metallothionein and Disease-Models--Animal

Impaired zinc homeostasis is observed in diabetes mellitus (DM2) and its complications. Zinc has a specific role in pancreatic β-cells via insulin synthesis, storage, and secretion. Intracellular zinc homeostasis is tightly controlled by zinc transporters (ZnT and Zip families) and metallothioneins (MT) which modulate the uptake, storage, and distribution of zinc. Several investigations in animal models demonstrate the protective role of MT in DM2 and its cardiovascular or renal complications, while a copious literature shows that a common polymorphism (R325W) in ZnT8, which affects the protein's zinc transport activity, is associated with increased DM2 risk. Emerging studies highlight a role of other zinc transporters in β-cell function, suggesting that targeting them could make a possible contribution in managing the hyperglycemia in diabetic patients. This article summarizes the current findings concerning the role of zinc homeostasis in DM2 pathogenesis and development of diabetic cardiomyopathy and nephropathy and suggests novel therapeutic targets. © 2017 BioFactors, 43(6):770-784, 2017.

Topics: Animals; Biological Transport; Diabetes Mellitus, Type 2; Diabetic Cardiomyopathies; Diabetic Nephropathies; Dietary Supplements; Disease Models, Animal; Gene Expression Regulation; Homeostasis; Humans; Insulin; Insulin-Secreting Cells; Metallothionein; Zinc; Zinc Transporter 8

Inflammatory bowel diseases (IBDs) are a group of chronic, relapsing, immune-mediated disorders of the intestine, including Crohn's disease and ulcerative colitis. Recent studies underscore the importance of the damaged epithelial barrier and the dysregulated innate immune system in their pathogenesis. Metallothioneins (MTs) are a family of small proteins with a high and conserved cysteine content that are rapidly upregulated in response to an inflammatory stimulus. Herein, we review the current knowledge regarding the expression and potential role of MTs in IBD. MTs exert a central position in zinc homeostasis, modulate the activation of the transcription factor nuclear factor (NF)-kappaB, and serve as antioxidants. In addition, MTs could be involved in IBD through their antiapoptotic effects or through specific immunomodulating extracellular effects. Reports on MT expression in IBD are contradictory but clearly demonstrate a deviant MT expression supporting the idea that these aberrations in IBD require further clarification.

Topics: Animals; Colitis; Disease Models, Animal; Humans; Inflammatory Bowel Diseases; Metallothionein; Models, Biological

Oxidative stress is considered to be the main cause for several chronic diseases including diabetes. Through hyperglycemia, hyperlipidemia, hypertension and possible iron dyshomeostasis, diabetes induces oxidative stress that causes damage to multiple organs, leading to various complications. Therefore, antioxidant therapy may be an interesting approach to prevent diabetes and diabetic complications. Metallothionein as a potent antioxidant was found to significantly protect heart and kidney against diabetes-induced pathophysiological changes. Zinc as an important trace element and a metallothionein inducer was found to have same protective function. Since diabetes would impair defensive system, including growth factor reduction, exogenous supplementation of fibroblast growth factor (FGF) significantly prevented diabetes-induced cardiac oxidative damage and wound healing impairment. These studies suggest that protective agents such as metallothionein, zinc and FGFs play an important role in preventing the development of diabetes and diabetic complications.

Topics: Animals; Antioxidants; Diabetes Complications; Diabetes Mellitus; Disease Models, Animal; Fibroblast Growth Factors; Humans; Iron Overload; Metallothionein; Oxidative Stress; Trace Elements; Zinc

Parkinson's disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta, and in other sub-cortical nuclei associated with a widespread occurrence of Lewy bodies. The cause of cell death in Parkinson's disease is still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative and nitrative stresses have been proposed. We have studied control(wt) (C57B1/6), metallothionein transgenic (MTtrans), metallothionein double gene knock (MTdko), alpha-synuclein knock out (alpha-syn(ko)), alpha-synuclein-metallothionein triple knock out (alpha-syn-MTtko), weaver mutant (wv/wv) mice, and Ames dwarf mice to examine the role of peroxynitrite in the etiopathogenesis of Parkinson's disease and aging. Although MTdko mice were genetically susceptible to 1, methyl, 4-phenyl, 1,2,3,6-tetrahydropyridine (MPTP) Parkinsonism, they did not exhibit any overt clinical symptoms of neurodegeneration and gross neuropathological changes as observed in wv/wv mice. Progressive neurodegenerative changes were associated with typical Parkinsonism in wv/wv mice. Neurodegenerative changes in wv/wv mice were observed primarily in the striatum, hippocampus and cerebellum. Various hallmarks of apoptosis including caspase-3, TNFalpha, NFkappaB, metallothioneins (MT-1, 2) and complex-1 nitration were increased; whereas glutathione, complex-1, ATP, and Ser(40)-phosphorylation of tyrosine hydroxylase, and striatal 18F-DOPA uptake were reduced in wv/wv mice as compared to other experimental genotypes. Striatal neurons of wv/wv mice exhibited age-dependent increase in dense cored intra-neuronal inclusions, cellular aggregation, proto-oncogenes (c-fos, c-jun, caspase-3, and GAPDH) induction, inter-nucleosomal DNA fragmentation, and neuro-apoptosis. MTtrans and alpha-Syn(ko) mice were genetically resistant to MPTP-Parkinsonism and Ames dwarf mice possessed significantly higher concentrations of striatal coenzyme Q10 and metallothioneins (MT 1, 2) and lived almost 2.5 times longer as compared to control(wt) mice. A potent peroxynitrite ion generator, 3-morpholinosydnonimine (SIN-1)-induced apoptosis was significantly attenuated in MTtrans fetal stem cells. These data are interpreted to suggest that peroxynitrite ions are involved in the etiopathogenesis of Parkinson's disease, and metallothionein-mediated coenzyme Q10 synthesis may provide neuroprotection.

