RESUMO
The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the induction of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducible MT-isoform isolated from Tetrahymena after exposure to a non-toxic dose of copper. The cDNA sequence was determined utilising the partial peptide sequence of purified protein. The Cu-MT cDNA encodes 96 amino acids containing 28 cysteine residues (29%) arranged in motifs characteristic of the metal-binding regions of vertebrate and invertebrate MTs. Both the amino acid and nucleotide sequences differ, not only from other animal MTs, but also from the previously characterised Tetrahymena Cd-MT. Both MTs contain the structural pattern GTXXXCKCXXCKC, which may be proposed as a conservative sequence of Tetrahymena MTs. Cu-dependent regulation of MT expression was also investigated by measuring MT-mRNA and MT levels. MT synthesis occurs very quickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological functions, are expressed according to the inducing metal, Cu or Cd.
Assuntos
Expressão Gênica , Metalotioneína/genética , Tetrahymena/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cobre/metabolismo , DNA Complementar , DNA de Protozoário , Metalotioneína/isolamento & purificação , Metalotioneína/metabolismo , Dados de Sequência Molecular , Peptídeos , RNA Mensageiro , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND AND AIM: Zinc enhances cell protection against infection and injury and the healing processes themselves. We evaluated the effect of zinc supplementation at different doses on a model of experimental colitis in the rat. METHODS: Colitis, induced by intra-rectal instillation of dinitrobenzen-sulphonic acid, was assessed at 1 week by examining: general outcome and macroscopic damage, myeloperoxidase activity, mucosal zinc, iron and metallothionein concentrations. Rats received zinc sulphate, 2 mg/kg or 30 mg/kg, twice a day by gavage for 9 days, starting 3 days before the induction of colitis, or intrarectal instillation of zinc (20 mg/kg) once daily starting 8 hours after the induction of colitis and for 6 days thereafter RESULTS: Zinc-treated rats had less diarrhoea, higher body weight and lower colonic weight than untreated rats but no effect was observed on macroscopic inflammation, adhesions, colonic distension and neutrophil infiltration of the colonic mucosa. Zinc supplementation did not affect mucosal iron and zinc concentrations or plasma zinc levels in colitic rats. Metallothionein synthesis was induced in control rats and to a lesser extent in colitic rats. CONCLUSION: Zinc administration induces metallothionein synthesis but has little effect on the short-term course of experimental colitis.
Assuntos
Colite/tratamento farmacológico , Mucosa Intestinal/química , Metalotioneína/análise , Oligoelementos/análise , Zinco/administração & dosagem , Administração Oral , Administração Retal , Análise de Variância , Animais , Colite/metabolismo , Colite/fisiopatologia , Suplementos Nutricionais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Mucosa Intestinal/patologia , Masculino , Metalotioneína/biossíntese , Peroxidase/análise , Peroxidase/biossíntese , Probabilidade , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
The treatment of Tetrahymena thermophila with cadmium causes a reduction in growth rate according to dose; almost all the metal is accumulated in the cytosol where the Zn content is also increased threefold. Bio-Gel and Water 160 (HPLC) column chromatography show that Cd and Zn are bound to a protein with an ultraviolet (UV) spectrum that appears to be similar to that of Cd-metallothioneins isolated by higher organisms, but its molecular weight is greater: about 28 000 D, comparable to that of metallothionein isolated from Tetrahymena pyriformis. Further purification of these proteins by ion exchange chromatography revealed the presence of two peaks, considered as two isoforms of the metallothioneins present in both T. thermophila and T. pyriformis (MT 1 and MT 2). Their amino acid analyses confirmed that they are different isometallothioneins, MT 1 and MT 2, with about 30% cysteine, and aspartic acid, glycine and lysine as major amino acids. From our analyses we may conclude that Tetrahymena pyriformis MTs are similar to those present in invertebrates and vertebrates, while Tetrahymena thermophila MTs are peculiar in that they have cyclic amino acid histidine in both MT 1 and MT 2; furthermore, aromatic amino acid phenylalanine is also present in MT 2.
