Presence Of Metallothionein Research Articles

Morphostructural and immunohistochemical study on the role of metallothionein in the detoxification of heavy metals in Apis mellifera L., 1758.

Honey is a valuable food produced by bees from sugary substances that they gather in nature. The transformation the nectar into honey, by bees, is long and complex. Except for honey, where heavy metals are absent or are found only in traces, the bees and their products have always been considered excellent biomarkers of such contaminants. We have assumed that the absence of heavy metals in honey is due to the presence of a detoxification system in the digestive system of bees, which involves metallothioneins, proteins that have a role in the homeostatic control of essential and non-essential metals. We have placed the beehives in three different zones: industrial, urban and rural. Investigations were carried out with ICP-MS method for the detection of heavy metals in the guts of honey bees and honey. The metallothioneins have been identified by Immunohistochemical and Western-blotting analisys. The investigations have shown the presence of heavy metals only in bees guts but not in honey, while the presence of metallothionein has been highlighted only in epithelium of the honey sac, demonstrating the existence of an efficient system of detoxification of heavy metals.

Unravelling the Role of Metallothionein on Development, Reproduction and Detoxification in the Wall Lizard Podarcis sicula.

Metallothioneins (MTs) are an evolutionary conserved multigene family of proteins whose role was initially identified in binding essential metals. The physiological role of MT, however, has been revealed to be more complex than expected, since not only are MTs able to bind to toxic heavy metals, but many isoforms have shown specialized and alternative functions. Within this uncertainty, the information available on MTs in non-mammalian vertebrates, particularly in neglected tetrapods such as the reptiles, is even more scant. In this review, we provide a summary of the current understanding on metallothionein presence and function in the oviparous lizard Podarcis sicula, highlighting the results obtained by studying MT gene expression in most representative adult and embryonic tissues. The results demonstrate that in adults, cadmium induces MT transcription in a dose- and tissue-specific manner. Thus, the MT mRNAs appear, at least in some cases, to be an unsuitable tool for detecting environmental ion contamination. In early embryos, maternal RNAs sustain developmental needs for MT protein until organogenesis is well on its way. At this time, transcription starts, but again in a tissue- and organ-specific manner, suggesting an involvement in alternative roles. In conclusion, the spatiotemporal distribution of transcripts in adults and embryos definitively confirms that MT has deserved the title of elusive protein.

Bacterial metallothioneins

Abstract Heavy metals are found in all living organisms where, as indispensable microelements (e.g. zinc, iron, copper), are involved in endless metabolic processes. However, living organisms are also at a risk of exposure to highly toxic metals, including cadmium or lead, which do not play any physiological role. Among multiple mechanisms associated with the maintenance of micronutrient homeostasis and detoxification of unwanted metals, there is a family of low-molecular-weight, cysteine-rich proteins, able to chelate multiple metal ions i.e. the metallothioneins (MTs). They are widely distributed among Eucaryota, however, they have also been found in some limited Procaryota, including cyanobacteria, pseudomonads and mycobacteria. These bacterial MTs differ in terms of primary structure, the number and type of metal ions they bind, as well as with regard to their physiological functions. The expression of bacterial MTs is regulated by metals via metalosensors. MTs from cyanobacteria seem to be involved in zinc homeostasis, while in Pseudomonas they are linked to cadmium detoxification. In Mycobacterium, MTs bind copper ions and may play a pivotal role in the virulence of these bacteria. The presence of MTs in other groups of bacteria remains questionable. Problems with identification of new bacterial MTs are mainly associated with low level of homology between MT amino acid sequences of different bacterial groups. Further research is needed to evaluate the physiological functions of metallothioneins in Procaryota. 1. Introduction. 2. The history of discoveries of bacterial metallothioneins. 3. Structure and metal-binding properties of bacterial MTs. 4. Functions of bacterial metallothioneins. 5. Regulation of metallothionein gene expression. 6. Presence of metallothioneins in bacteria. 7. Summary 1. Wstęp. 2. Historia odkryć metalotionein u bakterii. 3. Budowa i sposób wiązania jonów metali ciężkich przez bakteryjne MT. 4. Funkcje metalotionein bakteryjnych. 5. Regulacja ekspresji bakteryjnych metalotionein. 6. Obecność metalotionein u bakterii. 7. Podsumowanie

Cadmium resistance and uptake by bacterium, Salmonella enterica 43C, isolated from industrial effluent.

