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Introduction: Colon cancer is a common and malignant cancer featuring high morbidity and poor prognosis., Aims: This study was performed to explore the regulatory role of MT1G in colon cancer as well as its unconcealed molecular mechanism., Methods: The expressions of MT1G, c-MYC, and p53 were assessed with the application of RT-qPCR and western blot. The impacts of MT1G overexpression on the proliferative ability of HCT116 and LoVo cells were measured by CCK-8 and BrdU incorporation assays. Additionally, transwell wound healing, and flow cytometry assays were employed to evaluate the invasive and migrative capacities as well as the apoptosis level of HCT116 and LoVo cells. Moreover, the activity of the P53 promoter region was assessed with the help of a luciferase reporter assay., Results: It was found that the expressions of MT1G at both mRNA and protein levels were greatly decreased in human colon cancer cell lines, particularly in HCT116 and LoVo cell lines. After transfection, it was discovered that the MT1G overexpression suppressed the proliferation, migration and invasion but promoted the apoptosis of HCT116 and LoVo cells, which were then partially reversed after overexpressing c-MYC. Additionally, MT1G overexpression reduced c-MYC expression but enhanced the p53 expression, revealing that the MT1G overexpression could regulate c-MYC/P53 signal. Elsewhere, it was also shown that c-MYC overexpression suppressed the regulatory effects of MT1G on P53., Conclusion: To conclude, MT1G was verified to regulate c-MYC/P53 signal to repress the proliferation, migration and invasion but promote the apoptosis of colon cancer cells, which might offer a novel targeted-therapy for the improvement of colon cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: Parkinson's disease (PD) is the second most common neurodegenerative disease caused by selective degeneration of dopaminergic neurons in the substantia nigra. Metallothionein has been shown to act as a neuroprotectant in various brain injury. Thus, this study aims to identify the effects of full-length human metallothionein 2 peptide (hMT2) in paraquat-induced brain injury in the zebrafish., Methodology: A total of 80 adult zebrafish were divided into 4 groups namely control, paraquat-treated, pre-hMT2-treated, and post-hMT2-treated groups. Fish were treated with paraquat intraperitoneally every 3 days for 15 days. hMT2 were injected intracranially on day 0 (pre-treated group) and day 16 (post-treated group). Fish were sacrificed on day 22 and the brains were collected for qPCR, ELISA and immunohistochemistry analysis., Results: qPCR analysis showed that paraquat treatment down-regulated the expression of genes related to dopamine activity and biosynthesis ( dat and th1 ) and neuroprotective agent ( bdnf ). Paraquat treatment also up-regulated the expression of the mt2 , smtb and proinflammatory genes ( il-1α , il-1β , tnf-α and cox-2 ). hMT2 treatment was able to reverse the effects of paraquat. Lipid peroxidation decreased in the paraquat and pre-hMT2-treated groups. However, lipid peroxidation increased in the post-hMT2-treated group. Paraquat treatment also led to a reduction of dopaminergic neurons while their numbers showed an increase following hMT2 treatment., Conclusion: Paraquat has been identified as one of the pesticides that can cause the death of dopaminergic neurons and affect dopamine biosynthesis. Treatment with exogenous hMT2 could reverse the effects of paraquat in the zebrafish brain.

Cadmium (Cd) is a heavy metal with various human exposure sources. It accumulates in the liver, forming a complex with metallothionein protein and progresses to other organs. As a heavy metal, cadmium can replace calcium and other divalent ions and disturb their cascades, ultimately affecting the vital organs. Since cadmium acetate (CA) is considered more lethal than other Cd compounds, the current study examines the effect of different concentrations of CA doses in drinking water for different exposure times in murine models (Mus musculus). After the exposure period, the murine models were then examined histopathologically and biochemically. The histopathological examination of the heart, liver, and kidneys of the experimental group showed extensive degenerative effects. Atomic absorption spectroscopy was used to determine the quantity of cadmium in serum, kidney, and hepatic tissues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hepatic proteins, especially metallothionein, directly related to Cd administration. The biochemical parameters, including creatine kinase, alanine aminotransferase, aspartate aminotransferase, total proteins, glucose, urea, uric acid, and creatinine, were also analyzed. After thorough histochemical and biochemical analysis, it was concluded that even low dose exposure of CA is hazardous to murine models with damaging effects., (© 2023 Wiley Periodicals LLC.)

Parkinson's disease (PD) is a progressive neurodegenerative disease of both the central and peripheral / enteric nervous systems. Oxidative stress and neuroinflammation are associated with the pathogenesis of PD, suggesting that anti-oxidative and anti-inflammatory compounds could be neuroprotective agents for PD. Eucommia ulmoides (EU) is a traditional herbal medicine which exerts neuroprotective effects by anti-inflammatory and anti-oxidative properties. Our previous study showed that treatment with chlorogenic acid, a component of EU, protected against neurodegeneration in the central and enteric nervous systems in a PD model. In this study, we examined the effects of EU extract (EUE) administration on dopaminergic neurodegeneration, glial response and α-synuclein expression in the substantia nigra pars compacta (SNpc), and intestinal enteric neurodegeneration in low-dose rotenone-induced PD model mice. Daily oral administration of EUE ameliorated dopaminergic neurodegeneration and α-synuclein accumulation in the SNpc. EUE treatment inhibited rotenone-induced decreases in the number of total astrocytes and in those expressing the antioxidant molecule metallothionein. EUE also prevented rotenone-induced microglial activation. Furthermore, EUE treatment exerted protective effects against intestinal neuronal loss in the PD model. These results suggest that EU exerts neuroprotective effects in the central and enteric nervous systems of rotenone-induced parkinsonism mice, in part by glial modification., Competing Interests: No potential conflict of interest relevant to this article was reported.

Broadly distributed species must cope with diverse and changing environmental conditions, including various forms of stress. Cosmopolitan populations of Drosophila melanogaster are more tolerant to oxidative stress than those from the species' ancestral range in sub-Saharan Africa, and the degree of tolerance is associated with an insertion/deletion polymorphism in the 3' untranslated region of the Metallothionein A (MtnA) gene that varies clinally in frequency. We examined oxidative stress tolerance and the transcriptional response to oxidative stress in cosmopolitan and sub-Saharan African populations of D. melanogaster, including paired samples with allelic differences at the MtnA locus. We found that the effect of the MtnA polymorphism on oxidative stress tolerance was dependent on the genomic background, with the deletion allele increasing tolerance only in a northern, temperate population. Genes that were differentially expressed under oxidative stress included MtnA and other metallothioneins, as well as those involved in glutathione metabolism and other genes known to be part of the oxidative stress response or the general stress response. A gene coexpression analysis revealed further genes and pathways that respond to oxidative stress including those involved in additional metabolic processes, autophagy, and apoptosis. There was a significant overlap among the genes induced by oxidative and cold stress, which suggests a shared response pathway to these two stresses. Interestingly, the MtnA deletion was associated with consistent changes in the expression of many genes across all genomic backgrounds, regardless of the expression level of the MtnA gene itself. We hypothesize that this is an indirect effect driven by the loss of microRNA binding sites within the MtnA 3' untranslated region., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Naringenin (Nar) is a flavanone that has been suggested to provide human health benefits such as anti-inflammatory, anti-oxidant and anti-cancer properties. However, the mechanisms underlying these benefits are complex and still not fully understood. In this study, we investigated the effect of Nar on the inflammatory response of macrophages and its underlying mechanism. In lipopolysaccharide (LPS)-stimulated human macrophages, Nar inhibited the activation of NF-κB pathway and suppressed the downstream expression of pro-inflammatory factors. In addition, Nar was also able to induce metallothionein 1 G (MT1G) expression, and the inhibitory effects of Nar on the production of pro-inflammatory cytokines was dependent on MT1G. Mechanistically, we found that MT1G-mediated inhibition of pro-inflammatory cytokines responses might be through repressing NF-κB activation via zinc chelation. Overall, this study reveals a novel mechanism of Nar on inflammatory responses, the suppression of NF-κB activation through upregulation of MT1G., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Growth hormone (GH) transgenic fish often exhibit remarkable transformations in growth rate and other phenotypes relative to wild-type. The 5750A transgenic coho salmon strain exhibits strong sexually dimorphic growth, with females possessing growth stimulation at a level typical of that seen for both sexes in other strains harbouring the same gene construct (e.g. M77), while males display a modest level of growth stimulation. GH mRNA levels were significantly higher in females than in males of the 5750A strain but equivalent in the M77 strain, indicating sex and transgene insertion locus altered transgene expression. We found that acute estradiol treatments did not influence GH expression in either strain (5750A and M77) or the transgene promoter (metallothionein-B), suggesting that estradiol level was not a significant factor influencing transgene activity. The feminization of XX and XY fish of the 5750A and M77 strains generated all-female groups and resulted in equalized growth of the two genetic sexes, suggesting that the presence of the Y chromosome was not directly capable of influencing the GH transgene-mediated growth in a physiological female conditions. These data suggest that the difference in growth rate seen between the sexes in the 5750A strain arises from non-estradiol-mediated sex influences on gene regulation at the transgene locus. This study shows how genetic factors and transgene insertion sites can influence transgene expression with significant consequent effects on phenotype.