RESUMO
The aims of the work presented here were to determine the effect of long term treatment with zinc (Zn) on both total metallothionine (MT) and, in particular, oxidized MT (MTox) concentrations in Long-Evans Cinnamon (LEC) rat liver. We also evaluated semi-quantitatively the cell death index using TUNEL assay as it is a useful method to localize the nuclear fragmentation occurring in oxidative stress conditions. The results demonstrate there were no statistically different MT concentrations between Zn-treated and untreated rats, whereas the Zn treatment was very effective in reducing the percentage of oxidized MT (MTox). MTox is not able to bind metals, so it does not perform its "scavenger" action against copper (Cu) accumulation in LEC rats. The intensity and quantity of fluorescent staining observed in untreated rat sections decreased compared to the treated ones. These findings suggest that in LEC rats one of zinc's roles is to protect from oxidative stress, however, its mode of action remains partially unknown: a hypothesis is competition for Cu binding sites. A new insight is that Zn induced MT can protect efficiently against DNA damage by free radicals.
Assuntos
Dano ao DNA/efeitos dos fármacos , Degeneração Hepatolenticular/tratamento farmacológico , Fígado/efeitos dos fármacos , Metalotioneína/metabolismo , Zinco/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Contagem de Células , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Degeneração Hepatolenticular/metabolismo , Degeneração Hepatolenticular/patologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Fígado/metabolismo , Fígado/patologia , Masculino , Metalotioneína/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Endogâmicos LEC , TempoRESUMO
The present study was designed to investigate the effects of Zn administration on metallothionein concentrations in the liver, kidney, and intestine of copper-loaded rats. Male CD rats were fed a diet containing 12 mg Cu and 67 mg Zn/kg body wt. They were divided into either acute or chronic experimental protocols. Rats undergoing acute experiments received daily ip injections of either Cu (3 mg/kg body wt) or Zn (10 mg/kg body wt) for 3 d. Chronic experiments were carried out on rats receiving Cu ip injections on d 1, 2, 3, 10, 17, and 24, Cu injections plus a Zn-supplemented diet containing 5 g Zn/kg solid diet, or a Zn-supplemented diet alone. Rats injected Zn or Cu had increased MT concentrations in liver and kidney. Zn produced the most important effects and the liver was the most responsive organ. Rats fed a Zn-supplemented diet had significantly higher MT concentrations in liver and intestine with respect to controls. Increased MT synthesis in the liver may contribute to copper detoxification; the hypothesis of copper entrapment in enterocytes cannot be confirmed.
Assuntos
Cobre/metabolismo , Metalotioneína/biossíntese , Zinco/farmacologia , Animais , Mucosa Intestinal/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Ratos , Zinco/metabolismoRESUMO
OBJECTIVE: In this study, we have attempted to explore the possible protection afforded by Zn with regard to its antioxidant potential properties in the iron-induced toxicity. METHODS: Rat hepatoma cell line H4-II-E-C3 was treated with 150 muM ZnSO4, 200 muM FeSO4 or 150 muM ZnSO4+ 200 muM FeSO4 for 24 h. We studied the effect of metallothionein (MT), glutathione and metal (Fe and Zn) accumulation. We evaluated the levels of both MT-1 and MT-2 mRNAs, activities of antioxidant enzymes, and also determined the distribution of MT and cell death index. RESULTS: The cotreatment produced highest levels of MT. Fe concentration was significantly lower in the Zn-Fe-treated cells compared with Fe-treated cells. In both Zn treatments, the superoxide dismutase/glutathione peroxidase ratio was similar to control. The cell death index was lower in the Zn-Fe-treated cells compared with Fe-treated cells. CONCLUSIONS: Zn may act as antioxidant by competitive inhibition of iron uptake by the divalent metal transporter and/or by MT induction.
Assuntos
Antioxidantes/farmacologia , Carcinoma Hepatocelular/metabolismo , Ferro/toxicidade , Zinco/farmacologia , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Metalotioneína/metabolismo , Metais Pesados/farmacologia , Ligação Proteica , RNA Mensageiro/metabolismo , Ratos , Superóxido Dismutase/metabolismoRESUMO
The interactions between two essential metals, Cu and Zn, and the localization and concentration of metallothionein have been studied in rat liver and kidney. Rats receiving daily intraperitoneal injections of Cu for 3 days, or Zn for 2 days, or Cu for 3 days followed by Zn for 2 days, were sacrificed 24, 72, 120 h after the final injection. Our data indicate that Cu and Zn are both good inductors of metallothionein synthesis in rat tissues. Synergism between Cu and Zn in metallothionein synthesis was also observed as indicated by immunocytochemical experiments and chemical analysis. Moreover, in rats injected with Cu followed by Zn, the localization of metallothionein and the concentrations of both metallothionein and metal differed over time according to the organs considered. In rat kidney, a delay in the excretory process was also observed and metallothionein was present 120 h after the last injection.