Cadmium resistant bacterium, isolated from industrial wastewater, was characterized as Salmonella enterica 43C on the basis of biochemical and 16S rRNA ribotyping. It is first ever reported S. enterica 43C bared extreme resistance against heavy metal consortia in order of Pb2+>Cd2+>As3+>Zn2+>Cr6+>Cu2+>Hg2+. Cd2+ stress altered growth pattern of the bacterium in time dependent manner. It could remove nearly 57 % Cd2+ from the medium over a period of 8 days. Kinetic and thermodynamic studies based on various adsorption isotherm models (Langmuir and Freundlich) depicted the Cd2+ biosorption as spontaneous, feasible and endothermic in nature. Interestingly, the bacterium followed pseudo first order kinetics, making it a good biosorbent for heavy metal ions. The S. enterica 43C Cd2+ processivity was significantly influenced by temperature, pH, initial Cd2+ concentration, biomass dosage and co-metal ions. FTIR analysis of the bacterium revealed the active participation of amide and carbonyl moieties in Cd2+ adsorption confirmed by EDX analysis. Electron micrographs beckoned further surface adsorption and increased bacterial size due to intracellular Cd2+ accumulation. An overwhelming increase in glutathione and other non-protein thiols levels played a significant role in thriving oxidative stress generated by metal cations. Presence of metallothionein clearly depicted the role of such proteins in bacterial metal resistance mechanism. The present study results clearly declare S. enterica 43C a suitable candidate for green chemistry to bioremediate environmental Cd2+.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0225-9) contains supplementary material, which is available to authorized users.

Arsenic metalation of seaweed Fucus vesiculosus metallothionein: the importance of the interdomain linker in metallothionein.

The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protecting agent against toxic metals in the contaminated waters it can grow in. We report the first kinetic pathway data for A3+ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time and temperature dependence of the relative concentrations of apo-rfMT and the five As-containing species have been determined following mixing of As3+ and apo-rfMT using electrospray ionization mass spectrometry (ESI MS). Kinetic analysis of the detailed time-resolved mass spectral data for As3+ metalation allows the simulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consumption of As1- to As4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed here provides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and the Cys9 domain. The rate constants (M(-1) s(-1)) calculated from the fits for the 7-cysteine gamma domain are k1gamma, 19.8, and k2gamma, 1.4, and for the 9-cysteine beta domain are k1beta, 16.3, k2beta, 9.1, and k3beta, 2.2. The activation energies and Arrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which as we show from analysis of the ESI MS data, allows each of its two domains to bind As3+ independently of each other. The analysis provides for the first time an explanation of the differing metal-binding properties of two-domain MTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers) and mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker.

Arsenic metabolism in multiple myeloma and astrocytoma cells

Arsenic trioxide (As2O3, Trisenox) is used to treat patients with refractory or relapsed acute promyelocytic leukemia (APL). Its ability to induce apoptosis in various malignant cell lines has made it a potential treatment agent for other malignancies and many clinical trials are currently in progress to evaluate its clinical usefulness for multiple myeloma and glioblastoma cancer. In the present study, we investigated the metabolism of As2O3 regarding its cellular biotransformation and interaction with metallothionein (MT) as a possible protective responses of cells to arsenic-induced cytotoxicity. The study was performed on two types of cell treated with As2O3: (1) human astrocytoma (glioblastoma) cell line U87MG treated with 0.6 microM arsenic for 0, 3, 12, 24, and 48 h or 12 microM arsenic for 3, 6, 12, 24, and 48 h and (2) bone marrow cells (BM) from two patients with multiple myeloma (MM) treated with 7 microM arsenic for 0, 43, and 67 h. Cotreatment with vitamin C (1 mg/mL) was tested in longer exposure of MM BM cells. Traces of methylation products (mainly monomethylarsenic acid) were detected in cell lysates of both cell types and in pellets of U87 MG cells, although we found problems with column-sample interactions in cases where methanol pretreatment of the sample was not used. Pentavalent inorganic arsenic (AsV) was identified in both cell types, and up to 80% of total As in MM bone marrow cell lysates was present as AsV. Such an occurrence (generation) of pentavalent arsenic after As2O3 treatment demonstrates the presence of biological oxidation of trivalent arsenic, which could represent an additional protective mechanism of the cell. Vitamin C decreased As cell content and increased the percentage of pentavalent inorganic arsenic (in the growth medium and cells). The presence of metallothionein (MT) and its response to arsenic treatment was checked in all U87 MG cells, in the control, and in one exposed sample of MM BM cells. During 48 h exposure to 0.6 or 12 muM arsenic MTI/II levels increased in U87 MG cells, but with variable Zn levels, increased Cu levels, and As binding observed in traces only. Involvement of the MT-III isoform was negligible. In contrast, 43 h exposure to 7 microMarsenic did not increase MT content in multiple myeloma cells, and the levels even decreased with respect to the control. To evaluate the importance of the observed processes, MTs in U87 and AsIII-AsV conversion in MM BM cells, which could represent a resistance response of cancer cells treated by As2O3, longer-term observation with different arsenic concentrations should be performed.