Downregulation of the myeloid master regulator Spi1 /PU.1 plays a pivotal role in leukemogenesis, and we previously showed that Spi1 /PU.1 directly represses metallothionein (MT)-1G through the epigenetic activity of PU.1. Furthermore, we recently demonstrated that overexpression of MT-1G inhibits retinoic acid-induced differentiation of acute promyelocytic leukemia NB4 cells. As PU.1 is a master regulator of growth and differentiation in myeloid cells, we examined its effects on cell proliferation of MT-1G -overexpressing NB4 (NB4MTOE) cells in the present study. Although there were no significant differences in total viable cell numbers between NB4MTOE cells and control cells during the time course examined, the proportion of S-phase cells was obviously increased in all NB4MTOE cells at 16-24 h after serum stimulation. Consistent with these findings, real-time PCR analyses revealed marked increases in the expression of cyclin E (G1/S-phase cyclin) and cyclin A (S-phase cyclin) in NB4MTOE cells during the same time period. Furthermore, NB4MTOE cells were significantly resistant to cytosine arabinoside (Ara-C), an S-phase-specific chemotherapeutic drug. Collectively, these findings suggest a role for MT-1G in G1/S transition during the growth phase of NB4 cells., (Copyright © 2021 by the Association of Clinical Scientists, Inc.)

Cadmium (Cd), a substance with one of the most critical health hazard indices, can cause damage to both the blood and kidneys and accumulates in the body at last. The present work studied the toxicological effects of Cd and the therapeutic effects of metallothionein (MT) and vitamin E (VE) on the trunk kidney and blood of freshwater grass carp (Ctenopharyngodon idellus). Grass carp were divided into three groups: Cd + phosphate-buffered saline (PBS) group, Cd + VE group, and the Cd + MT group. Fish were injected with CdCl 2 on the first day and then VE, MT, or PBS was administered 4 days post-injection. Fish not injected with Cd were used as a negative control. The blood and trunk kidney amassed Cd and suffered severe damage in the forms of organ toxicity cytotoxicity, and immunotoxicity. However, the MT reduced the Cd content in the trunk kidney and blood and partially stabilized the damaged organs. Treatment with VE, however, only demonstrated weaker protection against on Cd-induced toxicity. The results indicate that exogenous MT may play an essential role in restoring homeostasis of the Cd-poisoned urinary and circulatory system and that it may help eliminate Cd in aquatic animals.

Background The aim of this study was to investigate the effects of selenium, zinc, insulin, and metallothionein on oxidative damage and metallothionein (MT) gene expression levels in streptozotocin (STZ)-induced type 1 diabetic rats exposed to Cd. Methods Rats were categorized under eight groups (control, STZ, Cd, STZ + Cd, Group 5, Group 6, Group 7, and STZ + Cd + MT [n:8/group]) were used. After diabetes was induced by STZ (55 mg/kg, i.p.), Cd was administered (1 mg/kg CdCl, orally) for 4 weeks. In cadmium-treated groups selenium (Na2SeO3 1.5 mg/kg, i.p.), zinc (ZnSO4 10 mg/kg via oral gavage), insulin (insulin glargine, 2U/day, s.c.), and MT (1mg/kg, every other 10 days, s.c.) were administered. MT gene expression levels, MDA levels, GPx, SOD, and CAT activity levels were determined in liver and kidney tissues. Results MT gene expression and MDA levels increased (p < 0.05) while GPx and SOD activity levels decreased (p < 0.05) in STZ, Cd, and STZ + Cd groups. In Group 5, Group 6, Group 7, and Group 8 groups MT gene expression and MDA levels were decreased while GPx and SOD activity levels were increased (p < 0.05). CAT activity significantly increased (p < 0.05) in STZ + Cd group while there were no significance in other groups (p > 0.05). Compared to the control, Group 5, Group 6, Group 7, and Group 8 groups provided no difference for alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen and creatinine levels (p > 0.05). Conclusions Our results suggest that Se, insulin, Zn and MT may have protective effects against hepatotoxicity and nephrotoxicity caused by Cd exposure in diabetic rats by reducing oxidative stress and MT gene expression levels.

Cadmium (Cd) pollution is an important issue affecting the food safety of aquatic products. Cd can impair the immune system and cause irreversible damage to fish and other aquatic organisms. The immunoprotection activities of exogenous metallothionein (MT) and vitamin E (VE) were investigated in Cd poisoned grass carp, Ctenopharyngodon idellus, in the present study. C. idellus were divided into three groups: Cd+phosphate-buffered saline (PBS) group; Cd+MT; and Cd+VE. All fish were injected with cadmium chloride (CdCl 2 ) on the first day and then treated with PBS, MT or VE four days post-injection. Fish not injected with Cd were used as a negative control. Cd exposure caused severe head-kidney and splenic injury in C. idellus, mainly expressed as an increase in Cd content, histological damage, percentage of head-kidney and splenic cells apoptosis and decreases in immune-related gene mRNA transcript expression. However, MT and VE treatments protected against Cd-induced immunotoxicity in C. idellus by decreasing Cd contents, lessening histological damage, reducing the percentage of apoptosis and recovering immune-related mRNA transcript expression. Our results demonstrate that MT and VE can alleviate Cd-induced immunotoxicity and that MT has a more powerful effect than VE, indicating that MT could be a potential antidote in cases of Cd poisoning., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Metallothioneins (MTs) is a metal ion binding protein to detoxify heavy metal stress in plant cells. This study examines involvement of MTs in metal chelation and ROS scavenging in rice seedling under Cr induction either Cr(VI) or Cr(III) at three different effective concentrations using Agilent 44K rice microarray and real-time PCR technology. Results showed that the concentration of Cr was higher in roots than in shoots in both Cr treatments. Accumulation of both H 2 O 2 and O 2 - in rice tissues was evident, but the fluctuation of H 2 O 2 was more remarkable than O 2 - . Both Cr exposures resulted in enhancement of MTs in plant tissues. Results from PCR analysis confirmed that ten specific OsMT genes responsible for regulating ROS removal were expressed differentially in plant tissues as well as in Cr variants, suggesting that their different regulation and responsiveness strategies. Expression patterns of metal chelation-related OsMT genes, after Cr exposure were also inconsistent in rice tissues. Longer exposure periods caused more transcriptional changes in both Cr treatments. We also noticed that OsMT1b might carry more weight during Cr chelation in roots rather than in shoots, while OsMT2c had more important role in eliminating H 2 O 2 accumulation in shoots than roots. These results suggest that different speciation of Cr in rice tissues resulted in inconsistent transcriptional changes of OsMT genes, which functioned in different regulation and responsiveness pathways responsible for metal ions chelating and ROS scavenging during Cr detoxification., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

