Metallothionein Synthesis Research Articles

Depuration of Cadmium from Blue Mussel (Mytilus edulis) by Protein Hydrolysate-Fe2+ Complex: The Role of Metallothionein.

Metallothionein (MT) is a low-molecular-weight protein with high metal-ion affinity. If the intracellular concentrations of metals are too high or if toxic metals are present within the cell, then the synthesis of MTs is induced and generated. In our previous work, it was found that the prepared hydrolysate-metal element complex showed obvious depuration activity of heavy metals (Cd2+ ) from blue mussel (Mytilus edulis). This study provided further the depuration mechanisms of Cd2+ from mussel (M. edulis), in particular to the role of MT and its chelate during the depuration process.

Interrelationships among trace metals and metallothionein in digestive glands and gills for field samples of Merceneria merceneria.

More widespread use of metallothionein (MT) as a biomarker for trace metal pollution continues to be partly dependent on obtaining reliable baseline concentrations and identifying increased induction of the enzyme with only modest increases in metal concentrations. In this study, new data on metals and MT levels in whole clams tissue, gills, and digestive glands from field samples and in sediments are presented. Concentrations of Cd, Cu, Fe, and Zn in depurated (24h) clam samples of digestive glands, gills, and the whole clam Merceneria merceneria from the Indian River Lagoon, Florida, varied with location and showed moderate to strong correlations among Zn, Cu, and Fe. Concentrations of metallothionein (dry wt.) ranged from 34─270μg/g in gills and 150-440μg/g in digestive glands and showed moderate to strong correlations between organs and with metal concentrations in those organs. Observed trends support increased synthesis of metallothionein with only moderate increases in metal values and in response to statistically higher sediment metal concentrations.

Impairments of cadmium on vitellogenin accumulation in the hepatopancreas of freshwater crab Sinopotamon henanense.

During ovary maturation of crabs, vitellogenin (Vg), a precursor molecule of vitellin (Vn) needed for embryogenesis, can be produced in large quantities in the hepatopancreas and then transported to the ovary by the hemolymph. In the present study, effects of Cd on Vg accumulation in the hepatopancreas and Vg transportation of the freshwater crab Sinopotamon henanense were investigated. We also studied the impacts of Cd on the mRNA expression of genes involved in energy metabolism, protein metabolism, and metallothionein (MT) and glutathione (GSH) synthesis. After Cd treatment, the Vg concentration and the Vg mRNA expression in the hepatopancreas were downregulated. Pearson's correlation coefficient showed that the Vg level in the hepatopancreas correlated positively with those of the ovary and hemolymph (correlation coefficients 0.844 and 0.749, respectively), suggesting that the Vg transport from the hepatopancreas to the ovary can be impaired by Cd. The levels of carbohydrate and protein in the hepatopancreas of Cd-exposed crabs were decreased, and an inhibited protein metabolism was also observed. Energy production related isocitrate dehydrogenase and cytochrome C oxidase mRNA expressions, and MT and GSH synthesis increased after 10days of Cd treatment and decreased after 20days. Cd also caused a time-dependent upregulation of malondialdehyde. Our findings showed that Cd decreased Vg accumulation in the hepatopancreas due to partially excessive energy consumption and an activated defense system in the hepatopancreas, suggesting a possible regulatory mechanism in S. henanense which is the competitive advantage of energy reserves in metabolic Cd stress responses over the high-energy flux during vitellogenesis to ensure a continuous supply of metabolic energy. Moreover, the damage of Vg accumulation in the hepatopancreas caused by Cd could lead to an insufficient accumulation of Vn in the ovary and cause a retardation of oocyte development.

Zinc and Oxidative Stress: Current Mechanisms.

Oxidative stress is a metabolic dysfunction that favors the oxidation of biomolecules, contributing to the oxidative damage of cells and tissues. This consequently contributes to the development of several chronic diseases. In particular, zinc is one of the most relevant minerals to human health, because of its antioxidant properties. This review aims to provide updated information about the mechanisms involved in the protective role of zinc against oxidative stress. Zinc acts as a co-factor for important enzymes involved in the proper functioning of the antioxidant defense system. In addition, zinc protects cells against oxidative damage, acts in the stabilization of membranes and inhibits the enzyme nicotinamide adenine dinucleotide phosphate oxidase (NADPH-Oxidase). Zinc also induces the synthesis of metallothioneins, which are proteins effective in reducing hydroxyl radicals and sequestering reactive oxygen species (ROS) produced in stressful situations, such as in type 2 diabetes, obesity and cancer. Literature provides strong evidence for the role of zinc in the protection against oxidative stress in several diseases.