Metallothionein expression in benign and malignant canine mammary gland tumours

The presence of metallothioneins (MTs) were demonstrated immunohistochemically using a monoclonal antibody (E9) against a conserved epitope of I and II isoforms in canine mammary tumours. In a semiquantitative analysis MT expression in the tumour cells was observed in 54/54 cases of benign and 32/40 malignant mammary neoplasms. A statistically significant difference at the level of P < 0.01 was observed for MT expression between benign and malign mammary tumours in terms of immunoreactivity score. It is concluded that immunohistochemically demonstrated MT expression is significantly associated with benign canine mammary tumours.

Hepatic metallothioneins in two neotenic salamanders, Proteus anguinus and Necturus maculosus (Amphibia, Caudata)

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12 000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.

Distribution of zinc-binding metallothionein in cirrhotic liver of rats administered zinc.

This study was undertaken in order to investigate the effect of zinc (Zn) administration on induction of Zn-binding metallothionein in rat liver with thioacetamide-induced cirrhosis, and the localization of metallothionein in the liver. Normal and cirrhotic rats received intraperitoneal injections with or without Zn. Subsequently, metal analyses, purification of metallothionein by gel filtration and immunohistochemical assessments of metallothionein were carried out. Although in Zn-injected cirrhotic rats, the Zn contents in the liver and plasma increased significantly depending upon the dose of Zn, the Zn contents in the liver and plasma of the cirrhotic rats were lower than those of normal rats after the same dose of Zn. The results of gel filtration also showed that the levels of Zn-metallothionein in the cirrhotic liver were reduced in comparison with those of the normal liver. By the immunohistochemical method, the presence of metallothionein in the parenchymal areas but not in the fibrotic areas of the cirrhotic liver was confirmed. These results suggested that the induced metallothionein was only located in the parenchymal areas. The metallothionein induced in the parenchymal areas was considered to play a role in protecting the parenchymal cells against the progression of fibrosis, because metallothionein has been thought to be involved in the cellular defense against oxidative stress.

Growth and Heavy Metal Binding Properties of Transgenic Chlamydomonas Expressing a Foreign Metallothionein Gene

ABSTRACT We have compared the growth rates and cadmium binding capacity of wild-type and transgenic Chlamydomonas reinhardtii cells expressing a foreign class-II metallothionein. We observed that cells expressing metallothionein grew to significantly higher cell densities than wild-type cells in the presence of a toxic cadmium concentration (40 μM). When grown at a low (5 μM) cadmium concentration, cells expressing metallothionein bound twofold more cadmium (0.43 μg Cd)mg Ch1) than wild-type. At cadmium concentrations (40 μM), which induce phytochelatin synthesis in wild-type cells the cadmium binding capacity of both wild-type (79.6 μg Cd)mg Ch1) and transformed cells (86.4 μg Cd)mg Ch1) was similar; however, the transformed cells grew to higher densities than the wild type. These results suggest that under conditions that apparently induce phytochelatin expression, the presence of metallothionein in the cytoplasm reduces heavy metal toxicity. Furthermore, because cells expressing metallothionein grow to higher densities than wild-type cells at a toxic cadmium concentration (40 μM), the transgenic cells sequester more total cadmium (9% of total Cd) from the medium than the wild type (5.5% of total Cd). These results indicate that the trace-metal binding properties of Chlamydomonas can be enhanced through the expression of trace-metal-specific binding proteins.