It is now well accepted that an imbalance between the Th17 and regulatory T-cell responses is closely associated with the development of rheumatoid arthritis (RA). However, the precise regulatory mechanism for the differentiation of Th17 and Treg in RA is not well characterized. The present study showed that metallothionein-1 (MT-1), which is a low molecular weight protein that is involved in the detoxification of heavy metals and scavenging of free radicals, was upregulated in RA. Furthermore, the synovial inflammation and pathologic symptoms in collagen-induced arthritis and collagen antibody-induced arthritis mice were significantly suppressed when MT-1 was expressed intraarticularly. Further investigation revealed that MT-1 inhibited the differentiation of Th17 cells but enhanced that of Treg cells. Furthermore, it markedly decreased both STAT3 and RAR-related orphan receptor gamma t (RORγt) expression in vitro and in vivo. Collectively, our studies demonstrated that MT-1 might manifest as a protein involved in immunosuppression of RA pathogenesis by shifting Th17/Treg balance and may prove to be a potential therapeutic target for RA autoimmune diseases., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Metallothioneins (MTs) are small cysteine-rich proteins that play important roles in metal homeostasis and protection against heavy metal toxicity, DNA damage, and oxidative stress. In humans, MTs have four main isoforms (MT1, MT2, MT3, and MT4) that are encoded by genes located on chromosome 16q13. MT1 comprises eight known functional (sub)isoforms (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X). Emerging evidence shows that MTs play a pivotal role in tumor formation, progression, and drug resistance. However, the expression of MTs is not universal in all human tumors and may depend on the type and differentiation status of tumors, as well as other environmental stimuli or gene mutations. More importantly, the differential expression of particular MT isoforms can be utilized for tumor diagnosis and therapy. This review summarizes the recent knowledge on the functions and mechanisms of MTs in carcinogenesis and describes the differential expression and regulation of MT isoforms in various malignant tumors. The roles of MTs in tumor growth, differentiation, angiogenesis, metastasis, microenvironment remodeling, immune escape, and drug resistance are also discussed. Finally, this review highlights the potential of MTs as biomarkers for cancer diagnosis and prognosis and introduces some current applications of targeting MT isoforms in cancer therapy. The knowledge on the MTs may provide new insights for treating cancer and bring hope for the elimination of cancer.

An excess of reactive oxygen species (ROS) relative to the antioxidant capacity causes oxidative stress, which plays a role in the development of Parkinson's disease (PD). Because mitochondria are both sites of ROS generation and targets of ROS damage, the delivery of antioxidants to mitochondria might prevent or alleviate PD. To transduce the antioxidant protein human metallothionein 1A (hMT1A) into mitochondria, we computationally designed a cell-penetrating artificial mitochondria-targeting peptide (CAMP). The recombinant CAMP-conjugated hMT1A fusion protein (CAMP-hMT1A) successfully localized to the mitochondria. Treating a cell culture model of PD with CAMP-hMT1A restored tyrosine hydroxylase expression and mitochondrial activity and reduced ROS production. Furthermore, injection of CAMP-hMT1A into the brain of a mouse model of PD rescued movement impairment and dopaminergic neuronal degeneration. CAMP-hMT1A delivery into mitochondria might be therapeutic against PD by alleviating mitochondrial damage, and we predict that CAMP could be used to deliver other cargo proteins to the mitochondria.

As an environmental and industrial pollutant, cadmium (Cd) can cause a broad spectrum of toxicological effects. Multiple organs, especially the liver, are considerably affected by Cd in both humans and animals. We investigated the protective effects of metallothionein (MT) and vitamin E (VE) supplementation on Cd-induced apoptosis in the grass carp ( Ctenopharyngodon idellus ) liver. Grass carp were divided into four groups: the control group, Cd + phosphate-buffered saline (PBS) group, Cd + VE group, and Cd + MT group. All fish were injected with CdCl 2 on the first day and then VE, MT, and PBS were given 4 days postinjection, respectively. The results showed that Cd administration resulted in liver poisoning in grass carp, which was expressed as an increase in Cd contents, malondialdehyde (MDA) concentration, percentage of hepatocyte apoptosis, and apoptosis-related gene mRNA transcript expression. However, VE and MT treatments protected against Cd-induced hepatotoxicity in grass carp by decreasing Cd contents, lipid peroxidation, and histological damage and reducing the percentage of hepatocyte apoptosis by regulating related mRNA transcript expression. These data demonstrate that oxidative stress and activation of the caspase signaling cascade play a critical role in Cd-induced hepatotoxicity. However, VE and MT alleviate Cd-induced hepatotoxicity through their antioxidative and antiapoptotic effects, and MT has a more powerful effect than VE.

Background: The extracellular environment plays an important role in supporting the regeneration of axons after injury. Metallothionein-II (MTII) is a metal-binding protein known for its neuroprotective effect by directly stimulating the growth of axons after injury. Previous studies have shown that MTII also modulates the response of astrocytes and microglia after injury. However, a detailed analysis describing how MTII modulates the interaction between microglia and neurons is lacking., Methods: We introduced fluorescently labelled MTII into the cortex at the time of needlestick injury to investigate the cellular uptake of MTII using immunohistochemistry with antibodies against cell-type-specific markers. The role of MTII in modulating the effect of microglia on axon outgrowth following an inflammatory response is further investigated using a co-culture model involving primary rodent microglia pre-treated with TNFα and primary rodent cortical neurons. The axon lengths were assessed 24 h after the plating of the neurons onto treated microglia. We also utilised siRNA to knockdown the expression of LRP1, which allows us to investigate the role of LRP1 receptors in the MTII-mediated effect of microglia on axon outgrowth., Results: Fluorescently labelled MTII was found to be associated with neurons, astrocytes and microglia following injury in vivo. Microglia-neuron co-culture experiments demonstrated that exogenous MTII altered the response of microglia to TNFα. The neurons plated onto the TNFα-stimulated microglia pre-treated with MTII have shown a significantly longer axonal length compare to the TNFα-stimulated microglia without the MTII treatment. This suggested that MTII reduce cytokine-stimulated activation of microglia, which would ordinarily impair neurite outgrowth. This inhibitory effect of MTII on activated microglia was blocked by siRNA-mediated downregulation of LRP1 receptor expression in microglia, suggesting that MTII acts via the LRP1 receptor on microglia., Conclusions: This study demonstrates that exogenous MTII acts via the LRP1 receptor to alter the inflammatory response of microglia following TNFα stimulation, providing a more supportive environment for axon growth.