Complex effect of zinc oxide nanoparticles on cadmium chloride-induced hepatotoxicity in mice: protective role of metallothionein.

The wide range of applications of ZnO nanoparticles (nano ZnO) in commercial products and the ubiquitous cadmium (Cd) contamination in the natural environment increase the chance of co-existence of nano ZnO with Cd in the surroundings. To investigate the effects of nano ZnO on CdCl2-induced hepatotoxicity in mice, the histopathologic changes, metallothionein expression, oxidative stress responses and serum biochemical parameters were determined after oral administration of bulk or nano ZnO and/or CdCl2 for seven consecutive days. Bulk or nano ZnO had low toxicity in mice. In contrast, CdCl2 led to significant hepatic oxidative damage, as indicated by hepatic histopathological abnormalities and dysfunction. Bulk and nano ZnO had nearly identical influences on the hepatotoxicity of CdCl2 in mice. Although co-administration of bulk or nano ZnO with CdCl2 had a positive cooperative effect on the hepatic uptake of Cd and Zn, both bulk and nano ZnO significantly attenuated CdCl2-caused hepatic damage via the reduction of oxidative stress. The increase in metallothionein synthesis and the reduction of Cd-induced perturbation of Zn2+ homeostasis after co-administration of bulk or nano ZnO with CdCl2 play two important roles in the protective effect of bulk or nano ZnO on CdCl2-caused hepatic oxidative damage.

Origin, Function, and Fate of Metallothionein in Human Blood.

Toxic heavy metals, toxic organic compounds, reactive oxygen species (ROS), infections, and temperature are well-known metallothionein (MT) inducers in human blood. The current review aims to summarize synthesis, function, and fate of human blood MT in response to the known MT inducers. Part of the MTs that are synthesized in different organs such as the liver, kidney, and spleen is transported and stored in different blood cells and in plasma. Cells of the circulatory system also synthesize MT. From the circulation, MT returns to the kidney where the metal-bound MTs are degraded to release the metal ion that in turn induces MT expression therein. The blood MTs play important roles in metal detoxification, transportation, and storage. By neutralizing ROS, MTs protect blood cells from oxidative stress-induced cytotoxicity and genotoxicity. Arguably, MTs are also involved in immune suppression. Given the permeating distribution of blood MT throughout the body as well as its diverse role in the protection against harmful environmental factors and in metal homeostasis, MT could be better recognized as a major public health protein.

Immunolocalization of metallothionein in hepatocytes of guppy fish (Poecilia reticulata) exposed to tannery effluent: A biomarker study

Composite tannery effluent (TE) contaminated with a load of metals, especially chromium, is used in East Calcutta Wetland Ecosystem (ECW; a Ramsar site of West Bengal; No 1208) after natural stabilization for pisciculture and agriculture. Quantity and synergistic and antagonistic activities of both essential and nonessential metals in the effluents can induce a variety of complex changes in animal physiology. High fish yield and no apparent adverse influence on the fish biosystems thriving under pollutant stress at ECW allowed the practice to thrive sustainably over the last nearly 100 years. Thus, the present study was conducted to investigate the role of metallothionein (MT) as a potential biomarker in a fish biosystem following acute and chronic exposure to sublethal concentrations of tannery wastewater. MT localization in the liver tissue of guppy fish (Poecilia reticulata) was studied by the immuno gold-labeled electron microscopic technique. The data demonstrated increased metal content in fish body upon exposure to TE. Electron micrographs showed significant induction of hepatic MT in exposed P. reticulata over control specimens. Liver, being the detoxification site of an organism, plays a crucial role in the synthesis of MT and arrest of excess metal. The method employed in the present study for demonstrating the induction and localization of MT is innovative and could be used as a biomarker in fish exposed to metal stress. Our study also indicated that the metalloprotein complexes in fish tissue sequestered excess load of metals and thereby arrested unwanted interferences of excess metal loads in metabolic processes.