Evidence that the reactions of cadmium in the presence of metallothionein can produce hydroxyl radicals.

A variety of Cu reconstituted metallothioneins (MTs) containing different amounts of copper ions together with Cd7-MT free of copper were prepared and used in spin trapping experiments designed to show cadmium is not a Fenton active metal. A significant increase of the DMPO/.OH adduct was observed, with increased concentrations of the copper containing MTs, H2O2 enhanced DMPO/.OH adduct formation, catalase and the Cu (I) specific chelating agent bathocuproine, reduced DMPO/.OH adduct formation. These results suggest that Cu (I) and H2O2 both have important roles in the production of active species in these systems and cause DMPO/.OH formation. However, Cd7-MT showed no ability to cause generation of DMPO adducts with H2O2 seeming to indicate cadmium is not a Fenton metal. To test this hypothesis further trapping studies were run with added sulphite and lipid peroxide using both commercial MT and Cd7-MT since cadmium causes peroxidation in vivo. Commercial MT generates radicals with added sulphite and peroxide, Cd7-MT does not, demonstrating that cadmium is not a Fenton metal. These results help to explain the oxidative damage to DNA observed in the presence of MT and cadmium in vitro.

Temporal changes of 210Po in temperate coastal waters

The temporal variation of Polonium-210 ( 210Po) was examined in coastal sea water, the mussel Mytilus edulis, the winkle Littorina littorea and green alga Ulva lactuca in order to investigate the entry of 210Po into the marine food chain. More than 99% of 210Po in the water column occurred in the particulate phase. Dissolved 210Po concentrations peaked during the spring phytoplankton bloom and it is suggested this is related to preferential scavenging of 210Po by the increased numbers of bacteria, viruses and small dissolved particulates. Changes in L. littorea 210Po specific activity are thought not to be related to food, but to a drop in body weight following spawning. Much of the 210Po accumulated by M. edulis was located in the digestive gland. The specific activity of 210Po in the digestive gland of M. edulis was shown to be strongly correlated with changes in sea water suspended particulate specific activity. Examination of other trace metal (Ag, Al, As, Ca, Cd, Cr, Co, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, Sb, Se, Sn and Zn) variations in the digestive gland revealed that class B and borderline metals had a strong positive correlation with 210Po. On-going work is investigating whether the accumulation and loss of 210Po is affected by the presence of metallothioneins.

Metallothionein in the ovaries of laying hens exposed to cadmium

The presence of metallothionein (MT) and its mRNA in the ovaries of laying hens is reported. In laying hens injected with cadmium (Cd), Cd accumulated in the follicle walls of the ovaries not in the follicle yolks. In the follicle walls, (Cd, Zn)- MT and its mRNA were found. This indicates that MT bound to Cd is biosynthesized in the follicle walls. We suggest that MT might play a role in sequestering Cd. On the other hand, zinc (Zn) was found in the follicle walls and yolks in the Cd-treated laying hens. The relationship between Zn and (Cd, Zn)-MT is also discussed.

Metal-binding proteins in eggs of various sea urchin species.

Metallothionein presence and amount were determined in the unfertilized eggs of six sea urchin species by silver saturation assay and gel-chromatography of cell extracts. The results showed high levels of metallothionein in the egg cytoplasm of the two Mediterranean species Paracentrotus lividus and Sphaerechinus granularis. No metallothionein was found either in the eggs of Arbacia lixula, or in those of the three Eastern species Strongylocentrotus intermedius, Temnopleurus hardwickii and Clypeaster japonicus. However, the extracts of the latter three species revealed the presence of zinc bound in a macromolecular form, thus suggesting the existence of metal-binding proteins distinct from metallothioneins.