Metallothionein is cysteine-rich low molecular mass protein. The involvement of MT in many physiological and pathophysiological processes such as apoptosis, proliferation, angiogenesis, and the detoxification of heavy metals suggested participation of this protein in carcinogenesis and tumor therapy. Depending on the type of tissue and classification of carcinoma various it was observed relation between MT expression and tumor type, stage, grade, poor prognosis and body resistance to radiotherapy and chemotherapy. MT in tumor cell plays important role in defense mechanism against the effect of radiation by inhibiting the processes that lead to the apoptosis. A number of studies have shown an increased expression of MT in various human tumors of larynx, pancreas, kidney, uterus and breast, whereas lower MT expression was detected in liver tumors. Variable MT expression was detected in case of thyroid, prostate, lung, stomach and central nervous system tumors. Also MT plays crucial role in the cytostatics treatment. MT can bind cis-platinum compounds and removes them from the cells, which may lead to multidrug resistance. However, the same functions of MT protect against the negative effects of chemotherapeutic treatment. It is especially important in case of heart cells. Analysis of MT expression in tumor cells may be useful in choosing method of treatment. It is difficult to determine whether increased expression of MT is only a inducing factor of the development of the carcinogenesis, its malignances and multidrug resistance, or it is a factor inhibiting the induction and development of cancer.

Lou Gehrig's disease, a synonym of amyotrophic lateral sclerosis, is an adult-onset lethal neurodegenerative disorder. Irrespective of extensive efforts to elucidate the pathogenesis of the disease and searches for therapies, no favorable pharmacotherapeutic strategies have yet to be proposed. In a popular rodent model of ALS, G93A SOD1 strain of mouse, intracellular copper conditions were geared toward copper accumulation inside cells, resulting in an acceleration of oxidative stress and apoptotic process. Disruption of intracellular copper homeostasis was common to transgenic mice expressing human mutant SOD1s. In this review, the novel hypothesis that disruption of intracellular copper homeostasis could be involved in the development of the disease was introduced. Based upon the hypothesis, therapeutic outcomes of agents that are capable of correcting and/or modifying intracellular copper homeostasis are described. Administration of ammonium tetrathiomolybdate, a selective intracellular copper chelator, delayed onset, slowed progression, and prolonged survival of a rodent model of the disease (G93A SOD1 mice). Metallothionein is a low molecular weight, cysteine-rich, metal-binding cytoplasmic protein that has beneficial properties in detoxification of toxic heavy metals, homeostatic regulation of intracellular essential trace elements, including copper, antioxidant, and antiapoptotic roles. In animal experiments of the G93A SOD1 mice, an increase of metallothionein proteins by means of induction by exercise or dexamethasone, genetic overexpression, or intraperitoneal administration, all resulted in a preferable outcome. The therapeutic effects were not inferior to those of approved drugs for ALS in humans. These observations suggest that metallothionein could be worth investigating the therapeutic potential in clinical use., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The low-density lipoprotein receptor-related protein receptors 1 and 2 (LRP1 and LRP2) are emerging as important cell signaling mediators in modulating neuronal growth and repair. We examined whether LRP1 and LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were chemorepulsive: α-2-macroglobulin, tissue plasminogen activator, and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction toward a gradient of metallothionein II was calcium-dependent, required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fiber regeneration so that it was complete within 7 days compared with 14 days in saline-treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction and that they may form part of a signaling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signaling system that has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cardiotoxicity is a serious complication of anticancer therapy by anthracycline antibiotics. Except for intercalation into DNA/RNA structure, inhibition of DNA-topoisomerase and histone eviction from chromatin, the main mechanism of their action is iron-mediated formation of various forms of free radicals, which leads to irreversible damage to cancer cells. The most serious adverse effect of anthracyclines is, thus, cardiomyopathy leading to congestive heart failure, which is caused by the same mechanisms. Here, we briefly summarize the basic types of free radicals formed by anthracyclines and the main processes how to scavenge them. From these, the main attention is paid to metallothioneins. These low-molecular cysteine-rich proteins are introduced and their functions and properties are reviewed. Further, their role in detoxification of metals and drugs is discussed. Based on these beneficial roles, their use as a new therapeutic agent against oxidative stress and for cardioprotection is critically evaluated with respect to their ability to increase chemoresistance against some types of commonly used cytostatics.

Metallothionein-II (MT-II) is an ubiquitously expressed small-molecular-weight protein and highly induced in various species and tissues upon stress, inflammation, and ischemia. MT-deficiency exacerbates ischemic injury in rodent stroke models in vitro and in vivo. However, there is conflicting data on the potential neuroprotective effect of exogenously applied metallothionein. Thus, we applied MT-II in an in vitro stroke model and intraperitoneally (i.p.) in two in vivo standard models of transient middle cerebral artery occlusion (MCAO) (a 'stringent' one [60 min MCAO/48 h reperfusion] and a 'mild' one [30 min MCAO/72 h reperfusion]), as well as i.v. together with recombinant tissue plasminogen activator (rtPA) to evaluate if exogenous MT-II-application protects against ischemic stroke. Whereas MT-II did not protect against 60 min MCAO, there was a significant reduction of direct and indirect infarct volumes and neurological deficit in the MT-II (i.p.) treated animals in the 'mild' model at 3d after MCAO. Furthermore, MT-II also improved survival of the mice after MCAO, suppressed TNF-α mRNA induction in ischemic brain tissue, and protected primary neuronal cells against oxygen-glucose-deprivation in vitro. Thus, exogenous application of MT-II protects against ischemic injury in vitro and in vivo. However, long-term studies with different species and larger sampling sizes are required before a clinical use can be envisaged.

Sulforaphane (SFN) prevents diabetic nephropathy (DN) in type 1 diabetes via up-regulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, it has not been addressed whether SFN also prevents DN from type 2 diabetes or which Nrf2 downstream gene(s) play(s) the key role in SFN renal protection. Here we investigated whether Nrf2 is required for SFN protection against type 2 diabetes-induced DN and whether metallothionein (MT) is an Nrf2 downstream antioxidant using Nrf2 knockout (Nrf2-null) mice. In addition, MT knockout mice were used to further verify if MT is indispensable for SFN protection against DN. Diabetes-increased albuminuria, renal fibrosis, and inflammation were significantly prevented by SFN, and Nrf2 and MT expression was increased. However, SFN renal protection was completely lost in Nrf2-null diabetic mice, confirming the pivotal role of Nrf2 in SFN protection from type 2 diabetes-induced DN. Moreover, SFN failed to up-regulate MT in the absence of Nrf2, suggesting that MT is an Nrf2 downstream antioxidant. MT deletion resulted in a partial, but significant attenuation of SFN renal protection from type 2 diabetes, demonstrating a partial requirement for MT for SFN renal protection. Therefore, the present study demonstrates for the first time that as an Nrf2 downstream antioxidant, MT plays an important, though partial, role in mediating SFN renal protection from type 2 diabetes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aims: Endoplasmic reticulum (ER) stress exerts myocardial oxidative stress, apoptosis, and contractile anomalies, although the precise interplay between ER stress and apoptosis remains elusive. This study was designed to examine the impact of the cysteine-rich free radical scavenger metallothionein on ER stress-induced myocardial contractile defect and underlying mechanisms., Methods and Results: Wild-type friendly virus B and transgenic mice with cardiac-specific overexpression of metallothionein were challenged with the ER stress inducer tunicamycin (1 mg/kg, intraperitoneal, 48 h) prior to the assessment of myocardial function, oxidative stress, and apoptosis. Our results revealed that tunicamycin promoted cardiac remodeling (enlarged left ventricular end systolic/diastolic diameters with little changes in left ventricular wall thickness), suppressed fractional shortening and cardiomyocyte contractile function, elevated resting Ca(2+), decreased stimulated Ca(2+) release, prolonged intracellular Ca(2+) clearance, and downregulated sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, the effects of which were negated by metallothionein. Treatment with tunicamycin caused cardiomyocyte mitochondrial injury, as evidenced by decreased mitochondrial membrane potential (∆Ѱm, assessed by JC-1 staining), the effect of which was negated by the antioxidant. Moreover, tunicamycin challenge dramatically facilitated myocardial apoptosis as manifested by increased Bax, caspase 9, and caspase 12 protein levels, as well as elevated caspase 3 activity. Interestingly, metallothionein transgene significantly alleviated tunicamycin-induced myocardial apoptosis., Conclusion: Taken together, our data favor a beneficial effect of metallothionein against ER stress-induced cardiac dysfunction possibly associated with attenuation of myocardial apoptosis.