Determination and Evaluation of Metallothionein and Metals in Mugil cephalus (Mullet) from Pontal Bay, Brazil.

To assess environmental contamination, studies have been increasingly carried out using biomarkers to diagnose the effects of toxic metal exposure, such as metallothionein (MT). In this context, the aims of the present study were to determine Cd, Cu, Ni and Pb concentrations, by ICP-MS, in liver samples from Mugil cephalus from the municipality of Ilhéus, located in North-Southeastern Brazil, in the state of Bahia, which has been increasingly suffering from anthropogenic pressure, and to quantify total MT in liver and gills. Among essential metals, copper (199.2μgg-1) presented concentrations approximately 7 times higher than allowed by the World Health Organization (30μgg-1). Liver samples showed higher concentrations than gills. Metallothionein concentrations indicate differential contamination along the study area. The first sampling showed higher induction of MT synthesis overall. Differences between liver and gill MT concentration trends were relatively minor, which may indicate the adequacy of analyzing gills in an environmental monitoring context.

A baseline study of the metallothioneins induction and its reversibility in Neohelice granulata from the Bahía Blanca Estuary (Argentina)

Contamination by heavy metals causes serious effects in marine systems. Nowadays, the combination of chemical and biological data is recommended in monitoring programs. Metallothioneins (MT) are early-warning signals of metal exposure and are widely used in biomonitoring. The present research evaluates the heavy metals levels in sediments and the MT synthesis in the crab Neohelice granulata from the Bahía Blanca Estuary (BBE). Then, the recovery capabilities of N. granulata followed by a depuration phase are assessed. Results demonstrate a slow decline in the level of metals in the study area. In relation to MT, female crabs showed elevated MT when compared to males. Furthermore, MT synthesis diminished after the depuration phase. These data constitute the first MT information in organisms from the BBE and may be considered as baseline for future studies in this matter. Although pollution level found was low, further biomonitoring considering both types of data is recommended.

Toxicity Evaluation of New Engineered Nanomaterials in Zebrafish.

The effect of the nanoparticles on the marine organisms, depends on their size, chemical composition, surface structure, solubility and shape. In order to take advantage from their activity, preserving the surrounding environment from a possible pollution, we are trying to trap the nanoparticles into new nanomaterials. The nanomaterials tested were synthesized proposing a ground-breaking approach by an upside-down vision of the Au/TiO2 nano-system to avoid the release of nanoparticles. The system was synthesized by wrapping Au nanoparticles with a thin layer of TiO2. The non-toxicity of the nano-system was established by testing the effect of the material on zebrafish larvae. Danio rerio o zebrafish was considered an excellent model for the environmental biomonitoring of aquatic environments and the Zebrafish Embryo Toxicity Test (ZFET) is considered an alternative method of animal test. For this reason zebrafish larvae were exposed to different concentrations of nanoparticles of TiO2 and Au and new nanomaterials. As biomarkers of exposure, we evaluated the expression of metallothioneins by immunohistochemistry analysis and western blotting analysis also. The results obtained by toxicity test showed that neither mortality as well as sublethal effects were induced by the different nanomaterials and nanoparticles tested. Only zebrafish larvae exposed to free Au nanoparticles showed a different response to anti-MT antibody. In fact, the immunolocalization analysis highlighted an increase of the metallothioneins synthesis.

Differential metallothionein, reduced glutathione and metal levels in Perna perna mussels in two environmentally impacted tropical bays in southeastern Brazil

Mussel farming is an important economic activity in Brazil, and these organisms are consumed by the majority of the population in most coastal zones in the country. However, despite the increasing pollution of aquatic ecosystems in Brazil, little is known about the biochemical activity in mussels in response to metal exposure. In this context, the aim of the present study was to investigate metal and metalloid exposure effects in Perna perna mussels, by determining metal levels, the induction of metallothionein (MT) synthesis, and oxidative stress, in the form of reduced glutathione (GSH) in 3 contaminated areas from the Guanabara Bay in comparison to a reference site, Ilha Grande Bay, both in summer and winter. Metal and metalloid concentrations were also compared to Brazilian and international guidelines, to verify potential health risks to human consumers. Mussels from all sampling sites were shown to be improper for human consumption due to metal contamination, including Ilha Grande Bay, which has previously been considered a reference site. Several statistically significant correlations and seasonal differences were observed between MT, GSH and metals and metalloids in both analyzed tissues. A Discriminant Canonical Analysis indicated that the digestive gland is a better bioindicator for environmental contamination by metals and metalloids in this species and offers further proof that MT variations observed are due to metal exposure and not oxidative stress, since GSH influence for both muscle tissue and the digestive glands was non-significant in this analysis. These results show that P. perna mussels are an adequate sentinel species for metal contamination with significant effects on oxidative stress and metal exposure biomarkers. To the best of our knowledge, this is the first study to report metals, metalloids, MT and GSH levels in the muscle tissue of this species.

