone at a rate similar to that in young cells.

Chem Biol Interact 1992 Mar;82(1):99-110

Glutathione is the antioxidant responsible for resistance to oxidative stress in V79 Chinese hamster fibroblasts rendered resistant to cadmium.

Chubatsu LS, Gennari M, Meneghini R

Department of Biochemistry, University of Sao Paulo, Brazil.

By manipulation of Cd and Zn concentrations in the medium, several phenotypes, differing in the contents of glutathione (GSH) and metallothionein (Mt), were derived from a parental clone of V79 Chinese hamster fibroblast. In some of these phenotypes, resistance to Cd and cross-resistance to oxidative stress was developed. The highest levels of GSH and Mt were found in cells which were rendered resistant to Cd by stepwise increases of Cd and Zn in the cell medium for over 50 passages. Upon removal of Cd/Zn from the medium of these cells or addition of Cd/Zn to the parental cell medium, changes of cellular GSH and Mt levels occurred to different extents. At the same time, changes in the resistance to Cd and H2O2 were observed. Good linear correlations were observed for Mt levels x resistance to Cd and for GSH levels x resistance to H2O2. Poor linear correlations were found for Mt levels x resistance to H2O2 or for GSH levels x resistance to Cd. Moreover, addition of Zn to the medium produced an increase in Mt content without affecting the GSH content. In this case no cross-resistance to oxidative stress was developed. Therefore, Mt which has been shown to be an excellent antioxidant in in vitro experiments, does not seem to play any major role against oxidative stress in Zn and Cd challenged cells. Most of the cross-resistance to oxidative stress in Cd challenged cells seems to be accounted for by the parallel increase in GSH.

J Invest Dermatol 1998 Jan;110(1):24-8

Inhibitory effect of beta-thujaplicin on ultraviolet B-induced apoptosis in mouse keratinocytes.

Baba T, Nakano H, Tamai K, Sawamura D, Hanada K, Hashimoto I, Arima Y

Department of Dermatology, Hirosaki University School of Medicine, Japan.

Sunburn cells are thought to represent ultraviolet B-induced apoptotic keratinocytes. It has been demonstrated that enzymatic and nonenzymatic antioxidants effectively suppress sunburn cell formation, indicating that reactive oxygen species may play a role in the progression of ultraviolet B-induced apoptosis. Metallothionein, a cytosol protein, has antioxidant activity, and overexpression of metallothionein has been reported to reduce the number of sunburn cells in mouse skin. We have also demonstrated that overexpression of metallothionein inhibits ultraviolet B-induced DNA ladder formation in mouse keratinocytes. These findings support the hypothesis that cellular metallothionein may play an important role in the inhibition of ultraviolet B-induced apoptosis in keratinocytes through its antioxidant activity. In the present study, we investigated the effects of beta-thujaplicin, an extract from the woods of Thuja plicata D. Don. and Chamaecyparis obtuse, Sieb. et Zucc., on ultraviolet B-induced apoptosis in keratinocytes and on metallothionein induction. Topical application of beta-thujaplicin decreased the number of ultraviolet B-mediated sunburn cells and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling-positive cells in mouse ear skin. Incubation with beta-thujaplicin suppressed ultraviolet B-induced DNA ladder formation in cultured mouse keratinocytes. Histochemical analysis showed that topical application of beta-thujaplicin induced metallothionein protein in mouse skin. Northern analysis and western blotting revealed significant induction of metallothionein mRNA and metallothionein protein, respectively, in beta-thujaplicin-treated cultured mouse keratinocytes. These findings indicate that beta-thujaplicin inhibits ultraviolet B-induced apoptosis in keratinocytes and strongly suggest that the inhibitory mechanism is due to the antioxidant activity of metallothionein induced by the agent.

 

Comp Biochem Physiol C 2000 Mar 1;125(3):325-332

Cloning and sequencing of cDNAs encoding for a novel copper-specific metallothionein and two cadmium-inducible metallothioneins from the blue crab Callinectes sapidus.