Background: Diabetic nephropathy (DN) is thought to be partially due to the injury of renal cells and the renal micro-environment by free radicals. Free radial scavenging agents that inhibit free radical damage may well prevent the development of underlying conditions such as mesangial expansion (by inhibiting extracellular matrix expression) in these patients., Methods: Using techniques for intra-cellular delivery of peptides, we made metallothionein (MT) and superoxide dismutase (SOD), potent endogenous antioxidants, readily transducible into cell membrane and tested their protective effect against the development of DN in OLETF rats. Herein, we study antioxidant peptides for their ability to prevent oxidative damage to primary rat mesangial cells (MCs), which are important constituents of renal glomeruli., Results: Intraperitoneal administration of these antioxidants resulted in delivery to the kidney and decreased ROS and the expression of downstream signals in renal cells and postponed the usual progression to DN. In in vitro experiments, MT and SOD were efficiently transferred to MCs, and the increased removal of ROS by MT and SOD was proportional to the degree of scavenging enzymes delivered. MT and SOD decreased three major oxidative injuries (hyperglycemia, AGE and ROS exposure) and also injuries directly mediated by angiotensin II in MCs while changing downstream signal transduction., Conclusions: The protective effects of MT and SOD for the progression of DN in experimental animals may be associated with the scavenging of ROS by MT and SOD and correlated changes in signal transduction downstream. Concomitant administration of these antioxidant peptides may prove to be a new approach for the prevention and therapy of DN.

Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

Alzheimer's disease (AD) is characterized by the deposition of amyloid-β (Aβ) peptide in the brains of AD patients. Such a process is linked to the binding of metal ions (e.g., Cu, Fe and Zn) with Aβ. As a result, metal chelation could be used as a rational therapeutic pathway for the treatment of AD. In this review, we address some noteworthy advances on the utilization of metal chelators, such as native metallothioneins and synthetic compounds, as potential therapeutic agents for AD. In addition, the future design and utility of metal chelating drugs as well as the strategy pursued to transport metal chelators into the brain are highlighted. We believe that this contribution will be valuable for the design of metal-chelating drugs for AD treatment.

After transient cerebral ischemia and reperfusion (I/R), damaging mechanisms, such as excitotoxicity and oxidative stress, lead to irreversible neurological deficits. The induction of metallothionein-II (MT-II) protein is an endogenous mechanism after I/R. Our aim was to evaluate the neuroprotective effect of MT-II after I/R in rats. Male Wistar rats were transiently occluded at the middle cerebral artery for 2 h, followed by reperfusion. Rats received either MT (10 μg per rat i.p.) or vehicle after ischemia. Lipid peroxidation (LP) was measured 22 h after reperfusion in frontal cortex and hippocampus; also, neurological deficit was evaluated after ischemia, using the Longa scoring scale. Infarction area was analyzed 72 hours after ischemia. Results showed increased LP in frontal cortex (30.7%) and hippocampus (26.4%), as compared to control group; this effect was fully reversed by MT treatment. Likewise, we also observed a diminished neurological deficit assessed by the Longa scale in those animals treated with MT compared to control group values. The MT-treated group showed a significant (P < 0.05) reduction of 39.9% in the infarction area, only at the level of hippocampus, as compared to control group. Results suggest that MT-II may be a novel neuroprotective treatment to prevent ischemia injury.

Ischemic heart diseases caused by insufficient oxygen supply to the cardiac muscle require pharmaceutical agents for the prevention of the progress and recurrence. Metallothionein (MT) has a potential as a protein therapeutic for the treatment of this disease due to its anti-oxidative effects under stressful conditions. In spite of its therapeutic potential, efficient delivery systems need to be developed to overcome limitations such as low transduction efficiency, instability and short half-life in the body. To enhance intra-cellular transduction efficiency, Tat sequence as a protein transduction domain (PTD) was fused with MT in a recombinant method. Anti-apoptotic and anti-oxidative effects of Tat-MT fusion protein were evaluated under hyperglycemia and hypoxia stress conditions in cultured H9c2 cells. Recovery of cardiac functions by anti-apoptotic and anti-fibrotic effects of Tat-MT was confirmed in an ischemia/reperfusion (I/R) rat myocardial infarction model. Tat-MT fusion protein effectively protected H9c2 cells under stressful conditions by reducing intracellular ROS production and inhibiting caspase-3 activation. Tat-MT fusion protein inhibited apoptosis, reduced fibrosis area and enhanced cardiac functions in I/R. Tat-MT fusion protein could be a promising therapeutic for the treatment of ischemic heart diseases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Zinc exerts a wide range of important biological roles. The present study was carried out to investigate the effects of zinc threoninate chelate in blood glucose levels, lipid peroxidation, activities of antioxidant defense systems and nitrite concentration, and histology of the pancreas in diabetic rats. Wistar rats were intravenously injected with a single dose of streptozotocin to induce diabetes. Then, diabetic rats were administrated orally with zinc threoninate chelate (3, 6, and 9 mg/kg body weight) once daily for 7 weeks. Fasting blood glucose was monitored weekly. At the end of the experimental period, the diabetic rats were killed, and levels of serum insulin, malondialdehyde, and nitric oxide, activities of glutathione peroxidase, total superoxide dismutase, copper/zinc-superoxide dismutase, and nitric oxide synthase were determined; pancreas was examined histopathologically as well. Zinc threoninate chelate significantly reduced the blood glucose levels and significantly increased the serum insulin levels in diabetic rats. In addition, zinc threoninate chelate caused a significant increase in activities of antioxidant enzymes and significant decrease in nitrite concentration and malondialdehyde formation in the pancreas and serum of diabetic rats. These biochemical observations were supplemented by histopathological examination of the pancreas. These results suggested that the antidiabetic effect of zinc threoninate chelate may be related to its antioxidative stress ability in diabetic rats.

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.

Metallothionein (MT) is a small molecular and multi-functional protein containing four atoms of copper (Cu) and three atoms of zinc (Zn) per molecule. It was isolated from the horse kidney in 1957 and half a century has passed since then. Although MT was found to work as a modulator of Zn and induce anti-oxidant reaction, the precise functions and its functional mechanisms remain to be elucidated. Over the years, a new isoform of MT, MT-III (also called growth inhibitory factor (GIF)), has been found in the brain, which was markedly diminished in the brain of Alzheimer's disease (AD). Many new findings on MT have been discovered in neurodegenerative diseases other than AD such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), prion disease, brain trauma, brain ischemia, and psychiatric diseases. In ALS in particular, MTs were markedly diminished in the spinal cord of patients with ALS. Initially, MT, which easily binds to cadmium (Cd) and copper (Cu), was considered to be toxic to our bodies. Molecular biological technologies enabled the production of recombinant MT saturated with zinc (Zn). MT has a high potential for the treatment of neurodegenerative diseases such as ALS, AD, and PD owing to its various functions including anti-oxidant properties and modulators not only for Zn but for Cu in the extra- and intracellular spaces. On the other hand, there are still various problems on MT to be elucidated in detail, including their binding proteins and functional mechanisms.

