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The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that nonspecific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 1010-11, that are dependent on sulfhydryl ligands to achieve high-affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase, and also considered to be insignificant in cells. Adding the low molecular weight fraction of cell supernatant to the proteome markedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione (GSH). In agreement, inclusion of GSH accelerated the reaction in a concentration-dependent manner. The implications of abundant high-affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with GSH in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking., (© The Author(s) 2021. Published by Oxford University Press.)

Zinc is an essential trace element that is often reduced under the type 1 diabetic condition. Previous studies demonstrated that zinc deficiency enhanced type 1 diabetes-induced liver injury and that zinc supplementation significantly helped to prevent this. Due to the differences in pathogenesis between type 1 and type 2 diabetes, it is unknown whether zinc supplementation can induce a beneficial effect on type 2 diabetes-induced liver injury. This possible protective mechanism was investigated in the present study. A high-fat diet, along with a one-time dose of streptozotocin, was applied to metallothionein (MT) knockout mice, nuclear factor-erythroid 2-related factor (Nrf) 2 knockout mice, and age-matched wild-type (WT) control mice, in order to induce type 2 diabetes. This was followed by zinc treatment at 5 mg/kg body weight given every other day for 3 months. Global metabolic disorders of both glucose and lipids were unaffected by zinc supplementation. This induced preventive effects on conditions caused by type 2 diabetes like oxidative stress, apoptosis, the subsequent hepatic inflammatory response, fibrosis, hypertrophy, and hepatic dysfunction. Additionally, we also observed that type 2 diabetes reduced hepatic MT expression, while zinc supplementation induced hepatic MT expression. This is a crucial antioxidant. A mechanistic study showed that MT deficiency blocked zinc supplementation-induced hepatic protection under the condition of type 2 diabetes. This suggested that endogenous MT is involved in the hepatic protection of zinc supplementation in type 2 diabetic mice. Furthermore, zinc supplementation-induced hepatic MT increase was unobserved once Nrf2 was deficient, indicating that Nrf2 mediated the upregulation of hepatic MT in response to zinc supplementation. Results of this study indicated that zinc supplementation prevented type 2 diabetes-induced liver injury through the activation of the Nrf2-MT-mediated antioxidative pathway., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2021 Lechu Yu et al.)

The Pacific oyster, Crassostrea gigas, is a traditional food worldwide. The soft body of the oyster can easily accumulate heavy metals such as cadmium (Cd). To clarify the molecular mechanism of Cd accumulation in the viscera of C. gigas, we identified Cd-binding proteins. 5,10,15,20-Tetraphenyl-21H,23H-porphinetetrasulfonic acid, disulfuric acid, tetrahydrate, and Cd-binding competition experiments using immobilized metal ion affinity chromatography revealed the binding of water-soluble high molecular weight proteins to Cd, including C. gigas protein disulfide isomerase (cgPDI). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed two CGHC motifs in cgPDI. The binding between Cd and rcgPDI was confirmed through a Cd-binding experiment using the TPPS method. Isothermal titration calorimetry (ITC) revealed the binding of two Cd ions to one molecule of rcgPDI. Circular dichroism (CD) spectrum and tryptophan fluorescence analyses demonstrated that the rcgPDI bound to Cd. The binding markedly changed the two-dimensional or three-dimensional structures. The activity of rcgPDI measured by a PDI Activity Assay Kit was more affected by the addition of Cd than by human PDI. Immunological analyses indicated that C. gigas contained cgPDI at a concentration of 1.0 nmol/g (viscera wet weight). The combination of ITC and quantification results revealed that Cd-binding to cgPDI accounted for 20% of the total bound Cd in the visceral mass. The findings provide new insights into the defense mechanisms of invertebrates against Cd.

Metallothioneins (MTs) are a family of cysteine-rich metal-binding proteins that are important in the chelating and detoxification of toxic heavy metals. Until now, the short length and the low sequence complexity of MTs have hindered the inference of robust phylogenies, hampering the study of their evolution. To address this longstanding question, we applied an iterative BLAST search pipeline that allowed us to build a unique dataset of more than 300 MT sequences in insects. By combining phylogenetics and synteny analysis, we reconstructed the evolutionary history of MTs in insects. We show that the MT content in insects has been shaped by lineage-specific tandem duplications from a single ancestral MT. Strikingly, we also uncovered a sixth MT, MtnF, in the model organism Drosophila melanogaster . MtnF evolves faster than other MTs and is characterized by a non-canonical length and higher cysteine content. Our methodological framework not only paves the way for future studies on heavy metal detoxification but can also allow us to identify other previously unidentified genes and other low complexity genomic features.

Aims: To evaluate the role of metallothionein (MT) in sex differences of obesity, we examined the effect of MT on regulation of lipid accumulation in female and male wild type (WT) and MT1/MT2-null (MT-KO) mice., Main Methods: Male and female WT and MT-KO mice fed standard diet (SD) or high-fat diet (HFD) for 35 weeks. Surgical castration in male mice was also performed to examine the effects of androgen on fat accumulation under HFD condition., Key Findings: The fat mass and size of adipocytes in white adipose tissue (WAT) was greater in adult MT-KO mice than in WT mice after 35 weeks of SD feeding without gender differences, suggesting a role of MT in limiting WAT development during normal growth in both sexes. In female mice fed HFD, weights of WAT and body were greater in MT-KO mice than in WT mice, indicating that MT had a preventive role against excess fat accumulation. In male mice fed HFD, WAT weight hardly increased in MT-KO mice compared to the increase in WT mice. Surgically castrated WT males fed HFD had lower WAT weight compared with sham-treated mice, although castrated MT-KO males fed HFD had greater increases in WAT weight compared with sham-treated mice and castrated WT males., Significance: These data suggest that MT could enhance the preventive action of estrogen against excess fat accumulation, on the contrary, MT augmented the ability of androgen to increase fat accumulation. MT may act to modify the susceptibility to obesity under sex hormones., (Copyright © 2019. Published by Elsevier Inc.)

Oxidative stress affects all the structures of the human eye, particularly the retina and its retinal pigment epithelium (RPE). The RPE limits oxidative damage by several protective mechanisms, including the non-enzymatic antioxidant system zinc-metallothionein (Zn-MT). This work aimed to investigate the role of Zn-MT in the protection of RPE from the oxidative damage of reactive oxygen intermediates by analytical and biochemical-based techniques. The Zn-MT system was induced in an in vitro model of RPE cells and determined by elemental mass spectrometry with enriched isotopes and mathematical calculations. Induced-oxidative stress was quantified using fluorescent probes. We observed that 25, 50 or 100 μM of zinc induced Zn-MT synthesis (1.6-, 3.6- and 11.9-fold, respectively), while pre-treated cells with zinc (25, 50, and 100 μM) and subsequent 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) treatment increased Zn-MT levels in a lesser extent (0.8-, 2.1-, 6.1-fold, respectively), exerting a stoichiometric transition in the Zn-MT complex. Moreover, AAPH treatment decreased MT levels (0.4-fold), while the stoichiometry remained constant or slightly higher when compared to non-treated cells. Convincingly, induction of Zn-MT significantly attenuated oxidative stress produced by free radicals' generators. We conclude that the stoichiometry of Zn-MT plays an important role in oxidative stress response, related with cellular metal homeostasis.