Syring RA, Hoexum Brouwer T, Brouwer M

Department of Coastal Sciences, Institute of Marine Sciences, University of Southern Mississippi, 703 East Beach Blvd., Ocean Springs, MS, USA

[Record supplied by publisher]

Metallothioneins (MTs) are cysteine-rich metal-binding proteins found in micro-organisms, plants and all invertebrate and vertebrate animals. Unicellular eukaryotes such as yeast have a copper-MT whose synthesis is induced by a copper-activated transcription factor. Most higher organisms have two major cadmium/zinc MT isoforms, whose synthesis is controlled by a zinc-activated transcription factor. The blue crab, Callinectes sapidus, has two cadmium-inducible isoforms, CdMT-I and CdMT-II, and a third isoform, CuMT-II, which is induced by copper, but not by cadmium. The cDNA sequence of the copper-specific MT, along with those of the two CdMTs, was determined utilizing 3' and 5' rapid amplification of cDNA ends (RACE). CuMT-II cDNA encodes a 63 amino acid protein containing 21 cysteine residues. CdMT-I and CdMT-II cDNA encode a 58 and 57 amino acid protein, respectively, each with 18 cysteines. Molecular phylogeny analysis shows that the CdMT isoforms cluster with other crustacean CdMTs, whereas the copper-specific MT is more closely related to mollusk MTs. CuMT-II shows considerable homology to a copper-specific, non-cadmium inducible, MT from the snail, Helix pomatia. The presence of copper-specific MTs in mollusks and crustaceans, both of which are dependent on hemocyanin for oxygen transport, suggests that CuMT-II is involved in copper homeostasis associated with the synthesis and degradation of hemocyanin.

 

Cell Mol Biol (Noisy-le-grand) 2000 Mar;46(2):465-88

Metallothionein in human disease.

Simpkins CO

Hamot Research Center and Tri-State Emergency System, Erie, PA, USA. phippsdian@aol.com

[Medline record in process]

Evidence concerning a role for metallothionein (MT) in human disease is reviewed. Current knowledge of MT is juxtaposed with our understanding of the pathogenesis of disease. MT is known to modulate three fundamental processes: 1) the release of gaseous mediators such as hydroxyl radical or nitric oxide; 2) apoptosis, and 3) the binding and exchange of heavy metals such as zinc, cadmium or copper. The capability to specifically manipulate MT levels in cells and in mice is beginning to provide answers regarding how MT could impact complex disease scenarios. Associations among MT and several diseases, including cancer, circulatory and septic shock, coronary artery disease, and Alzheimer's disease have been made. Strong evidence exists that MT modulates the immune system. The primary function of MT remains unknown.

 

 

Cell Mol Biol (Noisy-le-grand) 2000 Mar;46(2):451-63

Toxicological aspects of metallothionein.

Nordberg M, Nordberg GF

Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. monica.nordberg@imm.ki.se

[Medline record in process]

Metallothionein (MT) is expressed to a certain extent in almost all mammalian tissues. The biological significance of MT is related to its various forms MT-1, MT-2, MT-3 and MT4. For MT-1 several isoforms of the protein exist and it is likely that these isoforms are related to various functions involved in developmental processes occurring at various stages of gestation. Toxicokinetics and biochemistry of essential and toxic metals such as cadmium, zinc, mercury and copper in organs e.g. kidney, CNS, are often related to metallothionein. It is debated whether there is a relation or not for other metals e.g. selenium and bismuth. For the toxicokinetics of cadmium, MT plays an important role. By expanding techniques from experimental toxicology and biochemistry to include molecular biology methods, more specific and relevant studies can be performed of the actual role and biological function of MT. The present paper on toxicological aspects of metallothionein, presents an overview and evaluation of present knowledge concerning differences among organs and within organs of the expression of MT and how this affects tissue sensitivity to toxicity.

 

 

 

Life Sci 1999;64(11):PL145-50

Simian virus 40-transformed metallothionein null cells showed increased sensitivity to cadmium but not to zinc, copper, mercury or nickel.

Kondo Y, Yanagiya T, Himeno S, Yamabe Y, Schwartz D, Akimoto M, Lazo JS, Imura N

Department of Urology, Nippon Medical School, Tokyo, Japan.

Primary cultured embryonic cells derived from mice with disrupted metallothionein (MT) I and II genes and from control mice were transformed with a plasmid encoding the simian virus 40 (SV40) large T antigen. The resulting MT-/- and MT+/+ cell strains showed similar cell morphology, cell cycle and no significant differences in glutathione levels or in the activities of glutathione-related enzymes and antioxidant enzymes. The MT-/- cells were more sensitive to Cd than MT+/+ cells, though no increase in the sensitivity to Zn, Cu, Hg or Ni were observed in MT-/- cells. MT+/+ cells accumulated more Cd than MT-/- cells but showed less lesion, suggesting the role of MT induced by Cd in MT+/+ cells as a scavenger of toxic Cd ion. These results suggest a dominant protective role of MT against Cd compared with other metals. SV40-transformed MT-/- cells seem to be a useful tool for the investigation of cellular function of MT.