Objectives: Numerous studies indicate that certain genetic polymorphisms modify lead toxicokinetics. Metallothioneins are protective against the toxicity of many metals, including lead. The aim of this study was to determine whether the maternal metallothionein 2A (MT2A) -5 A/G single-nucleotide polymorphism is related to the lead levels in maternal blood, placental tissue and cord blood in 91 pregnant women and their newborns., Methods: Venous blood from the mother was collected to investigate lead levels and MT2A polymorphism. Cord blood and placenta were collected for lead levels. Analyses were made using an Atomic Absorption Graphite Furnace Spectrophotometer. Standard PCR-RFLP technique was used to determine MT2A polymorphism., Results: Blood lead levels of heterozygote genotype (AG) mothers were statistically higher than those of homozygote genotype (AA) (P < 0.05). Maternal lead levels were significantly associated with cord blood lead levels for pregnant women with AA genotype (P < 0.001). This association was not statistically significant for pregnant women with AG. In contrast, the mean value of cord blood lead level for newborns with mothers of AG genotype was slightly higher than others, though the difference was not significant. No significant difference existed in placenta lead levels between the groups., Conclusion: This study suggests that pregnant women with AG genotype for MT2A polymorphism might have high blood lead levels and their newborns may be at risk of low-level cord blood lead variation.

Burn injury can lead to abnormal sensory function at both the injury and at distant uninjured sites. Here, we used a mouse model to investigate return of nociceptive function and reinnervation of the skin at the wound and uninjured distant sites following a 3% total burn surface area full-thickness burn injury. We have previously shown that topical application of zinc-metallothionein-IIA (Zn(7) -MT-IIA) accelerates healing following burn injury, and here, we investigated the potential of Zn(7) -MT-IIA to enhance reinnervation and sensory recovery. In all burn-injured animals, there was a significant reduction in nociceptive responses (Semmes-Weinstein filaments) at locations near and distant to the wound up to 8 weeks following injury. Cutaneous nerve reinnervation (assessed using protein gene product 9.5 immunohistochemistry) of the wound center was slow in the epidermis but rapid in the dermis. In the dermis, nerves subsequently degenerated both at the wound center and in distant uninjured areas. In contrast, epidermal nerve densities in the distant uninjured areas returned to normal, uninjured levels. Zn(7) -MT-IIA did not influence return of nociceptive function nor reinnervation. We conclude that burn injury compromises nociceptive function and nerve regeneration both at the injury site and systemically; thus, therapies in addition to Zn(7) -MT-IIA should be explored to return normal sensory function., (© 2012 by the Wound Healing Society.)

After spinal cord injury (SCI), a complex cascade of pathophysiological processes rapidly damages the nervous tissue. The initial damage spreads to the surrounding tissue by different mechanisms, including oxidative stress. We have recently reported that the induction of metallothionein (MT) protein is an endogenous rapid-response mechanism after SCI. Since the participation of MT in neuroprotective processes after SCI is still unknown, the aim of the present study was to evaluate the possible neuroprotective effect of exogenously administered MT-II during the acute phase after SCI in rats. Female Wistar rats weighing 200-250g were submitted to spinal cord contusion by means of a computer-controlled device (NYU impactor). Rats received several doses of MT-II (3.2, 10 and 100μg) at 2 and 8h after SCI. Results of the BBB scale were statistically analysed using an ANOVA of repeated-measures, followed by Tukey's test. Among the three doses tested, only 10 and 100μg were able to significantly increase (p<0.05) BBB scale scores eight weeks after SCI from a mean of 7.88 in the control group, to means of 12.63 and 10.88 for the 10 and 100μg doses of MT-II, respectively. The amount of spared tissue was also higher in the groups treated with 10 and 100μg, as compared to the control group values. Results from the present study demonstrate a significant neuroprotective effect of exogenously administered MT-II. Further studies are needed in order to characterize the mechanisms involved in this neuroprotective action., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Prior studies have reported that metallothionein I/II (MT) promote regenerative axonal sprouting and neurite elongation of a variety of central nervous system neurons after injury. In this study, we evaluated whether MT is capable of modulating regenerative axon outgrowth of neurons from the peripheral nervous system. The effect of MT was firstly investigated in dorsal root ganglion (DRG) explants, where axons were scratch-injured in the presence or absence of exogenous MT. The application of MT led to a significant increase in regenerative sprouting of neurons 16 h after injury. We show that the pro-regenerative effect of MT involves an interaction with the low-density lipoprotein receptor megalin, which could be blocked using the competitive antagonist RAP. Pre-treatment with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 also completely abrogated the effect of exogenous MT in promoting axonal outgrowth. Interestingly, we only observed megalin expression in neuronal soma and not axons in the DRG explants. To investigate this matter, an in vitro injury model was established using Campenot chambers, which allowed the application of MT selectively into either the axonal or cell body compartments after scratch injury was performed to axons. At 16 h after injury, regenerating axons were significantly longer only when exogenous MT was applied solely to the soma compartment, in accordance with the localized expression of megalin in neuronal cell bodies. This study provides a clear indication that MT promotes axonal regeneration of DRG neurons, via a megalin- and MAPK-dependent mechanism., (© Springer Basel AG 2011)

Since the seminal discoveries of Bert Vallee regarding zinc and metallothioneins (MTs) more than 50 years ago, thousands of studies have been published concerning this fascinating story. One of the most active areas of research is the involvement of these proteins in the inflammatory response in general, and in neuroinflammation in particular. We describe the general aspects of the inflammatory response, highlighting the essential role of the major cytokine interleukin-6, and review briefly the expression and function of MTs in the central nervous system in the context of neuroinflammation. Particular attention is paid to the Tg2576 Alzheimer disease mouse model and the preliminary results obtained in mice into which human Zn(7)MT-2A was injected, which suggest a reversal of the behavioral deficits while enhancing amyloid plaque load and gliosis.

Objective: [corrected] To assess the effects of metallothionein on myocyte apoptosis and energy supply of isolated rabbit heart muscle during perfusion with ropivacaine.., Methods: Sixty New Zealand white male rabbits were randomized into 3 equal groups. In group I, the rabbits received a intreaperitioneal injection of distilled water 24 h before isolation of the heart with perfusion by Langendoff model; in group II, distilled water was injected intreaperitioneally, and 24 h later the heart was isolated and perfused with Langendoff model and ropivacaine; in group III, 3.6% ZnSO(4) was injected intreaperitioneally and the isolated heart was perfused with Langendoff model and ropivacaine. The myocardial metallothionein content, myocyte apoptosis, and myocardial ATP, ADP and AMP content were detected., Results: The myocardial metallothionein content was significantly higher in group III than in the other two groups; the percent of myocyte apoptosis was the highest in group II, and was significantly higher in group III than in group I. The myocardial content of ATP was the highest in group I, and was significantly higher in group III than in group II., Conclusion: Metallothionein can significantly inhibit myocyte apoptosis and alleviate energy supply disorder induced by ropivacaine.

Objective: Metallothionein 3 (MT-3) has been shown to protect against apoptotic neuronal death in the brains of patients with Alzheimer's disease. Zinc is a potent inhibitor of caspase-3 and its deficiency was found to promote apoptosis. Here, we measured the zinc and copper content in the brains of senescence-accelerated mouse/PRONE8 (SAMP8) and sought to investigate the effect of MT-3 on the apoptosis of neurons in the hippocampal CA1 region of these mice., Method: The zinc and copper content in the brain samples of SAMP8 and normal control SAMR1 mice were determined using an atomic absorption spectrophotometer. The mice were administered intraperitoneally for four weeks with MT-3 or MT1 and thereafter apoptosis was measured using the TUNEL method and the expression of anti-apoptotic protein Bcl-2 and proapoptotic protein Bax was examined by immunohistochemistry., Results: Compared with that in SMAR1 mice, the content of zinc in the brains of SAMP8 mice was significantly reduced (P<0.05). Moreover, significant levels of apoptosis of neurons were observed in the hippocampus of SAMP8 mice, which, compared with those in SMAR1 mice, also showed significantly lower levels of Bcl-2 and higher levels of Bax (P<0.05). MT-3 increased zinc concentration in the hippocampus of SAMP8 mice and also significantly decreased apoptosis in these neurons dose-dependently and increased the levels of Bcl-2 and decreased the levels of Bax., Conclusion: MT-3 could attenuate apoptotic neuron death in the hippocampus of SAMP8, suggesting that the protein may lessen the development of neurodegeneration.