Inorganic mercury is a harmful heavy metal that causes severe kidney damage. Glutathione (GSH), a tripeptide comprising L-glutamic acid, glycine and L-cysteine, and metallothionein (MT), a cysteine-rich and metal-binding protein, are biologically important protective factors for renal toxicity by inorganic mercury. However, the relationship between GSH and MT for the prevention of renal toxicity by inorganic mercury is unknown. We examined the sensitivity of the mice depleted in GSH by treatment with L-Buthionine-SR-sulfoximine (L-BSO), and MT-I/II null mice genetically deleted for MT-I and MT-II, to inorganic mercury (HgCl 2 ). Kidney damage was not induced in the wild-type mice treated with HgCl 2 (30 µmol/kg). In the MT-I/II null mice, renal toxicity was induced by HgCl 2 at a dose of 30 µmol/kg but not 1.0 µmol/kg. All GSH-depleted mice of both strains were dead following the injection of HgCl 2 (30 µmol/kg). GSH-depleted wild-type mice treated with HgCl 2 (1.0 µmol/kg) developed kidney damage similar to MT-I/II null mice treated with HgCl 2 (30 µmol/kg). Moreover, renal toxicity induced by HgCl 2 (1.0 µmol/kg) was more severe in GSH-depleted MT-I/II null mice compared with GSH-depleted wild-type mice. The present study found that GSH and MT-I/II play cooperatively an important role in the detoxification of severe kidney damage caused by inorganic mercury. In addition, GSH may act as a primary protective factor against inorganic mercury-induced acute renal toxicity, because GSH-depleted mice were more sensitive to inorganic mercury than MT-I/II null mice.

Cadmium is a nonessential heavy metal and an industrial and environmental pollutant. It has been known that cadmium must enter cells to cause damage. To understand the transport systems responsible for cadmium entry into cells, it is important to determine the precise mechanisms underlying cadmium toxicity. Numerous studies have sought to unravel the exact pathways by which cadmium enters various cells and the mechanisms by which it causes toxicity in the organs of human and animals. The purpose of this review is to present the progress made regarding the mechanisms of cadmium transport in various cells and the mechanisms underlying cadmium toxicity in organs.

Knockout of metallothionein (MT) genes contributes to a heavier body weight in early life and the potential to become obese through the intake of a high fat diet (HFD) in mice. It has thus been suggested that MT genes regulate the formation of adipose tissue, which would become the base for later HFD-induced obesity. We evaluated the fat pads of mice during the lactation stage. The fat mass and adipocyte size of MT1 and MT2 knockout mice were greater than those of wild type mice. Next, we assayed the ability of small interfering RNA (siRNA) to silence MT genes in the 3T3-L1 cell line. The expressions of MT1 and MT2 genes were transiently upregulated during adipocyte differentiation, and the siRNA pretreatment led to the suppression of the expression of both MT mRNAs and proteins. The MT siRNA promoted lipid accumulation in adipocytes and caused proliferation of post-confluent preadipocytes; these effects were suppressed by an inhibitor of phosphatidylinositol 3-kinase (LY294002). In addition, MT siRNA promoted insulin-stimulated phosphorylation of Akt, a downstream kinase of the insulin signaling pathway. Enhanced lipid accumulation in 3T3-L1 cells resulting from MT-gene silencing was inhibited by pretreatment with an antioxidant, N-acetylcysteine, used as a substitute for antioxidant protein MTs. These results suggest that interference in MT expression enhanced the activation of the insulin signaling pathway, resulting in higher lipid accumulation in 3T3-L1 adipocytes.

Metallothioneins (MTs) are small molecular weight stress response proteins that play a central role as reservoir of essential divalent heavy metal cations such as zinc and copper, and also can diminish the effects of toxic heavy metals such as mercury and cadmium. Historically, MT has been considered to be an intracellular protein with roles to play in the management of heavy metals, as a regulator of cellular redox potential, and as a buffer of free radicals. Our recent studies have highlighted immunomodulatory role of MT in inflammatory diseases and also in the progression of metastatic cell movement. Hence, manipulation and detection of MT is essential for its possible use as a diagnostic and in therapeutic interventions of chronic inflammation. This review describes procedures used to detect MT using techniques such as western immunoblot, competition ELISA, flow cytometry and immunohistochemistry. Additionally, it also describes the use of a colorimetric cell proliferation assay (CellTiter 96 AQ ueous One Solution/MTS) to study the proliferative effect of MT. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

Metallothionein (MT) plays a central role in cellular defense against heavy metals and oxidative stress. Since the induction of MT requires the activation of metal response element (MRE)-binding transcription factor-1 (MTF-1) by binding of zinc ions, inorganic zinc is regarded as a typical MT inducer. However, in a previous report, we showed that inorganic zinc could not induce MT in vascular endothelial cells. While it is suggested that endothelial MT presents mechanisms different from those of other cell types, these remain unclear. In this study, we investigated whether the induction of endothelial MT expression involves the Nrf2-ARE pathway using copper(II) bis(diethyldithiocarbamate), termed Cu10, using a culture system of bovine aortic endothelial cells. Cu10 induced MT-1/2 protein expression and increased the expression of mRNAs for MT-1A, MT-1E, and MT-2, MT isoforms expressed in the cells. Cu10 activated not only the MTF-1-MRE, but also the Nrf2-ARE pathway. MTF-1 knockdown resulted in the repression of Cu10-induced MT-1 and -2 expression. Cu10-induced MT-1 expression was down-regulated by Nrf2 knockdown. However, MT-2 expression was not affected by Nrf2 knockdown. These results suggest that the expression of endothelial MT is up-regulated by the Nrf2-ARE pathway as well as by the MTF-1-MRE pathway. Moreover, MT-1 regulation mechanisms differ from that of MT-2. Specifically, the present data support the hypothesis that MT-1 participates in the biological defense system, while MT-2 mainly regulates intracellular zinc metabolism.

Vascular endothelial cells are in direct contact with blood. Inorganic zinc is thought to be incapable of inducing metallothionein, which protects cells from heavy metal toxicity and oxidative stress, in vascular endothelial cells. Here, we aimed to further characterize the induction of metallothionein in vascular endothelial cells. Our results confirmed that inorganic zinc could not induce metallothionein in vascular endothelial cells. Moreover, ZnSO4 could not activate both the metal response element (MRE) transcription factor 1 (MTF-1)/MRE and Nrf2/antioxidant response element (ARE) pathways and was incapable of inducing metallothionein. In addition, bis(L-cysteinato)zincate(II), a zinc complex that activates the MTF-1/MRE pathway, increased MRE promoter activity but failed to induce metallothionein, suggesting that vascular endothelial metallothionein was not induced only by activation of the MTF-1/MRE pathway. Further analysis of a library of zinc complexes showed that zinc(II) bis(diethyldithiocarbamate) activated the MTF-1/MRE pathway but not the Nrf2/ARE pathway, increased MT-1A, MT-1E, and MT-2A mRNA levels, and induced metallothionein proteins. These data indicated that zinc complexes may be excellent tools to analyze metallothionein induction in vascular endothelial cells.