Recovery capabilities of Xenopus laevis after exposure to Cadmium and Zinc

The present investigation evaluates the recovery capabilities of Xenopus laevis following 12days of exposure to 30μg CdL−1 and 1000μg ZnL−1 alone or mixed, followed by a depuration phase in laboratory conditions. Focused endpoints, which were investigated at different times of depuration, are bioaccumulation of Cd and Zn, micronucleus induction, quantification of metallothioneins (MTs), and expression of genes involved in metal toxicity mechanisms.The results show that at the end of the contamination phase, there was higher metal bioaccumulation capability and MT synthesis in remaining tissues than in the liver. An increased expression of genes involved in detoxification and oxidative stress mechanisms was observed, suggesting an additive effect of both metals and a higher Zn regulation in the liver. During the depuration phase, the results show the recovery capability of Xenopus from 7days of depuration related to metamorphosis processes, which were observed at the end of the experiment. The results confirm the relevance of the amphibian model and the complementarities between a marker of genotoxicity, MT production, bioaccumulation and transcriptional analysis in the evaluation of the ecotoxicological impact. The results also highlight the reversible effects of Cd and Zn toxicity.

Identification and expression of an atypical isoform of metallothionein in the African clawed frog Xenopus laevis

Exploiting the annotation of the western clawed frog Silurana tropicalis genome, we identified a new metallothionein (MT) gene, exhibiting all the features to be considered an active gene, but with an atypical coding region, showing only 17 cysteine residues instead of the canonical 20 cysteines of vertebrate metallothioneins and two anomalous cysteine triplets. However, the presence of a gene in the genome does not ensure its effective expression. By using conventional and Real-Time PCR analyses, we demonstrated that this atypical MT is constitutively expressed throughout the life cycle of the African clawed frog Xenopus laevis; moreover, this gene is highly expressed in the adult liver, the major site of MT expression and synthesis in vertebrates. To our knowledge, the X. laevis MT described in this paper is the first sequence of a vertebrate MT showing only 17 cysteine residues, arranged in two Cys–Cys–Cys motifs. Phylogenetic analyses also demonstrated that the atypical X. laevis MT merges in the anuran clade, but is the most derived sequence among tetrapods MTs. Finally, Tajima's Relative Rate Test suggested a different evolutionary rate between the canonical X. laevis MT and this novel isoform.

Mercury, cadmium, zinc and copper concentrations and stable isotope ratios of carbon and nitrogen in tiger sharks (Galeocerdo cuvier) culled off Ishigaki Island, Japan

We analyzed Hg, Cd, Zn and Cu in the liver as well as Hg and stable isotope ratios of carbon and nitrogen (δ13C and δ15N) in muscle from tiger sharks (Galeocerdo cuvier) in Japan. The Hg concentration in the muscle increased slightly and proportionally with increases in body length, but the Hg concentration in the liver increased markedly after maturation (exceeding 2.7m precaudal length). The Hg concentration in the liver of mature shark was higher than that in the muscle. The Cd concentration in the liver increased with increases in body length. On the other hand, the Zn and Cu concentrations in the liver decreased during the growth stage, but thereafter increased with increased Cd burden due to growth. The marked increase in hepatic Hg in mature sharks may be explained by the continuous intake of Hg via food, slower growth and Hg–Se complex formation. High concentrations of Zn and Cu in the liver of immature sharks and concomitant increases in Zn and Cu with the Cd burden in the liver of mature sharks may be explained by the physiological requirements of Zn and Cu during the growth stage, the induction of metallothionein synthesis due to the Cd burden and the subsequent binding of these metals to metallothionein. The δ15N and δ13C values decreased with increases in body length, suggesting a shift from coastal feeding to pelagic feeding with shark growth. The Hg and Cd concentrations tended to be negatively correlated with the δ15N and δ13C values as a result of the increase in Hg and Cd accumulation due to the growth and the decreases in δ15N and δ13C values due to the sift of feeding area.