Background: A major pathological hallmark of AD is the deposition of insoluble extracellular beta-amyloid (Abeta) plaques. There are compelling data suggesting that Abeta aggregation is catalysed by reaction with the metals zinc and copper., Methodology/principal Findings: We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Abeta1-40 and Abeta1-42. This action of MT-2A appears to involve a metal-swap between Zn7MT-2A and Cu(II)-Abeta, since neither Cu10MT-2A or carboxymethylated MT-2A blocked Cu(II)-Abeta aggregation. Furthermore, Zn7MT-2A blocked Cu(II)-Abeta induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons., Conclusions/significance: These results indicate that MTs of the type represented by MT-2A are capable of protecting against Abeta aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Abeta leaving a metal-free Abeta that can readily bind metals again, we believe that MT-2A might represent a different therapeutic approach as the metal exchange between MT and Abeta leaves the Abeta in a Zn-bound, relatively inert form.

Objective: To investigate the effect of metallothionein (MT) on rifampicin (RFP)-induced hepatotoxicity and the possible mechanisms., Methods: Male MT- I / II null (MT-/-) and wild type (MT+/+) mice were randomly divided into 4 groups, respectively, and were orally administered RFP (50, 100 or 200 mg/kg) or equal volumes of solvent daily for 15 consecutive days. Levels of plasma alanine aminotransferase (ALT), alkaline phosphatase (ALP) and direct bilirubin (DB) were determined. Liver indexes were calculated and liver histopathologic changes were examined by hematoxylin and eosin (HE) staining. The content of glutathione (GSH) as well as the activities of glutathione peroxydase (GPx) and glutathione reductases (GR) were measured in the liver homogenates., Results: RFP treatment induced significant increases in plasma ALT, AST and DB, as well as liver index. Significant histopathologic changes which were charactered as fatty degeneration in liver were noteced. Moreover, augmentations of GSH content and GR activity and attenuation of GPx activity were observed. More severe hepatic injuries in MT-/- mice were observed as compared to MT+/+ mice, which were evidenced by higher liver/body weight ratio and GR activity, lower GSH content and GPx activity, and more serious fatty degeneration., Conclusion: RFP-induced hepatotoxicity was associated with cholestasis and oxidative damage. MT -/- mice were more susceptible than MT +/+ mice to RFP-induced hepatotoxicity and the enhanced hepatotoxicity involves increased oxidative stress.

Objective: To evaluate the protective effects of N(G)-nitro-L-arginine methyl ester (L-NAME) and metallothioneins on excess nitric oxide toxicity in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis., Study Design: In this study, 70 rats were assigned to 7 groups of controls, and colitis was induced with 120 mg/kg TNBS, 35 mg/kg L-NAME, and 1 and 2 mg/kg metallothionein 1 (MT1) and metallothionein 2 (MT2), respectively. A day after the administration of TNBS, L-NAME, MT1 and MT2 were given intraperitoneally for 3 days to the experimental groups. After the administration of TNBS, dissections of the rats in the L-NAME, MT1 and MT2 groups were performed at 3-day periods under ether anesthesia, and whole blood, bone marrow and colon were obtained., Results: On the third day, red and white blood cell values were increased, while platelet and bone marrow granule cells decreased in the L-NAME- and TNBS-induced group. On the third day, all the blood values increased in MT1 (1 and 2 mg/kg) and MT2 (1 and 2 mg/kg) in the TNBS-administered groups. Histologic findings were macroscopic score, ulcer, loss of mucous cells, crypt abscess, inflammatory cyst, mucosa atrophy, edema, vascular dilatation and induced nitric oxide synthetase, which increased in the descending colon in the colitis rats, while it was decreased rats given L-NAME, MT1 and MT2 administration., Conclusion: The results suggest that MT1 and MT2 are more effective in protecting against the toxic effects of excess nitric oxide as compared with L-NAME in the colitis rats.

Metallothionein (MT) is a low-molecular weight sulfur-rich protein that plays role in metal homeostasis/detoxification and radical scavenging. The following study investigated the ability of exogenous MT to protect against oxidative damage induced by thallium (TI) in rat liver. Male Wistar rats were divided into four groups; a control and three experimental groups. The control group received physiological saline. Group 1 animals were injected with thallium acetate intraperitoneally (i.p.) at a single dose of LD(50) (32 mg/kg). In group 2 and group 3, metallothionein I was administrated once at two different doses (1 or 2.5mg/kg i.p., respectively) 1h before TI intoxication. Levels of endogenous antioxidants, oxidative stress markers were measured and histopathological examinations were performed 4 days after TI administration. TI accumulation in liver decreased related to the dose of MT. Mostly all of the alterations in the levels antioxidants restored to normal levels in MT administrated animals. H(2)O(2) levels and lipid peroxidation decreased, integrity of hepatocytes and membranous structures inside the cells were preserved. The toxic effects of TI were modulated in MT administrated animals particularly at the dose of 2.5mg/kg. These findings suggest an active role of exogenous MT against TI-induced oxidative stress in rat liver., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the US. Metallothionein (MT) expression attenuates APAP-induced liver injury. However, the mechanism of this protection remains incompletely understood. To address this issue, C57BL/6 mice were treated with 100 micromol/kg ZnCl2 for 3 days to induce MT. Twenty-four hours after the last dose of zinc, the animals received 300 mg/kg APAP. Liver injury (plasma ALT activities, area of necrosis), DNA fragmentation, peroxynitrite formation (nitrotyrosine staining), MT expression, hepatic glutathione (GSH), and glutathione disulfide (GSSG) levels were determined after 6 h. APAP alone caused severe liver injury with oxidant stress (increased GSSG levels), peroxynitrite formation, and DNA fragmentation, all of which were attenuated by zinc-induced MT expression. In contrast, MT knockout mice were not protected by zinc. Hydrogen peroxide-induced cell injury in primary hepatocytes was dependent only on the intracellular GSH levels but not on MT expression. Thus, the protective effect of MT in vivo was not due to the direct scavenging of reactive oxygen species. Zinc treatment had no effect on the early GSH depletion kinetics after APAP administration, which is an indicator of the metabolic activation of APAP to its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). However, MT was able to effectively trap NAPQI by covalent binding. We conclude that MT scavenges some of the excess NAPQI after GSH depletion and prevents covalent binding to cellular proteins, which is the trigger for the propagation of the cell injury mechanisms through mitochondrial dysfunction and nuclear DNA damage.

Following central nervous system injury, astrocytes rapidly respond by undergoing a stereotypical pattern of molecular and morphological alterations termed "reactive" astrogliosis. We have reported previously that metallothioneins (MTs) are rapidly expressed by reactive astrocytes and that their secretion and subsequent interaction with injured neurons leads to improved neuroregeneration. We now demonstrate that exogenous MT induces a reactive morphology and elevated GFAP expression in cultured astrocytes. Furthermore, these astrogliotic hallmarks were mediated via JAK/STAT and RhoA signalling pathways. However, rather than being inhibitory, MT induced a form of astrogliosis that was permissive to neurite outgrowth and which was associated with decreased chondroitin sulphate proteoglycan (CSPG) expression. The results suggest that MT has an important role in mediating permissive astrocytic responses to traumatic brain injury.