Transgenic mice overexpressing spermine oxidase (SMO) in the cerebral cortex (Dach-SMO mice) showed increased vulnerability to excitotoxic brain injury and kainate-induced epileptic seizures. To investigate the mechanisms by which SMO overexpression leads to increased susceptibility to kainate excitotoxicity and seizure, in the cerebral cortex of Dach-SMO and control mice we assessed markers for astrocyte proliferation and neuron loss, and the ability of kainate to evoke glutamate release from nerve terminals and astrocyte processes. Moreover, we assessed a possible role of astrocytes in an in vitro model of epileptic-like activity in combined cortico-hippocampal slices recorded with a multi-electrode array device. In parallel, as the brain is a major metabolizer of oxygen and yet has relatively feeble protective antioxidant mechanisms, we analyzed the oxidative status of the cerebral cortex of both SMO-overexpressing and control mice by evaluating enzymatic and non-enzymatic scavengers such as metallothioneins. The main findings in the cerebral cortex of Dach-SMO mice as compared to controls are the following: astrocyte activation and neuron loss; increased oxidative stress and activation of defense mechanisms involving both neurons and astrocytes; increased susceptibility to kainate-evoked cortical epileptogenic activity, dependent on astrocyte function; appearance of a glutamate-releasing response to kainate from astrocyte processes due to activation of Ca(2+)-permeable AMPA receptors in Dach-SMO mice. We conclude that reactive astrocytosis and activation of glutamate release from astrocyte processes might contribute, together with increased reactive oxygen species production, to the vulnerability to kainate excitotoxicity in Dach-SMO mice. This mouse model with a deregulated polyamine metabolism would shed light on roles for astrocytes in increasing vulnerability to excitotoxic neuron injury.

Hypertension is an independent risk factor for heart disease and is responsible for the increased cardiac morbidity and mortality. Oxidative stress plays a key role in hypertensive heart diseases although the precise mechanism remains unclear. This study was designed to examine the effect of cardiac-specific overexpression of metallothionein, a cysteine-rich antioxidant, on myocardial contractile and intracellular Ca(2+) anomalies in N(G)-nitro-l-arginine methyl ester (l-NAME)-induced experimental hypertension and the mechanism involved with a focus on autophagy. Our results revealed that l-NAME treatment (14 days) led to hypertension and myocardial anomalies evidenced by interstitial fibrosis, cardiomyocyte hypertrophy, increased LV end systolic and diastolic diameters (LVESD and LVEDD) along with suppressed fractional shortening. l-NAME compromised cardiomyocyte contractile and intracellular Ca(2+) properties manifested as depressed peak shortening, maximal velocity of shortening/relengthening, electrically-stimulated rise in intracellular Ca(2+), elevated baseline and peak intracellular Ca(2+). These l-NAME-induced histological and mechanical changes were attenuated or reconciled by metallothionein. Protein levels of autophagy markers LC3B and p62 were decreased and increased, respectively. Autophagy signaling molecules AMPK, TSC2 and ULK1 were inactivated while those of mTOR and p70s6K were activated by l-NAME, the effects of which were ablated by metallothionein. Autophagy induction mimicked whereas autophagy inhibition nullified the beneficial effect of metallothionein against l-NAME. These findings suggested that metallothionein protects against l-NAME-induced myocardial anomalies possibly through restoration of autophagy., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Metallothioneins (MTs) are cysteine-rich, low molecular weight, and heavy metal-binding protein molecules. MT participates in metallic homeostasis and detoxification in living animals due to its abundant cysteine. In order to investigate the functions of MT during spermiogenesis in the mudskipper (Boleophthalmus pectinirostris), we identified the MT complete which contains: an 83bp 5' untranslated region, a 110bp 3' untranslated region, and a 183bp open reading frame. The protein alignment between MT sequences of other species shows a high similarity and a strong identity in cysteine residues vital for the metal-binding affinity of MT. The localizations of MT were mainly in the cytoplasm of germinal cells, indicating a role in spermatogenesis and testis protection. After the cadmium (Cd) exposure, the testis presents abnormal morphology and MT mRNA expression, both of which indicate a sensitive response of testis MT to Cd. Therefore, we suggest that MTs play an important role in spermatogenesis and testes protection against Cd toxicity in B. pectinirostris., (Copyright © 2015 Elsevier Inc. All rights reserved.)

As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. Yeast CUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study, CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.

Paraquat (PQ) is a widely used herbicide, and lung is the primary target of PQ poisoning. Metallothionein (MT) is a potent antioxidant and free radical scavenger, and has been shown to play a protective role in lung injury induced by different stressors. This study was undertaken to evaluate the protective potential of MT against PQ-induced acute lung injury using MT-I/II null (MT(-/-)) mice. Wild-type (MT(+/+)) mice and MT(-/-) mice were given one intragastric administration of 50mg/kg PQ for 24h, and it was revealed that MT(-/-) mice were more susceptible to PQ-induced acute lung injury than MT(+/+) mice evidenced by the following findings. As compared with MT(+/+) mice, MT(-/-) mice presented more severe histopathological lesions in the lung, higher pulmonary malondialdehyde content, and more reduced pulmonary antioxidative enzymes activities. PQ also induced more apoptosis in pneumocytes from MT(-/-) mice, and the expressions of apoptosis-related proteins Bax, Bcl-2, cleaved-caspase-3, and the ratio of Bax/Bcl-2 were all more significantly increased in PQ-treated MT(-/-) mice. Our results clearly demonstrate that endogenous MT can attenuate PQ-induced acute lung injury, possibly through the mechanisms of anti-oxidation and anti-apoptosis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: The aim of this prospective multicenter study was to identify biomarkers that can be used to predict therapeutic responses to tocilizumab in patients with rheumatoid arthritis (RA)., Methods: We recruited patients with RA who were treated with tocilizumab for the first time, and determined therapeutic responses at 6 months. In the training cohort (n = 40), gene expression in peripheral blood mononuclear cells (PBMCs) at baseline was analyzed using genome-wide DNA microarray, with 41,000 probes derived from 19,416 genes. In the validation cohort (n = 20), expression levels of the candidate genes in PBMCs at baseline were determined using real-time quantitative polymerase chain reaction (qPCR) analysis., Results: We identified 68 DNA microarray probes that showed significant differences in signal intensity between nonresponders and responders in the training cohort. Nineteen putative genes were selected, and a significant correlation between the DNA microarray signal intensity and the qPCR relative expression was confirmed in 15 genes. In the validation cohort, a significant difference in relative expression between nonresponders and responders was reproduced for 3 type I interferon response genes (IFI6, MX2, and OASL) and MT1G. Receiver operating characteristic curve analysis of models incorporating these genes showed that the maximum area under the curve was 0.947 in predicting a moderate or good response to tocilizumab in the validation cohort., Conclusion: Using genome-wide DNA microarray analyses, we identified candidate biomarkers that can be used to predict therapeutic responses to tocilizumab in patients with RA. These findings suggest that type I interferon signaling and metallothioneins are involved in the pathophysiology of RA., (Copyright © 2014 by the American College of Rheumatology.)

Metallothioneins (MTs) are known to participate in protection against oxidative stress. This study assessed the effects of MT-I&II gene knockout on methotrexate (MTX)-induced bone damage in growing mice. MT-I&II knockout (MT⁻/⁻) and wild type (MT⁺/⁺) male mice were injected with saline or 12.5 mg kg⁻¹ MTX for three consecutive days. MTX treatment was shown to cause more severe damage in MT⁻/⁻ mice when compared to MT⁺/⁺ mice, as demonstrated by the more obvious thinning of growth plate, reduced proliferation and increased apoptosis of chondrocytes, and reduced metaphysis heights in the knockout mice. Analysis of total liver glutathione (the most abundant intracellular antioxidant) also revealed significant lower glutathione levels in all MT⁻/⁻ mice. In conclusion, MT⁻/⁻ mice were more susceptible than MT⁺/⁺ mice to MTX-induced bone damages, which may be associated with the reduction of basal antioxidant defence, suggesting a protective role of MTs in the growing skeleton against damages caused by MTX chemotherapy.