Assuntos
Cobre/administração & dosagem , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metalotioneína/metabolismo , Zinco/administração & dosagem , Animais , Cobre/metabolismo , Sinergismo Farmacológico , Imuno-Histoquímica , Masculino , Microscopia de Fluorescência , Ratos , Zinco/metabolismoRESUMO
The addition of copper (10 micrograms ml-1) or cadmium (5 micrograms ml-1) to the medium is well tolerated by Tetrahymena pyriformis GL. Both metals are accumulated by cells, cadmium to a greater extent than copper. The growth rate is not affected and from the micrographs it is evident that the ultrastructure is not altered by the treatments. After 3 days of culture the macronucleus contains dense masses of chromatin and numerous nucleolar fusion bodies. Granules, cytolysomes and many food vacuoles are present in both control and treated cells. Cadmium induces the formation of a chelating protein; the amino acid analyses and the ultraviolet spectrum indicate that it is similar to the metallothionein isolated by higher organisms. The molecular weight of native protein is about 27,000. After treatment by sulphitolysis or oxidation we obtained a peak of molecular weight at about 6,000. The treatment with copper does not appear to induce metallothioneins or other chelatins. The high tolerance of Tetrahymena towards cadmium is believed to be due to the formation of a Cd-Zn metallothionein. The different chelating proteins induced by copper and cadmium in other groups of Protozoa and the different detoxification mechanisms present in these organisms are discussed.
Assuntos
Cádmio/farmacologia , Cobre/farmacologia , Tetrahymena pyriformis/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células Cultivadas , Cromatografia em Gel , Metalotioneína/biossíntese , Microscopia Eletrônica , Tetrahymena pyriformis/metabolismo , Tetrahymena pyriformis/ultraestruturaRESUMO
UNLABELLED: The role of metallothionein (MT) in the scavenging of superoxide radicals (*O2-) generated by macrophages has been examined. The present work has focused on the effects of added cadmium, a known inducer of MT biosynthesis, on determined amounts of superoxide radicals produced by in vitro cultured rat peritoneal macrophages on their stimulation with phorbol-12-myristate-13-acetate (PMA). The levels of superoxide radicals (*O2-) have been found to decrease when cadmium was added to cells exposed to PMA. However, substantially lower levels of MT have been determined in this case compared to cells untreated with PMA. This effect could be reversed by incubation of the PMA and cadmium-treated cells with a reducing agent, 2-mercaptoethanol (2-ME). Results suggest that *O2- caused thiolate oxidation and subsequent metal loss, thus reducing the cellular MT content as quantified by the silver saturation METHOD: This conclusion is supported by cell-free experiments in which the oxidation of rabbit MT-I by a xanthine/xanthine-oxidase system could be reversed by its subsequent reduction with 2-ME. The data presented provide direct evidence of the involvement of MT in scavenging superoxide radicals in living cells.
Assuntos
Cádmio/metabolismo , Macrófagos/metabolismo , Metalotioneína/metabolismo , Explosão Respiratória/fisiologia , Superóxidos/metabolismo , Animais , Macrófagos/efeitos dos fármacos , Mercaptoetanol/farmacologia , Oxirredução , Ratos , Ratos Wistar , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
This work presents the further purification of a Cd-linking protein in Oxytricha granulifera by reverse-phase chromatography. This protein contains 25% cysteine and no aromatic amino acid. It may be considered as a chelatin with some similarity to metallothioneins. During the purification of another Cd-linking compound, we were able to demonstrate that the H protein precursor of glycine cleavage is present in Oxytricha. This is the first finding of the presence of this system in Protozoa.