Apoptotic cell death was found to play a critical role in the development of diabetic cardiomyopathy. As one of pathogenic components of diabetes angiotensin II (Ang II) induced cardiac cell death in vitro and in vivo through induction of reactive oxygen and nitrogen species. However, Ang II-induced cell death signaling in the heart remains unclear. The present study was to investigate whether Ang II induces p53 expression and activation and if so, whether Ang II-induced cardiac cell death is p53-dependent, and whether a potent antioxidant metallothionein (MT) prevents Ang II-induced p53 expression, and associate apoptotic cell death signaling. A cardiac cell line (H9c2) was exposed to Ang II. We found that exposure of H9c2 cells to Ang II at 10, 50 and 100 nM for 24 h induced a significant apoptotic effect, measured by DNA fragmentation and cleaved caspase-3. Induction of apoptotic cell death by Ang II can be completely blocked by p53 inhibitor Pitithrin-alpha. Exposure of H9c2 cells to Ang II also significantly increased p53 phosphorylation, DNA double strand breaks and Bax/Bcl-2 ratio. All these effects were not observed in H9c2MT7 cells that forcedly overexpresses human MT-IIA gene, suggesting the preventive effect of antioxidant MT against Ang II-induced p53 activation and its apoptotic death signaling. Furthermore, the in vitro finding was validated in animal models by supplying Ang II to wild-type mice (WT) and MT-TG mice that has cardiac-specifically overexpressed MT gene. Ang II-induced significant up-regulation of p53 expression along with an increase in Bax/Bcl-2 ratio in the hearts of WT mice, but not MT-TG mice. These results suggest that Ang II-induced cardiac apoptotic cell death is mediated by p53 apoptotic signaling pathway, which is related to oxidative stress. Antioxidant MT can completely prevent Ang II-induced p53 activation and associated apoptotic effect in the heart.

Metallothionein (MT) belongs to a family of metal-binding cysteine-rich proteins comprising several structurally related proteins implicated in tissue protection and regeneration after injuries and functioning as antiapoptotic antioxidants in neurological disorders. This has been demonstrated in animals receiving MT treatment and in mice with endogenous MT overexpression or null mutation during various experimental models of neuropathology, and also in patients with Alzheimer's disease and amyotrophic lateral sclerosis. Exogenously applied MT increases neurite outgrowth and neuronal survival in rat cerebellar, hippocampal, dopaminergic, and cortical neurons in vitro. In this study, the intraneuronal signaling involved in MT-mediated neuritogenesis was examined. The MT-induced neurite outgrowth in cultures of cerebellar granule neurons was dependent on activation of a heterotrimeric G-protein-coupled pathway but not on protein tyrosine kinases or on receptor tyrosine kinases. Activation of phospholipase C was necessary for MT-induced neurite outgrowth, and furthermore it was shown that inhibition of several intracellular protein kinases, such as protein kinase A, protein kinase C, phosphatidylinositol 3-kinase, Ca(2+)/calmodulin kinase-II, and mitogen-activated protein kinase kinase, abrogated the MT-mediated neuritogenic response. In addition, exogenously applied MT resulted in a decrease in phosphorylation of intraneuronal kinases implicated in proinflammatory reactions and apoptotic cell death, such as glycogen synthase-serine kinase 3alpha, Jun, and signal transducer and activator of transcription 3. This paper elucidates the intraneuronal molecular signaling involved in neuroprotective effects of MT., (2009 Wiley-Liss, Inc.)

Objective: Glycogen synthase kinase (GSK)-3beta plays an important role in cardiomyopathies. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice were highly resistant to diabetes-induced cardiomyopathy. Therefore, we investigated whether metallothionein cardiac protection against diabetes is mediated by inactivation of GSK-3beta., Research Design and Methods: Diabetes was induced with streptozotocin in both MT-TG and wild-type mice. Changes of energy metabolism-related molecules, lipid accumulation, inflammation, nitrosative damage, and fibrotic remodeling were examined in the hearts of diabetic mice 2 weeks, 2 months, and 5 months after the onset of diabetes with Western blotting, RT-PCR, and immunohistochemical assays., Results: Activation (dephosphorylation) of GSK-3beta was evidenced in the hearts of wild-type diabetic mice but not MT-TG diabetic mice. Correspondingly, cardiac glycogen synthase phosphorylation, hexokinase II, PPARalpha, and PGC-1alpha expression, which mediate glucose and lipid metabolisms, were significantly changed along with cardiac lipid accumulation, inflammation (TNF-alpha, plasminogen activator inhibitor 1 [PAI-1], and intracellular adhesion molecule 1 [ICAM-1]), nitrosative damage (3-nitrotyrosin accumulation), and fibrosis in the wild-type diabetic mice. The above pathological changes were completely prevented either by cardiac metallothionein in the MT-TG diabetic mice or by inhibition of GSK-3beta activity in the wild-type diabetic mice with a GSK-3beta-specific inhibitor., Conclusions: These results suggest that activation of GSK-3beta plays a critical role in diabetes-related changes in cardiac energy metabolism, inflammation, nitrosative damage, and remodeling. Metallothionein inactivation of GSK-3beta plays a critical role in preventing diabetic cardiomyopathy.

The kynurenine pathway has been implicated as a major component of the neuroinflammatory response to brain injury and neurodegeneration. We found that the neurotoxic kynurenine pathway intermediate quinolinic acid (QUIN) is rapidly expressed, within 24 h, by reactive microglia following traumatic injury to the rodent neocortex. Furthermore, administration of the astrocytic protein metallothionein attenuated this neuroinflammatory response by reducing microglial activation (by approximately 30%) and QUIN expression. The suppressive effect of MT was confirmed upon cultured cortical microglia, with 1 mug/ml MT almost completely blocking interferon-gamma induced activation of microglia and QUIN expression. These results demonstrate the neuroimmunomodulatory properties of MT, which may have therapeutic applications for the treatment of traumatic brain injury.

The present study was carried out to evaluate the effect of exogenously administered metallothionein (MT) against carmustine (BCNU)-induced lung toxicity in rats. A total of 60 rats were randomly divided into four groups (15/group): control group in which the animals received 0.5 ml physiologic saline containing 10% ethanol (IP) weekly, MT-administered group in which rats received MT (30 micromol/kg, IP) weekly, BCNU-administered group in which rats received BCNU (5 mg/kg, IP) weekly and MT + BCNU group in which rats received weekly doses of BCNU (5 mg/kg, IP) followed 24 h later by MT (30 micromol/kg, IP). At the end of the experiment (after 6 weeks), lung histological changes, collagen staining, the activity of glutathione reductase (GR) and contents of reduced glutathione (GSH) and hydroxyproline (Hpr) in the lung as well as serum level of tumor necrosis factor-alpha (TNF-alpha) were evaluated. The obtained data revealed that BCNU induced pathological changes and markedly increased lung collagen and level of Hpr but decreased GSH content and GR activity and increased serum TNF-alpha compared to both control and MT-administered rats. Administration of MT + BCNU markedly improved histological features and decreased staining of collagen along with increased GR activity, GSH content but decreased level of Hpr in lung tissue as well as decreased serum level of TNF-alpha compared with BCNU-treated rats. Based on our results, it is possible to postulate that exogenous MT can act against BCNU-induced lung toxicity by a mechanism related, at least in part, to its ability to decrease oxidative stress and fibrosis.