Organisms have evolved different strategies to respond to oxidative stress generated as a by-product of aerobic respiration and thus maintain the redox homeostasis within the cell. In particular, fungal pathogens are exposed to reactive oxygen species (ROS) when they interact with the phagocytic cells of the host which are the first line of defense against fungal infections. These pathogens have co-opted the enzymatic (catalases, superoxide dismutases (SODs), and peroxidases) and non-enzymatic (glutathione) mechanisms used to maintain the redox homeostasis within the cell, to resist oxidative stress and ensure survival within the host. Several virulence factors have been related to the response to oxidative stress in pathogenic fungi. The opportunistic fungal pathogen Candida glabrata (C. glabrata) is the second most common cause of candidiasis after Candida albicans (C. albicans). C. glabrata has a well defined oxidative stress response (OSR), which include both enzymatic and non-enzymatic mechanisms. C. glabrata OSR is controlled by the well-conserved transcription factors Yap1, Skn7, Msn2 and Msn4. In this review, we describe the OSR of C. glabrata, what is known about its core elements, its regulation and how C. glabrata interacts with the host. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012)., (Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Many Chinese medicines have the potential to be hepatoprotective and therefore can be used to treat acute and chronic liver diseases. The challenge is to identify the molecular target for their protective mechanism. This study investigated the induction of nuclear factor-erythroid 2(NF-E2)-related factor 2 (Nrf2) antioxidant genes and metallothionein as a common mechanism of hepatoprotective effects of Chinese medicines such as Piper puberulum. Mice were pretreated with Piper puberulum extract (PPE, 500 mg/kg, po) or vehicles for seven days, followed by intoxication with CCl 4 (25 μl/kg, ip for 16 h), D-galactosamine (800 mg/kg, ip for 8 h), or acetaminophen (400 mg/kg, ip for 8 h). Hepatotoprotection was evaluated by serum enzyme activities and histopathology. To determine the mechanism of protection, mice were given PPE (250-1000 mg/kg, po for seven days) and livers were collected to quantify the expression of Nrf2-targeted genes and metallothionein. Nrf2-null mice were also used to determine the role of Nrf2 in PPE-mediated hepatoprotection.PPE pretreatment protected against the hepatotoxicity produced by CCl 4, D-galactosamine, and acetaminophen, as evidenced by decreased serum enzyme activities and ameliorated liver lesions. PPE treatment increased the expression of hepatic Nrf2, NAD(P)H:quinone oxidoreductase1 (Nqo1), heme oxygenase-1 (Ho-1), glutamate-cysteine ligases (Gclc), and metallothionein (MT), at both transcripts and protein levels. PPE protected wild-type mice from CCl 4 and acetaminophen hepatotoxicity, but not Nrf2-null mice, fortifying the Nrf2-dependent protection. In conclusion, induction of the Nrf2 antioxidant pathways and metallothionein appears to be a common mechanism for hepatoprotective herbs such as PPE.

Metallothioneins (MTs) are small cysteine-rich proteins which are involved in e.g. metal homeostasis, metal detoxification and protection against oxidative stress. In addition, several MTs have been shown to regulate expression of proangiogenic growth factors like vascular endothelial growth factor. Detailed information about the expression and regulation of specific MT isoforms in endothelial cells (EC) is limited. We therefore performed extensive mRNA expression profiling of all known human MTs in EC. We found that the basal endothelial expression is restricted to MT1E, MT1X, MT2A, and MT3. Physiological activation of EC by exposure to serum increased the expression of MT1E and MT2A and induced the expression of MT1M. Furthermore, exposure to zinc or copper induced the expression of most MT1 isoforms, while hypoxia specifically increased the expression of MT1E, MT1M, MT1X, and MT3. Finally, knockdown of the dominant MT isoform in EC, i.e. MT2A, resulted in decreased proliferation and sprouting as well as in increased migration of human umbilical vein EC. Together, these findings provide a link between MTs and angiogenesis., (© 2014 S. Karger AG, Basel.)

Metallothionein (MT) is a kind of metal binding protein. As an important member in metallothionein family, MT-I/II regulates metabolism and detoxication of brain metal ion and scavenges free radicals. It is capable of anti-inflammatory response and anti-oxidative stress so as to protect the brain tissue. During the repair process of brain injury, the latest study showed that MT-I/II could stimulate brain anti-inflammatory factors, growth factors, neurotrophic factors and the expression of the receptor, and promote the extension of axon of neuron, which makes contribution to the regeneration of neuron and has important effect on the recovery of brain injury. Based on the findings, this article reviews the structure, expression, distribution, adjustion, function, mechanism in the repair of brain injury of MT-I/II and its application prospect in forensic medicine. It could provide a new approach for the design and manufacture of brain injury drugs as well as for age estimation of the brain injury.

In this study, the expression and functional role of metallothioneins I and II (MT-I/II) were evaluated in the spinal cord in rat models of inflammatory and neuropathic pain. Complete Freund's adjuvant (CFA) injection into the hindpaw induced an increase in MT-I/II protein expression in bilateral dorsal and ventral horns throughout the spinal cord, while chronic constriction injury (CCI) of the sciatic nerve induced an increase in MT-I/II expression in the ipsilateral dorsal and ventral horns of the lower lumbar spinal cord. Increased MT-I/II immunoreactivity was predominantly localized to vascular endothelial cells. CFA injection- and CCI-induced MT-I/II expression was inhibited by intrathecal administration of MT-I siRNA. Treatment with MT-I siRNA before CFA injection or at early time points after CCI resulted in a significant attenuation of mechanical allodynia and thermal hyperalgesia, while treatment at later time points had no effect on established pain behaviors. Our results suggest that endogenous MT-I/II might play an important role in the pathogenesis of pain behaviors, participating in the initiation of inflammatory and neuropathic pain rather than in their maintenance., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Zn is an essential trace element, involved in many different cellular processes. A relationship between Zn, pancreatic function and diabetes was suggested almost 70 years ago. To emphasise the importance of Zn in biology, the history of Zn research in the field of diabetes along with a general description of Zn transporter families will be reviewed. The paper will then focus on the effects of Zn on pancreatic β-cell function, including insulin synthesis and secretion, Zn signalling in the pancreatic islet, the redox functions of Zn and its target genes. The recent association of two 'Zn genes', i.e. metallothionein (MT) and Zn transporter 8 (SLC 30A8), with type 2 diabetes at the genetic level and with insulin secretion in clinical studies offers a potential new way to identify new drug targets to modulate Zn homeostasis directly in β-cells. The action of Zn for insulin action in its target organs, as Zn signalling in other pancreatic islet cells, will be addressed. Therapeutic Zn-insulin preparations and the influence of Zn and Zn transporters in type 1 diabetes will also be discussed. An extensive review of the literature on the clinical studies using Zn supplementation in the prevention and treatment of both types of diabetes, including complications of the disease, will evaluate the overall beneficial effects of Zn supplementation on blood glucose control, suggesting that Zn might be a candidate ion for diabetes prevention and therapy. Clearly, the story of the links between Zn, pancreatic islet cells and diabetes is only now unfolding, and we are presently only at the first chapter.