 

In the following study is was determined that cadmium, zinc, and copper all induce the same identical metallothionein isoform, MT1a.  I feel that this is important because this provides a mechanism by which each of these three metals can compete with the other two: by competition for binding locations on the metallothionein molecule.

 

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 1998 Aug;120(2):251-9

Induction and identification of cadmium-, zinc- and copper-metallothioneins in the shore crab Carcinus maenas (L.).

Pedersen SN, Pedersen KL, Hojrup P, Knudsen J, Depledge MH

Institute of Biology, University of Odense, Denmark.

Shore crabs Carcinus maenas were injected with either Cd, Cu or Zn to determine whether different metals could induce specific metallothionein (MT) isoforms in the midgut gland. Furthermore, the relative ability of the three metals to induce MT was quantified. Accumulation of the three metals in the midgut gland caused variable and in the case of Cd and Zn significant increases in MT levels. The increase in MT levels (pmol g-1 midgut gland) per nmol of metal accumulated was determined as 90, 60 and 4 pmol for Cd, Zn, and Cu respectively. The MT isoforms were purified using a combination of acetone precipitation, FPLC and reverse phase HPLC. In contrast to Cd and Zn induced MTs, the Cu induced MT was highly susceptible to oxidation during purification. The induced MT isoforms were characterized by N-terminal amino acid sequencing and mass-spectrometry. All three metals induced the same identical isoform MTIa.

 

Environ Health Perspect 1998 Sep;106(9):587-95

Exposure of human proximal tubule cells to cd2+, zn2+, and Cu2+ induces metallothionein protein accumulation but not metallothionein isoform 2 mRNA.

Garrett SH, Somji S, Todd JH, Sens DA

Robert C. Byrd Health Sciences Center, Department of Pathology, West Virginia University, Morgantown, WV 26506, USA.

The organization of the human metallothionein (MT) gene family is more complex than the commonly used mouse and rat models. The human MTs are encoded by a family of genes consisting of 10 functional and 7 nonfunctional MT isoforms. One objective of this study was to determine if the accumulation of MT protein in cultures of human proximal tubule (HPT) cells exposed to metals is similar to that expected from the knowledge base obtained from rodent models. To accomplish this objective, HPT cells were exposed to both lethal and sublethal concentrations of Cd2+, Zn2+, Cu2+, Ag2+, Hg2+, and Pb2+ and MT protein levels were determined. The results were in general agreement with animal model studies, although there were some exceptions, mainly in areas where the animal model database was limited. In clear agreement with animal models, Cd2+, Zn2+, and Cu2+ were demonstrated to be potent inducers of MT protein accumulation. In contrast to the similarity in MT protein expression, we obtained evidence that the human renal MT-2 gene has a unique pattern of regulation compared to both animal models and human-derived cell cultures. In the present study, we determined that MT-2A mRNA was not induced by exposure of HPT cells to Cd2+ or the other metals, a finding in contrast to studies in both animal models and other human cell culture systems in which a high level of MT-2 mRNA induction occurs upon exposure to Cd2+ or Zn2+. While MT protein expression may be similar between humans and animal models, this finding provides initial evidence that regulation of the genes underlying MT protein expression may be divergent between species.

 

Biol Trace Elem Res 1998 Jun;62(3):135-53

Dietary flavonoids interact with trace metals and affect metallothionein level in human intestinal cells.

Kuo SM, Leavitt PS, Lin CP

Nutrition Program, State University of New York at Buffalo, 14214, USA.

Flavonoids are natural compounds found in food items of plant origin. The study examined systematically the interaction of structurally diverse dietary flavonoids with trace metal ions and the potential impact of dietary flavonoids on the function of intestinal cells. Spectrum analysis was first performed to determine flavonoid-metal interaction in the buffer. Among the flavonoids tested, genistein, biochanin-A, naringin, and naringenin did not interact with any metal ions tested. Members of the flavonol family, quercetin, rutin, kaempferol, flavanol, and catechin, were found to interact with Cu(II) and Fe(III). On prolonged exposure, quercetin also interacted with Mn(II). Quercetin at 1:1 ratio to Cu(II) completely blocked the Cu-dependent color formation from hematoxylin. When quercetin was added to the growth medium of cultured human intestinal cells, Caco-2, the level of metal binding antioxidant protein, metallothionein, decreased. The effect of quercetin on metallothionein was dose- and time-dependent. Genistein and biochanin A, on the contrary, increased the level of metallothionein. The interaction between dietary flavonoids and trace minerals and the effect of flavonoids on metallothionein level imply that flavonoids may affect metal homeostasis and cellular oxidative status in a structure-specific fashion.