INCREASED RESISTANCE TO DOXORUBICIN IN HUMAN NON-SMALL-CELL LUNG CARCINOMAS WITH METALLOTHIONEIN EXPRESSION

Ninety-four human non-small cell lung carcinomas (NSCLC) of previously untreated patients were analyzed for the presence of metallothionein (MT) expression by means of immunohistochemistry. Of the tumors investigated, expression of MT was detected in 63%. The expression of MT was correlated with the results of doxorubicin resistance of the tumors in vitro. A significant relationship between MT expression and doxorubicin resistance was found (p=0.01). Significant correlations also exist between MT and P-glycoprotein or glutathione-S-transferase-pi expression respectively (p<0.005).

Accumulation of Fe, Zn, Cu, and Cd during the different stages of the reproductive cycle in Mytilus edulis

1. 1. Levels of zinc, cadmium, copper and iron were measured in the whole body of Mytilus edulis collected along the year, and the patterns throughout the reproductive cycle were established. 2. 2. The maxima for the accumulation of those metals occur in the stages of sexual maturity and spawning. The higher values for Cd and Zn occur in stage III B and for Fe and Cu in stages III A and III B. 3. 3. These data are discussed in relation to the known presence of metallothioneins in bivalves, and to the role of steroid hormones in protein synthesis during the reproductive cycle.

Immunohistochemical localization of metallothionein in the rat prostate gland during postnatal development.

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.

Quantitative immunohistochemistry of metallothionein in rat placenta.

The presence of metallothionein (MT) was demonstrated in placentae from cadmium-exposed and control rats by an immunohistochemical technique, using peroxidase as label and the diaminobenzidine procedure for the staining reaction. The protein was found in different regions of the placenta, i.e. in trophoblastic labyrinth, in spongiotrophoblast and in visceral yolk sac. Cytophotometric analysis of the final reaction product revealed that the amount of MT was increased in the placental labyrinth of cadmium-exposed rats. Increases were found in both nuclei and cytoplasm of trophoblast cells in the labyrinth region. Possible roles of MT in the transport of zinc and in the carbohydrate metabolism are discussed.

TRACE METALS AND METHYL MERCURY: ASSOCIATIONS AND TRANSFER IN HARP SEAL (PHOCA GROENLANDICA) MOTHERS AND THEIR PUPS

AbstractMilk samples from the stomachs of harp seal pups were analysed for Cu, Zn, Se, Cd and Hg, as were liver, kidney, and muscle from mother‐pup pairs. Tissues were also analysed for MeHg. Milk contained, in addition to essential trace metals, Cd and Hg (57 ng/g and 6.5 ng/g respectively).Pups had mercury in all three tissues. The percent methyl mercury in liver of pups was higher than in liver of mothers. Mercury in muscle was mostly methyl mercury in both mothers and pups. Total mercury in liver of mothers but not pups was correlated positively with selenium. Estimates of ingested mercury by pups indicated they had acquired most of their mercury during gestation.Although mothers had cadmium in liver and kidney, it was not detected in tissues of pups. Cadmium did not transfer across the placenta, while mercury did. Tissue concentrations of Cu and Zn were higher in pups than mothers. The presence of metallothionein in pup tissues was postulated.A strong positive correlation of copper and selenium between mothers and pups indicated transfer of these elements from mother to pup in direct proportion to their concentrations in maternal liver and kidney.

Thymic hormone-containing cells. V. Immunohistological detection of metallothionein within the cells bearing thymulin (a zinc-containing hormone) in human and mouse thymuses.

Using an immunofluorescence (IF) assay, the presence of metallothionein (MT) was investigated in sections of normal and pathologic human thymuses as well as in cultures of thymic epithelial cells. This protein, known to have a high binding affinity for class II B transitional metals, such as zinc, was detected in the epithelial component of the thymus. Moreover, double labeling experiments with the anti-MT and an anti-thymulin monoclonal antibody showed that all cells containing thymulin, a thymic hormone whose active structure is known to contain zinc, also exhibited large amounts of metallothionein. These results, together with the fact that zinc and thymulin have been detected in the same type of cell organelles, lead to the conclusion that the MT present in thymic epithelial cells might be involved in the mechanism of zinc storage in these cells, thus favoring the secretion of thymulin in its biologically active, zinc-containing form.