Background: Cardiovascular diseases remain a leading cause of the mortality world-wide, which is related to several risks, including the life style change and the increased diabetes prevalence. The present study was to explore the preventive effect of zinc on the pathogenic changes in the aorta., Methods: A genetic type 1 diabetic OVE26 mouse model was used with/without zinc supplementation for 3 months. To determine gender difference either for pathogenic changes in the aorta of diabetic mice or for zinc protective effects on diabetes-induced pathogenic changes, both males and females were investigated in parallel by histopathological and immunohistochemical examinations, in combination of real-time PCR assay., Results: Diabetes induced significant increases in aortic oxidative damage, inflammation, and remodeling (increased fibrosis and wall thickness) without significant difference between genders. Zinc treatment of these diabetic mice for three months completely prevented the above pathogenic changes in the aorta, and also significantly up-regulated the expression and function of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a pivotal regulator of anti-oxidative mechanism, and the expression of metallothionein (MT), a potent antioxidant. There was gender difference for the protective effect of zinc against diabetes-induced pathogenic changes and the up-regulated levels of Nrf2 and MT in the aorta., Conclusions: These results suggest that zinc supplementation provides a significant protection against diabetes-induced pathogenic changes in the aorta without gender difference in the type 1 diabetic mouse model. The aortic protection by zinc against diabetes-induced pathogenic changes is associated with the up-regulation of both MT and Nrf2 expression.

Silver (Ag) possesses antibacterial activity and has been used in wound dressings and deodorant powders worldwide. However, the metabolic behavior and biological roles of Ag in mammals have not been well characterized. In the present study, we exposed human bronchial epithelial cells (BEAS-2B) to AgNO3 and investigated uptake and intracellular distribution of Ag, expression of metallothionein (MT), generation of reactive oxygen species (ROS), and changes in mitochondrial respiration. The culture medium concentration of Ag decreased with time and stabilized at 12h. The concentration of both Ag and MT in the soluble cellular fraction increased up to 3h and then decreased, indicating that cytosolic Ag relocated to the insoluble fraction of the cells. The levels of mRNAs for the major human MT isoforms MT-I and MT-II paralleled with the protein levels of Ag-MT. The intensity of fluorescence derived from ROS was elevated in the mitochondrial region at 24h. Ag decreased mitochondrial oxygen consumption in a dose-dependent manner and the activity of mitochondrial complex I-IV enzymes was significantly inhibited following exposure to Ag. In a separate experiment, we found that hydrogen peroxide (H2O2) at concentrations as low as 0.001% (equivalent to the concentration of H2O2 in Ag-exposed cells) removed Ag from MT. These results suggest MT was decomposed by cytosolic H2O2, and then Ag released from MT relocated to insoluble cellular fractions and inhibited electron chain transfer of mitochondrial complexes, which eventually led to cell damage., (Crown Copyright © 2013. Published by Elsevier Ireland Ltd. All rights reserved.)

In ageing, the accumulation of damaged molecules provoked by oxidative stress and inflammation contributes to altered gene expressions and cellular dysfunction. The antioxidant system is crucial in order to prevent damage to intracellular molecules including DNA and, consequently, to avoid cellular dysfunction or neoplastic transformation. However, during serious DNA damage, the cells can activate a response characterized by cell cycle arrest and production of factors (mainly chemokines and cytokines) named "senescent associated secretory phenotype" (SASP) with the putative function to attract immune cells involved in the clearance of the senescent cells. This phenomenon named "cellular senescence" is, by one side, an important tumor suppressive mechanism but, on the other side, it contributes to impair tissue regenerative capacity and to possible transformation of neighbouring cells to cancer cells if a rapid clearance of the senescent cell doesn't occur. Therefore, preventing DNA damage via an optimal intracellular antioxidant defence is the key to reduce risk of cancer while keeping senescent changes at minimum. Zinc-bound Metallothioneins (MT), could play a key role in this prevention because they are antioxidant proteins and release zinc ions for several proteins and enzymes involved in antioxidant and DNA-repair responses. Reduced MT expression and intracellular zinc occur in some models of senescent cells. This process is of relevance since zinc ions released from MT could be implicated in the modulation of SASP. In chronic inflammation, such as in ageing, the dysfunction in zinc release from MT occurs, suggesting a potential contribution to the onset of senescent cells. Hence, MT could be directly or indirectly involved in the modulation of cellular senescent state and might represent a possible therapeutic target against the accumulation of dysfunctional senescent cells.

Low-ambient temperature environment exposure increased the risk of cardiovascular morbidity and mortality, although the underlying mechanism remains unclear. This study was designed to examine the impact of cardiac overexpression of metallothionein, a cysteine-rich heavy metal scavenger, on low temperature (4°C)-induced changes in myocardial function and the underlying mechanism involved, with a focus on autophagy. Cold exposure (4°C for 3 wk) promoted oxidative stress and protein damage, increased left ventricular end-systolic and -diastolic diameter, and suppressed fractional shortening and whole heart contractility, the effects of which were significantly attenuated or ablated by metallothionein. Levels of the autophagy markers LC3B-II, beclin-1, and Atg7 were significantly upregulated with unchanged autophagy adaptor protein p62. Fluorescent immunohistochemistry revealed abundant LC3B puncta in cold temperature-exposed mouse hearts. Coimmunoprecipitation revealed increased dissociation between Bcl2 and Beclin-1. Cold exposure reduced phosphorylation of the autophagy inhibitory signaling molecules Akt and mTOR, increased ULK1 phosphorylation, and dampened eNOS phosphorylation (without changes in their total protein expression). These cold exposure-induced changes in myocardial function, autophagy, and autophagy signaling cascades were significantly alleviated or mitigated by metallothionein. Inhibition of autophagy using 3-methyladenine in vivo reversed cold exposure-induced cardiomyocyte contractile defects. Cold exposure-induced cardiomyocyte dysfunction was attenuated by the antioxidant N-acetylcysteine and the lysosomal inhibitor bafilomycin A1. Collectively, these findings suggest that metallothionein protects against cold exposure-induced cardiac anomalies possibly through attenuation of cardiac autophagy.

We recently reported that administration of the non-selective cyclic GMP-phosphodiesterase (cGMP-PDE) inhibitor zaprinast to cortically cryoinjured rats results three days post-lesion in reduced neuronal cell death that was associated to decreased macrophage/microglial activation and oxidative stress and increased astrogliosis and angiogenesis. Similar effects have been observed in cryoinjured animals overexpressing metallothioneins I/II (MT-I/II), metal-binding cysteine-rich proteins that are up-regulated in response to injury. In this work we have examined the effect of administration of the selective PDE5 inhibitor sildenafil (10mg/kg, sc) 2h before and 24 and 48h after induction of cortical cryolesion in wild-type and MT-I/II-deficient mice. Our results show that in wild-type animals sildenafil induces similar changes in glial reactivity, angiogenesis and antioxidant and antiapoptotic effects in the cryolesioned cortex as those observed in rats with zaprinast, indicating that inhibition of PDE5 is responsible for the neuroprotective actions. However, these effects were not observed in mice deficient in MT-I/II. We further show that sildenafil significantly increases MT-I/II protein levels in homogenates of lesioned cortex and MT-I/II immunostaining in glial cells around the lesion. Taken together these results indicate that cGMP-mediated pathways regulate expression of MT-I/II and support the involvement of these proteins in the neuroprotective effects of sildenafil in focal brain lesion., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background and Aim: The pathogenesis of non-steroidal anti-inflammatory drugs (NSAIDs)-induced small intestinal lesions remains unclear, although it is considered to be quite different from that of upper gastrointestinal tract ulcers due to the absence of acid and the presence of bacteria and bile in the small intestine. The aim of this study was to characterize specific gene expression profiles of intestinal mucosa in indomethacin-induced small intestinal injury, and to investigate the effects of rebamipide on the expression of these genes., Methods: Intestinal injury was induced in male Wistar rats by subcutaneous administration of indomethacin. Total RNA of the intestinal mucosa was extracted 24 h after indomethacin administration, gene expression was investigated using microarray analysis, and the identified genes were confirmed by real-time polymerase chain reaction (PCR). In addition, we investigated whether the treatment with rebamipide altered the expression of these identified genes., Results: The administration of indomethacin induced small intestine injuries, and these lesions were significantly inhibited by the treatment with rebamipide. Microarray analysis showed that the genes for several matrix metalloproteinases (MMPs) and several chemokine-related genes were significantly upregulated, and metallothionein 1a (MT1a) was downregulated in the intestinal mucosa after administration of indomethacin. The expressions of these genes were reversed by the treatment with rebamipide., Conclusion: These data suggest that MMPs, chemokines, and MT1a may play an important role in the intestinal mucosal injury induced by indomethacin. In particular, the inhibition of MMP genes and chemokine-related genes by rebamipide may be important for the therapeutic effect against NSAIDs-induced small intestinal injury., (© 2012 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