Topics: alpha-Synuclein; Animals; Apoptosis; Brain; Coenzymes; Disease Models, Animal; Dopamine; Gene Expression Regulation; Humans; Metallothionein; Mice; Mice, Neurologic Mutants; Mice, Transgenic; MPTP Poisoning; Nerve Tissue Proteins; Parkinson Disease; Synucleins; Ubiquinone

Molecular and cellular studies have demonstrated several roles for zinc (Zn) in insulin production and the consequent actions of insulin on metabolism. Clinical and epidemiological studies suggest that reduced Zn status is associated with diabetes. Investigations of Zn in rodent models of diabetes have provided a valuable link for understanding the molecular, cellular, clinical and epidemiological observations in the context of inter-organ metabolism and the metabolic disturbances of diabetes. This review highlights some of the current knowledge and future research directions for the role of Zn in the pancreas and diabetes based on rodent studies and experimental manipulations of Zn. Overall, Zn supplementation is effective for preventing or ameliorating diabetes in several rodent models of Type 1 and Type 2 diabetes. Studies with chemically-induced Type 1 diabetes indicate that the protective effects of Zn involve antioxidant mechanisms whether it is Zn alone (as an antioxidant), Zn induction of metallothionein or Zn inhibition of redox-sensitive transcription factors. Further studies are needed to identify the mechanism(s) for Zn protection in Type 2 diabetes, including pancreatic and peripheral effects. Experimental manipulations of Zn status in rodent models of diabetes provide a valuable approach to explore mechanisms for the protective effects of Zn; however, long term clinical studies establishing safety (lack of toxicity) and efficacy are required before any recommendations can be made for people with diabetes.

Topics: Animals; Antioxidants; Diabetes Mellitus; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Disease Models, Animal; Humans; Insulin; Insulin-Secreting Cells; Metallothionein; Pancreas; Rats; Streptozocin; Zinc

In many research groups including our laboratory, metallothionein (MT)-I/II null mice have been used to clarify the biological function and physiological role of MT. Recent studies with MT-I/II null mice concerning the role of MT in the toxicity and distribution of metal, oxidative stress and chemical carcinogenesis were reviewed. Some reports, including our findings, showed that MT-I/II null mice have an increased sensitivity to harmful metals such as cadmium, mercury, zinc and arsenic. Moreover, it was clarified using MT-I/II null mice that MT plays a major role in the retention of cadmium, mercury and zinc in target tissues. MT-I/II null mice were found to be much more sensitive than wild-type mice to the toxicity caused by free radical-inducing factors, which include paraquat, acetaminophen, ethanol, X-ray, ultraviolet B, carbon tetrachloride, cisplatin, doxorubicin, cerulein and streptozotocin. In addition, MT-I/II null mice were highly susceptible to skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene and bladder carcinogenesis caused by N-butyl-N-(4-hydroxybutyl)nitrosamine. These results suggest that MT is an important protective factor against metal toxicity, oxidative stress and chemical carcinogenesis.

Topics: Animals; Carcinogens, Environmental; Disease Models, Animal; Environmental Pollutants; Metallothionein; Mice; Mice, Inbred Strains; Oxidative Stress

Treatment of patients with acute pancreatitis has greatly improved due to a better understanding of the pathophysiology of the disease. This pathophysiology includes the activation and release of pancreatic enzymes in the interstitium, the autodigestion of the pancreas, and a multiple organ dysfunction after their release into the systemic circulation. Moreover, significant evidence exists that synthesis and release of proinflammatory cytokines and chemokines are also responsible for the local injury and systemic dispersion of the inflammation. The use of knockout mice devoid of active pro- or anti-inflammatory mediators allows examination of the effects of a specific cytokine without any drawbacks induced by pharmacological manipulations. The results obtained from these genetically modified mice show that numerous mediators have a major role in the pathophysiology of acute pancreatitis. They also clearly demonstrate that a single genetic deletion cannot completely prevent the occurrence of pancreatic or distant organ injury. However, the fact that the immune system is characterized by redundancies of ligands and receptors complicates the full understanding of each report. The utility of such experimental models might have limitations, and a full extrapolation of experimental data from genetically modified mice to humans must be done with caution.