Assuntos
Cádmio/metabolismo , Glicina/metabolismo , Metalotioneína/isolamento & purificação , Metalotioneína/metabolismo , Oxytricha/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Metalotioneína/química , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência de AminoácidosRESUMO
Heavy metal pollution is known to be widespread in the sediments of the Lagoon of Venice. Therefore, physiological parameters influenced by this form of contamination were examined. The bivalve molluscs blue mussel (Mytilus galloprovincialis), ark clam (Scapharca inaequivalvis), and Japanese littleneck (Tapes philippinarum) were sampled in two sites (Marghera, Chioggia) every 3 months for 1 year. The digestive gland and gills of each bivalve were analyzed. The concentrations of essential and nonessential metals (including chromium, manganese, iron, cobalt, nickel, copper, zinc, and cadmium) were determined. Because glutathione and metallothioneins (MTs) are involved in metal homeostasis and detoxification, their concentrations were evaluated in relation to metal concentrations. Results show that (1) all three studied species accumulate metals to a considerable extent, with some species-specific differences between the digestive gland and gills; (2) gills are a good tissue to evaluate pollution by examining the MT content. In particular, the correlation between Zn and MT levels in the gills indicates that M. galloprovincialis and S. inaequivalvis are sentinel organisms and can be used specifically for Zn pollution; (3) T. philippinarum accumulates Cu in the digestive gland more readily than the other two bivalves and therefore has the highest MT.
Assuntos
Proteínas de Transporte/análise , Monitoramento Ambiental/métodos , Metais Pesados/farmacocinética , Moluscos/metabolismo , Animais , Sistema Digestório/química , Sistema Digestório/metabolismo , Brânquias/química , Brânquias/metabolismo , Glutationa/análise , Itália , Metalotioneína/análise , Especificidade da Espécie , Distribuição TecidualRESUMO
OBJECTIVE: Wilson's disease is effectively treated by zinc administration which, in vitro, increases metallothionein concentrations. To ascertain whether the latter also occurs in humans we measured metallothionein and trace element concentrations in the duodenal mucosa of 15 Wilson's disease patients: 12 treated with zinc sulphate, two treated with penicillamine, and one not yet on treatment. The control group consisted of 17 patients with dyspepsia, who underwent the same study protocol. METHODS: Metallothionein and trace element concentrations were measured in duodenal mucosa biopsies according to the silver-saturation hemolysate method and atomic absorption spectrophotometry. RESULTS: Metallothionein concentrations increased by 1500% after zinc and 150% after penicillamine in Wilson's disease patients, with respect to controls who had negative endoscopy and Wilson's disease patients who were not treated. A significant correlation was found between metallothionein and duodenal zinc concentrations. Mucosal iron concentration increased in Wilson's disease patients whether they were treated with zinc or penicillamine. Duodenum with duodenitis also had significantly increased iron levels compared with normal duodenum. CONCLUSIONS: Zinc administration increases intestinal metallothionein in Wilson's disease patients. The blockade of copper absorption and its elimination in the stools on desquamation of the intestinal cells probably explains one of the mechanisms underlying the effect of zinc treatment. Despite normal endoscopy, Wilson's disease patients present increased mucosal iron concentrations similar to those in controls with duodenitis. Metallothionein may therefore prevent oxidative damage caused by metal toxicity.
Assuntos
Duodeno/metabolismo , Degeneração Hepatolenticular/tratamento farmacológico , Ferro/metabolismo , Metalotioneína/metabolismo , Sulfato de Zinco/uso terapêutico , Adulto , Estudos de Casos e Controles , Quelantes/uso terapêutico , Cobre/metabolismo , Duodenite/metabolismo , Feminino , Degeneração Hepatolenticular/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Masculino , Penicilamina/uso terapêuticoRESUMO
BACKGROUND: The production of free radicals is increased in inflammatory bowel disease, and trace elements are crucial components of several antioxidants. Trace elements deficiency may therefore compromise the defense against oxidative damage. The aims of this study were to measure plasma and tissue concentration of trace elements and antioxidants and to relate this to disease activity. METHODS: A 10-ml blood sample and six colonic biopsy specimens were obtained from 24 patients with either active ulcerative colitis or in remission and 10 patients with irritable bowel syndrome for measurement of trace elements and trace element-dependent enzymes. RESULTS: Patients with moderately active disease had significantly lower plasma iron, selenium, and glutathione peroxidase levels than patients in remission and controls, whereas no significant differences were found between the zinc and copper values of patients and controls. Mucosal concentrations of zinc and metallothionein were reduced, whereas iron and glutathione peroxidase concentrations were increased in patients with endoscopically active disease as compared with controls and patients in remission. CONCLUSIONS: Patients with ulcerative colitis have altered plasma and tissue levels of trace elements and antioxidant-related enzymes. The resulting reduced protection against free radicals may contribute to the inflammatory process.