Members of the metallothionein (MT) family are short, cysteine-rich proteins involved in metal metabolism and detoxification, suggesting that MT proteins protect cells from damage caused by electrophilic carcinogens and thereby constitute a critical surveillance system against carcinogenesis. However, the roles of MT proteins in human hepatocellular carcinoma (HCC) are not fully understood. We identified a member of the MT family, termed MT1M. MT1M is expressed in various normal tissues with the highest level in the liver. MT1M expression can be induced by heavy metals and protect Escherichia coli from heavy metal toxicity. However, MT1M expression markedly decreased in human HCC specimens. A methylation profiling analysis indicated that the MT1M promoter is methylated in the majority of HCC tumors examined. Moreover, restored expression of MT1M in the HCC cell line Hep3B, which lacks endogenous MT1M expression, suppressed cell growth in vitro and in vivo and augmented apoptosis induced by tumor necrosis factor α. Furthermore, stable expression of MT1M in Hep3B cells blocked tumor necrosis factor α-induced degradation of IκBα and transactivation of NF-κB. We conclude that MT1M is a novel member of the MT family. Frequent downregulation of MT1M in human HCC may contribute to liver tumorigenesis by increasing cellular NF-κB activity.

Trace element concentrations (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn) were investigated in the liver, kidneys, muscle and feathers of 31 black-tailed godwits (Limosa limosa) accidentally killed during catches by mist net in the Pertuis Charentais, Atlantic coast of France. Analyses of carbon and nitrogen stable isotope ratios were carried out in liver, muscle and feathers in order to elucidate dietary patterns and to determine whether differences in diet explained the variation in elemental uptake. This study also aimed to have a preliminary assessment of sub-lethal effects triggered by trace elements through the investigation of gene expressions by quantitative real-time PCR, antioxidant enzyme activities (catalase, superoxide dismutase, glutathione peroxidase), and metallothionein (MT) levels. The results showed that Cr and Ni concentrations in tissues of adults were lower than in juveniles in part because adults may have eliminated these trace elements through moulting. Except for Cd and Ni, trace element concentrations were negatively correlated to the body mass of godwits. Ag, As, Hg and Se concentrations were positively linked with the trophic position of birds. The diet could be considered as a fundamental route of exposure for these elements demonstrating therefore the qualitative linkage between dietary habits of godwits and their contaminant concentrations. Our results strongly suggest that even though trace element concentrations were mostly below toxicity threshold level, the elevated concentrations of As, Ag, Cd, Cu, Fe and Se may however trigger sub-lethal effects. Trace elements appear to enhance expression of genes involved in oxidative stress defence, which indicates the production of reactive oxygen species. Moreover, birds with the highest concentrations appeared to have an increased mitochondrial metabolism suggesting that the fight against trace element toxicity requires additional energetic needs notably to produce detoxification mechanisms such as metallothioneins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The role of metallothioneins (MTs) in aging is not completely understood. Several studies have shown evidence that these proteins could represent a defense system against oxidative damage, but survival studies on mice overexpressing MTs are poor. Here we describe a survival study performed on old MT-1-overexpressing mice (MT-TG) and their respective controls (C57BL/6J) fed a standard or zinc (Zn)-supplemented diet. MT-TG mice had significantly increased survival compared with control. Zn supplementation affects the survival curves of MT-TG and C57BL/6J mice differently. This study poses the basis for intervention based on gene therapy with MTs to enhance the health span of laboratory mice.

Metallothioneins (MTs) are low-molecular weight cysteine- and metal-rich proteins with unquestionable metal binding capacity, antioxidant and anti-inflammatory properties, and a clear involvement in diverse physiological actions as inhibition of proapoptotic mechanisms, enhancement of cell survival, and tissue regeneration. Concurrent with this wide array of functions, MT-1/2 have been implicated in neuroprotection and neuroregeneration. The zinc binding capacity and antioxidant properties of MTs may account for most of their physiological features in the brain. However, some receptor-mediated actions of MT-1/2 have also been reported recently, a subject to be fully elucidated. This review analyses and updates the current knowledge on the actions of MTs related to neuroprotection and neuroregeneration in an effort to distinguish receptor-mediated actions of MTs from those arising from its zinc binding capacity and its antioxidant properties., (Copyright © 2011 Wiley Periodicals, Inc.)

Metallothionein (MT) is known to be involved in various physiological roles and diseases. However, a standard method for MT measurement has not been established until recently. Therefore, we have developed an easy and specific enzyme-linked immunosorbent assays (ELISA) to determine MT-1 and MT-2. In order to evaluate the method we developed, MT-1/2 in liver, kidney and brain was determined in wild type (WT), MT-1/2 knockout (KO) and MT-3 KO mice, with and without Cd treatment. MT 1/2 in urine was determined in genetically disordered LEC rats (an animal model of Wilson disease). MT-1/2 concentrations in the liver, kidney and brain in MT-1/2 KO mice were significantly lower compared to those of WT and MT-3 KO mice. MT-1/2 concentrations in the livers of WT mice significantly increased with Cd administration, but not in MT-1/2 KO mice. Similar results were observed by immunohistochemical staining. To confirm the molecular weight (MW) of MT detected in organs by the ELISA, analysis with a Sephadex G-75 was performed. Two peaks of MT-1/2 (MW small and large) were detected in WT and MT-3 KO mice. The small MT peak was mostly depleted in MT 1/2-KO mice, while a large MT peak remained. A significant increase in MT-1/2 concentration was detected in the urine of LEC rats with age and especially at the hepatitis stage. In conclusion, MT-1/2 ELISA and immunohistochemical staining was highly correlated with MT-1/2 determination in experimental animal specimens and could be a robust analytical tool for physiological and toxicological studies.

We recently reported that TCRP1, a novel multidrug-resistance associated human gene, can mediate cisplatin resistance in OSCC cells. However, the molecular mechanism underlying this role of TCRP1 remained to be elucidated. In this study, by using Human Toxicology and Drug Resistance Microarray, we identified 30 genes with significantly different expression levels between Tca/PYM and TCRP1 knockdown cell lines. Co-immunoprecipitation experiments and GST-pull down assays showed that metallothionein1X (MT1X) and Akt interact with TCRP1. siRNA-mediated knockdown of TCRP1 and MT1X was found to sensitize cells to cisplatin, leading to increased apoptosis and inhibition of cell proliferation. These functions of TCRP1 may be caused at least in part via activation of the PI3K/Akt/NF-κB signaling pathway. Taken together, our findings indicate that TCRP1 may be an important drug target for improvement of the treatment and survival of patients with oral squamous cell carcinoma.