Bioaccumulation of nickel and its biochemical and genotoxic effects on juveniles of the neotropical fish Prochilodus lineatus

Juveniles of the freshwater fish Prochilodus lineatus were exposed to three concentrations of nickel (Ni): 25, 250 and 2500µgL-1 or water only for periods of 24 and 96h to test for Ni bioaccumulation, its effects on antioxidant defenses and metallothioneins, and the occurrence of DNA damage. After exposure, the fish were sampled and tissue removed from the gills, liver, kidney and muscle to test for Ni accumulation and conduct biochemical (gills and liver) and genotoxic (blood cells and gills) analyses. The results showed that Ni accumulates in the organs in different proportions (kidney>liver>gills>muscle) and accumulation varied according to exposure time. Metallothionein (MT) levels increased in the liver and gills after exposure to Ni, implying that the presence of Ni in these tissues could induce MT synthesis. We also observed that Ni exposure affected antioxidant defenses, increasing lipid peroxidation in the liver of fish exposed to Ni for 96h at the highest concentration tested. DNA damage increased in both blood cells and gills of fish exposed to all Ni concentrations, indicating the genotoxic potential of Ni on fish. We therefore concluded that Ni accumulates in various tissues and results in oxidative and DNA damage in P. lineatus, and that the maximum permitted Ni concentration set in Brazilian legislation (25µgL−1) for freshwaters is not safe for this species.

Lead in the soil–mulberry (Morus alba L.)–silkworm (Bombyx mori) food chain: Translocation and detoxification

The translocation of lead (Pb) in the soil–mulberry–silkworm food chain and the process of Pb detoxification in the mulberry–silkworm chain were investigated. The amount of Pb in mulberry, silkworm, feces and silk increased in a dose-responsive manner to the Pb contents in the soils. Mulberry roots sequestered most of the Pb, ranging from 230.78 to 1209.25mgkg−1. Over 92% of the Pb in the mulberry leaves was deposited in the cell wall, and 95.29–95.57% of the Pb in the mulberry leaves was integrated with oxalic acid, pectates and protein, and it had low bioavailability. The Pb concentrations in the silkworm feces were 4.50–4.64 times higher than those in the leaves. The synthesis of metallothioneins in three tissues of the silkworms was induced to achieve Pb homeostasis under Pb stress. These results indicated the mechanism involved in Pb transfer along the food chain was controlled by the detoxification of Pb in different trophic levels. Planting mulberry and rearing silkworm could be a promising approach for the remediation of Pb-polluted soils due to the Pb tolerance of mulberry and silkworm.

Researches on mercurial preparations: The prime requirement for their acceptance in medical world.

Ayurvedic and Siddha medicinal preparations containing mercury have been used over centuries in India. The recent WHO guidelines on the use of mercurials as well as actions by other international organizations into eliminating mercury in all forms have put the people practicing Rasa Shastra in a quandary. Active research in the mechanism of curative actions of mercurials is very much essential, to have widespread acceptance of the ancient practice. The toxicity of a substance depends on its bio-availability; the chemical form in which it is present and the biochemical reactions it participates. Mercury is usually administered as mercuric sulfide (Rasasindura or Linga Chendooram) which has a KSP value of 10–54. Despite this extreme insolubility, how mercury becomes bio-available under enzymatic conditions needs to be studied. Its bioaccumulation in critical organs and excretory pathways are to be ascertained. Research is also needed to establish whether Rasasindura or equivalent medicines induce the (excess) synthesis of sulfur containing biomolecules in human systems, which act as cell protectors against free radical-induced cell damage. The antioxidants themselves could be the curative agents; mercury being just a catalyst. It may also be possible that the exposure to mercury, even in very small amounts, could lead to the synthesis of specific metallothioneins in the human system, helping to detoxify the mercury exposure. The author is of the opinion that Ayurvedic practitioners/researchers should carry out long-term follow-up studies on human patients. The superiority of mercury based Ayurvedic preparations, as against modern allopathic medicines, in providing rapid and long lasting cure for specific diseases needs to document and published. In the absence of such supportive research literature, the use of mercury will become untenable, even for medicinal purposes.