Metallothionein (MT), a low-molecular-weight protein with a high affinity for divalent heavy metal ions, is involved in many pathophysiological processes, including metal homeostasis, detoxification, cell proliferation and protection against oxidative damage. We previously found that MT in gastric mucosa plays a role in protecting against Helicobacter pylori (H. pylori)-induced gastritis at the early stage of infection. H. pylori-induced chronic gastric inflammation is shown to be associated with gastric carcinogenesis. Thus, to examine whether gastric MT contributes to protection against H. pylori-induced chronic inflammation, we compared histological changes in the gastric mucosa of MT-null and the wild-type mice at 53 weeks after inoculation three times with H. pylori SS1. As a result, we observed disruption of the gastric mucosa in MT-null mice, but not in the wild-type mice, even at the late stage of H. pylori-infection. Evaluation of pathological changes in gastric specimens by the updated Sydney grading system revealed that scores related to chronic inflammation and polymorphonuclear cell activity were higher in infected MT-null mice than those in the wild-type mice. Furthermore, a higher score for metaplasia was also observed in the MT-null stomach. These results suggested that MT might be involved in protecting against H. pylori-induced gastric chronic inflammation associated with carcinogenesis.

Active studies of skin science have gradually clarified the underlying mechanisms of skin problems regarding skin beauty. The major skin problems are the alterations in appearance such as the hyperpigmentation and wrinkling caused by age. Those skin alterations are accelerated by solar light, particularly by ultraviolet rays, and it has been reported that reactive oxygen species (ROS) also involves in most of those processes. Thus, the reduction of oxidative stress induced by intracellular ROS is one approach to prevent and improve hyperpigmentation and wrinkling. Zn(2+) is well-known as an inducer of MT (metallothionein) and γGCS (γ-glutamyl cysteinyl synthetase: a rate-limiting enzyme of glutathione synthesis) via the up-regulation of their mRNAs through a metal transcription factor. The inductions of both MT and glutathione are expected to reduce oxidative stress due to the more effective scavenging of intracellular ROS. Several complexes of Zn(2+) and amino acids were synthesized and then evaluated for effects on MT synthesis in HaCaT keratinocytes. Among the complexes tested, we found a superior induction by a Zn(2+) glycine complex, Zn(Gly)(2). The anti-pigmentation and anti-wrinkling effects of Zn(Gly)(2) are introduced in this paper.

The Metallothionein (MT) is a protein which has several interesting biological effects and has been demonstrated increase focus on the role of MT in various biological systems in the past three decades. The studies on the role of MT were limited with few areas like apoptosis and antioxidants in selected organs even fifty years after its discovery. Now acknowledge the exploration of various isoforms of MT such as MT-I, MT-II, MT-III and MT-IV and other isoforms in various biological systems.Strong evidence exists that MT modulates complex diseases and the immune system in the body but the primary function of MT still remains unknown. This review's main objective is to explore the capability to specifically manipulate MT levels in cells and in animals to provide answers regarding how MT could impact those complex disease scenarios.The experimental result mentioned in this review related among MT, zinc, cadmium, diabetic, heart disease, bone retardation, neuro toxicity, kidney dysfunction, cancer, and brain suggest novel method for exploration and contribute significantly to the growing scientist to research further in this field.

Our previous studies demonstrated the involvement of quinone formation in dopaminergic neuron dysfunction in the L-DOPA-treated parkinsonian model and in methamphetamine (METH) neurotoxicity. We further reported that the cysteine-rich metal-binding metallothionein (MT) family of proteins protects dopaminergic neurons against dopamine (DA) quinone neurotoxicity by its quinone-quenching property. The aim of this study was to examine MT induction in astrocytes in response to excess DA and the potential neuroprotective effects of astrocyte-derived MTs against DA quinone toxicity. DA exposure significantly upregulated MT-1/-2 in cultured striatal astrocytes, but not in mesencephalic neurons. This DA-induced MT upregulation in astrocytes was blocked by treatment with a DA-transporter (DAT) inhibitor, but not by DA-receptor antagonists. Expression of nuclear factor erythroid 2-related factor (Nrf2) and its binding activity to antioxidant response element of MT-1 gene were significantly increased in the astrocytes after DA exposure. Nuclear translocation of Nrf2 was suppressed by the DAT inhibitor. Quinone formation and reduction of mesencephalic DA neurons after DA exposure were ameliorated by preincubation with conditioned media from DA-treated astrocytes. These protective effects were abrogated by MT-1/-2-specific antibody. Adding exogenous MT-1 to glial conditioned media also showed similar neuroprotective effects. Furthermore, MT-1/-2 expression was markedly elevated specifically in reactive astrocytes in the striatum of L-DOPA-treated hemi-parkinsonian mice or METH-injected mice. These results suggested that excess DA taken up by astrocytes via DAT upregulates MT-1/-2 expression specifically in astrocytes, and that MTs or related molecules secreted specifically by astrocytes protect dopaminergic neurons from damage through quinone quenching and/or scavenging of free radicals., (Copyright © 2010 Wiley-Liss, Inc.)

The overexpression of metallothionein-2A (MT-2A) is frequently observed in invasive human breast tumors and has been linked with more aggressive breast cancers. MT-2A overexpression led to the induction of MDA-MB-231 breast cancer cell migratory and invasive abilities. The reduction of MT-2A expression through small interfering RNA (siRNA) targeting MT-2A in invasive MDA-MB-231 cells completely inhibited both cell invasion and migration. In addition, the expression of matrix metalloproteinase-9 (MMP-9) and the transcriptional activity of AP-1 and NF-κB were upregulated by MT-2A overexpression. Collectively, our results provide the first demonstration that MT-2A promotes breast cancer cell invasion by upregulating MMP-9 via AP-1 and NF-κB activation. Furthermore, we found that MT-2A silencing can inhibit breast cancer invasiveness., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In many developed countries, prostate cancer is the most common male tumour disease. The high incidence and mortality requires early diagnosis, differentiation of aggressive, highly malignant forms from clinically silent forms and understanding of the pathogenesis with its typical metabolic aberrancies (if any) in order to develop new targeted therapies. Prostate cells (including prostate cancer cells) are unique in their relation to zinc ions. Prostate tissue can accumulate these ions in up to tenfold higher concentration than other body cells. These ions influence many cellular processes incl. proliferation, differentiation and apoptosis. Prostate cancer cells lack ability to accumulate zinc. Therefore, zinc ions may be expected to play an important role in the disease pathogenesis, in its propagation and metastatic potential of tumour cells. Intracellular zinc levels are regulated by zinc-binding proteins, especially metallothioneins, and zinc transporters. Zinc level regulation dysfunction has been identified in prostate cancer cells and may thus play an important role in the prostate cancer pathogenesis. Moreover, due to its overproduction by prostate tissue, metallothionein serum levels are elevated and can be used as an important tumour marker.