Impact of cadmium on the level of hepatic metallothioneins, essential elements, and selected enzymes in the experimental rat model

The response of different strains of laboratory rats (Rattus norvegicus L.) on both acute (via intraperitoneal injection) and chronic (via drinking water and/or diet) cadmium intoxication was investigated in the model study. The rat strains Long Evans (LE), Spontaneously hypertensive rat (SHR), and Brown Norway (BN) were tested and compared, and total Cd levels and metallothionein (MT) concentrations were determined in the liver of experimental animals. The liver MT concentrations were determined by using adsorptive chronopotentiometry and modified Brdička reaction and were significantly correlated (r = 0.965) with the total liver Cd content. Moreover, the Cd application resulted in increasing zinc liver contents confirming intensive MT synthesis in the rat liver. In the blood plasma, specific enzymatic activity of glutathione reductase (GR) and glutathione-S-transferase (GST) was determined suggesting increasing activity of GR with the amount of applied Cd for all three strains, whereas ambiguous results have been found for the activity of GST. Therefore, MT concentrations seemed to be more sensitive indicators of the Cd intoxication compared to the assessment of the specific enzymatic activity.  

Copper oxide nanoparticles stimulate glycolytic flux and increase the cellular contents of glutathione and metallothioneins in cultured astrocytes.

Copper oxide nanoparticles (CuO-NPs) are frequently used for industrial or medical applications and are known for their high toxic potential. As little is known so far on the consequences of an exposure of brain cells to such particles, we applied CuO-NPs to cultured primary rat astrocytes and investigated whether such particles affect cell viability and alter their metabolic properties. Astrocytes efficiently accumulated CuO-NPs in a time- and concentration-dependent manner. The cells remained viable during a 24 h incubation with 100 µM copper in the form of CuO-NPs, while higher concentrations of CuO-NPs severely compromised the cell viability. Astrocytes that were exposed for 24 h to 100 µM CuO-NPs showed significantly enhanced extracellular lactate concentrations and increased cellular levels of glutathione and metallothioneins. The CuO-NP-induced increase in lactate release and metallothionein content were prevented by the presence of the membrane-permeable copper chelator tetrathiomolybdate, while this chelator increased already in the absence of CuO-NPs the cellular glutathione content. After removal of the CuO-NPs following a 24 h pre-incubation with 100 µM CuO-NPs, astrocytes maintained during a further 6 h incubation an elevated glycolytic lactate release and exported significantly more glutathione than control cells that had been pre-incubated without CuO-NPs. These data suggest that copper ions which are liberated from internalized CuO-NPs stimulate glycolytic flux as well as the synthesis of glutathione and metallothioneins in cultured viable astrocytes.

Cellular Mechanisms in Higher Plants Governing Tolerance to Cadmium Toxicity

Cadmium (Cd) is an inorganic mineral in the earth's crust. Cadmium entry into the environment occurs through geogenic and anthropogenic sources. Industrial activities including mining, electroplating, iron and steel plants, and battery production employ Cd during their processes and often release Cd into the environment. When disseminated into soil, Cd can be detrimental to agro-ecosystems because it is relatively mobile and phytotoxic even at low concentrations. Cadmium's phytotoxicity is due to reductions in the rate of transpiration and photosynthesis and chlorophyll concentration resulting in retardation of plant growth, and an alteration in the nutrient concentration in roots and leaves. In response to Cd toxicity, plants have developed protective cellular mechanisms such as synthesis of phytochelatins and metallothioneins, metal compartmentalization in vacuoles, and the increased activity of antioxidant enzymes to neutralize Cd-induced toxicity. While these direct protective mechanisms can help alleviate Cd toxicity, other indirect mechanisms such as microelements (zinc, iron, manganese, and selenium) interfering with Cd uptake may decrease Cd concentration in plants. This comprehensive review encompasses the significance of Cd, portals of contamination and toxicity to plants, and implications for crop production. Various mitigation strategies with the beneficial effects of zinc, iron, manganese, and selenium in activating defence mechanisms against Cd stress are discussed. Furthermore, this review systematically identifies and summarises suitable strategies for mitigating Cd-induced toxicity in plants.