Metallothionein (MT) is a low molecular weight protein with anti-apoptotic properties that has been demonstrated to scavenge free radicals in vitro. MT has not been extensively investigated within the context of aging biology. The purpose of this review, therefore, is to discuss findings on MT that are relevant to basic aging mechanisms and to draw attention to the possible role of MT in pro-longevity interventions. MT is one of just a handful of proteins that, when overexpressed, has been demonstrated to increase mouse lifespan. MT also protects against development of obesity in mice provided a high fat diet as well as diet-induced oxidative stress damage. Abundance of MT is responsive to caloric restriction (CR) and inhibition of the insulin/insulin-like signaling (IIS) pathway, and elevated MT gene expression has been observed in tissues from fasted and CR-fed mice, long-lived dwarf mice, worms maintained under CR conditions, and long-lived daf-2 mutant worms. The dysregulation of MT in these systems is likely to have tissue-specific effects on aging outcomes. Further investigation will therefore be needed to understand how MT contributes to the response of invertebrates and mice to CR and the endocrine mutations studied by aging researchers., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The aim of the present study was to investigate whether metallothionein was involved in the protection of lung ischemic preconditioning (IP) against lung ischemia-reperfusion (I/R) injury. Adult male Sprague-Dawley rats were randomly divided into 3 groups based upon the intervention (n=8): control group (C), lung I/R group (I/R), lung I/R+IP group (IP). At the end of the experiment, the content of metallothionein was tested in lung tissue. Blood specimens collected from the arteria carotis were tested for the contents of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and myeloperoxidase (MPO). The pneumocyte apoptosis index (AI) was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL). Ultrastructural changes of lung tissue were observed by using transmission electron microscope. The results showed that in I/R group, the content of metallothionein was decreased (P<0.05), the content of MDA and MPO activity were increased (P<0.01), and SOD activity was decreased (P<0.01), compared with those in control group. IP treatment significantly increased the content of metallothionein (P<0.01), attenuated the MDA level (P<0.05) and MPO activity (P<0.01), and improved SOD activity (P<0.01) in blood serum. The number of TUNEL-positive cells in IP group was significantly reduced compared with that in I/R group (P<0.01). There were abnormal ultrastructural changes in I/R group, which were markedly reversed in IP group. In conclusion, IP may protect lung against I/R injury by inducing the expression of metallothionein.

DNA methylation plays a major role in cancer by silencing tumour suppressor genes. In melanoma, only a discrete number of methylated genes have been identified so far. After the treatment of melanoma cells with a DNA methyltransferase inhibitor and subsequent transcriptomic profiling, we had identified earlier a cohort of melanoma progression-associated genes regulated by methylation. Here, we identified which of these genes are directly methylated in melanoma cell lines and tissues. First, we examined 16 genes by bisulphite sequencing in the WM793 isogenic cell line model series. Five of these genes (CYBA, FABP5, MT1E, TSPY1 and TAC1) displayed increased methylation in several invasive cell lines compared with the parental WM793 cells, indicating their involvement in progression. Next, we analyzed several matched primary/metastatic tumours using methylation-specific PCR, which revealed that MT1E (one of the five genes assessed) was methylated in the largest proportion of tumours. Examination of a larger cohort of samples showed that 1 of 17 (6%) of the benign naevi, 16 of 43 (37%) primary tumours and 6 of 13 (46%) of the metastases displayed MT1E methylation. In addition, ectopic over-expression of MT1E mediated sensitization to cisplatin-induced apoptosis. Overall, these studies suggest that MT1E is a potential tumour suppressor gene, whose loss may promote resistance to apoptosis-inducing therapies.

Purpose: Few quantitative analyses of the induction of metallothioneins (MTs) have been conducted, and there are no reports on the contribution of MTs to the protective mechanism against ultraviolet (UV) radiation in the lens. In this study, we quantitatively analyzed the induction of MTs and analyzed the resulting protective effects against both metal- and UV radiation-induced damage in the cultured lens epithelial cell line, alphaTN4-1., Methods: The induction profiles of MTs by ZnCl(2) treatment in alphaTN4-1 cells were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The cells in which MTs were induced were either treated with high concentrations of ZnCl(2) or CdCl(2), or irradiated with UV-C, UV-B, or UV-A radiation, followed by analysis of cell viability. The (3)H-thymidine incorporation rate was used as an indicator of cell viability., Results: mRNA expression of MT-I and MT-II, the main MT isoform classes, was induced by ZnCl(2) treatment in a dose-dependent manner. MT induction increased the protective effects against both metaland UV-A radiation-induced cell damage., Conclusions: Our results suggest that MTs play an important role in the protection against damage induced by both toxic metals and UV-A radiation in lens epithelial cells.

We have shown that zinc-thiolate moieties of the metal binding protein metallothionein (MT) are critical targets for nitric oxide (NO) with resultant increases in intracellular labile zinc. Such an NO-MT-Zn signaling pathway appears to participate in important cardiovascular functions associated with biosynthesis of NO including hypoxic vasoconstriction in the lung. Although downstream effector signaling molecules and critical contractile targets remain unclear, current investigations reveal a contributory role for zinc dependent protein kinases and cytoskeletal proteins in mediating hypoxic induced constriction of pulmonary endothelial cells.

Oxidative stress accelerates adipocyte differentiation and lipid accumulation, leading to endoplasmic reticulum (ER) stress, which causes insulin resistance. Because metallothionein (MT) has a role in prevention of oxidative and ER stress, we examined the effects of MT on the development of obesity induced by 27 wk of a high-fat diet (HFD) in female MT-I- and MT-II-null (MT(-/-)) and wild-type (MT(+/+)) mice. Body weight, fat mass, and plasma cholesterol increased at a greater rate in MT(-/-) mice fed an HFD than in MT(-/-) mice fed a control diet (CD) and MT(+/+) mice fed an HFD, indicating that MT(-/-) mice fed an HFD became obese and hypercholesterolemic and that MT could prevent HFD-induced obesity. The observed increases in the levels of plasma leptin and leptin mRNA in the white adipose tissue of MT(-/-) mice fed the HFD suggested a leptin-resistant state. Enhanced expression of a mesoderm-specific transcript, which regulates the enlargement of fat cells, was accompanied by enlarged adipocytes in the white adipose tissue of young MT(-/-) mice before obesity developed after 3 and 8 wk of feeding the HFD. Thus, MT may have a preventive role against HFD-induced obesity by regulating adipocyte enlargement and leptin signaling.

Previous studies revealed butenolide (BUT), a Fusarium mycotoxin distributes extensively, induced myocardial oxidative damage, which could be abated by antioxidants such as glutathione. Metallothionein (MT) has proved to attenuate several oxidative cardiomyopathies via its potent antioxidant property. The present study is therefore undertaken to investigate the protective potential of the endogenous expression of MT against BUT-induced myocardial toxicity. Primary cultures of neonatal cardiomyocytes from MT-I/II null mice along with the corresponding wild-type mice will be utilized to determine the possible mechanistic properties of MT. BUT treatment to the cardiomyocytes evoked significant cytotoxicity as evidenced by morphological changes and concentration- and time-dependent reductions in cell viability. Additionally, BUT treatment remarkably increased reactive oxygen species (ROS) production in the cardiomyocytes of both MT-I/II null and wild-type mice. As a result, noticeable DNA damage in both cardiomyocytes was detected by alkaline comet assay. Furthermore, the comparison between the MT-I/II null and wild-type cardiomyocytes indicated that ROS production in the cardiomyocytes from the MT-I/II null mice was higher than from wild-type mice. DNA damage as evaluated by percentage of comet tail DNA, tail length and tail moment was more severe in the MT-l/II null cardiomyocytes than in wild-type myocytes. And in agreement with those results mentioned above, the MT-l/II null cardiomyocytes were more sensitive to BUT-induced cytotoxicity than wild-type cardiomyocytes. Taken together, these findings clearly show that basal MT can efficiently attenuate BUT-induced cytotoxic injuries in cardiomyocytes via the inhibition of intracellular ROS production, and associated DNA damage., (2010 Elsevier Ltd. All rights reserved.)