Topics: Acute Disease; Animals; Cathepsin B; Cytokines; Disease Models, Animal; Intercellular Adhesion Molecule-1; Metallothionein; Mice; Mice, Knockout; Pancreatitis; Receptors, CCR1; Receptors, Chemokine; Receptors, Neurokinin-1; Superoxide Dismutase

Copper (Cu) is an essential trace element and constitutes the active center of the redox Cu enzymes such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD), ceruloplasmin and cytochrome c oxidase. Among hereditary diseases due to a defect in the metabolism of Cu, Menkes disease (caused by a Cu deficiency) and Wilson disease (caused by the excessive accumulation of Cu) have been shown to be caused by the mutation of genes encoding Cu-binding ATPase for the efflux of Cu, ATP7A and ATP7B, respectively. Following the identification of these causative genes, intracellular Cu transporters (Cu chaperones) specific for the Golgi apparatus, mitochondria and Cu, Zn-SOD were discovered, and these findings have facilitated the study of the underlying mechanisms of the biological regulation of Cu. Apart from these physiological and biochemical studies, toxicological studies have elucidated the underlying mechanisms of the occurrence of acute hepatitis caused by the accumulation of Cu accumulating in the liver of an animal model for Wilson disease, LEC rats. In these toxicological studies, two biological aspects of metallothionein (MT), i.e., antioxidant and prooxidant depending on the Cu/Zn ratio in Cu-containing MT have been proposed. The present article overviews the recent findings on the biological regulation of Cu and on the toxicological aspect of Cu. It is known that Cu forms a stable ternary complex with molybdenum and sulfur under reductive conditions in the body. On the basis of this observation, tetrathiomolybdate (TTM) has been applied to remove Cu from the liver of Long-Evans rats with a cinnamon-like coat color (LEC) rats. Precise mechanisms underlying the complex formation between Cu bound to MT and TTM were presented, and an appropriate protocol for the chelation therapy was also proposed together with the mechanisms underlying the occurrence of side-effects.

Topics: Adenosine Triphosphatases; Animals; Carrier Proteins; Cation Transport Proteins; Chelating Agents; Copper; Copper-Transporting ATPases; Disease Models, Animal; Hepatolenticular Degeneration; Humans; Liver; Metallothionein; Molybdenum; Rats; Rats, Inbred LEC; Recombinant Fusion Proteins

Disordered copper (Cu) metabolism in LEC rats, an animal model of Wilson disease, was characterized by specifying Cu in the liver, bloodstream and kidneys during the accumulation process and at the onset of jaundice; Cu accumulates in the liver with age in a form bound to metallothionein (MT). Massive Cu is liberated from MT when Cu accumulates beyond the capacity of MT synthesis. Cu bound to MT is not supplied to ceruloplasmin (Cp) during its maturation process, while the metal is transferred to Cu,zinc (Zn)-superoxide dismutase (SOD) directly from MT. Cu ions not bound to MT are transferred to Cp and the holo-Cp is excreted into the bloodstream near and at the onset of jaundice. Cu accumulated in the liver in a form bound to MT is removed selectively by tetrathiomolybdate (TTM), and the animal at the beginning of the onset of jaundice recovers by the chelation therapy. Mechanisms of the removal reaction were proposed to involve formation of MT/TTM, Cu/TTM, and/or polymeric Cu/TTM complexes according to the stoichiometry of TTM to Cu. The Cu/TTM complex was assumed to be effluxed into the bloodstream and to bind specifically to albumin. TTM is taken up by the liver in accordance with the Cu content. The toxicity of Cu was explained by the active oxygen species produced in Cu-mediated reactions, and the participation of MT.

Topics: Animals; Copper; Disease Models, Animal; Hepatolenticular Degeneration; Metallothionein; Rats; Rats, Mutant Strains

Topics: Acrodermatitis; Cadmium; Chromium; Copper; Disease; Disease Models, Animal; Growth; Hemochromatosis; Hepatolenticular Degeneration; Humans; Iron; Magnesium; Male; Menkes Kinky Hair Syndrome; Metallothionein; Milk, Human; Nucleic Acids; Selenium; Testis; Trace Elements; Zinc

Metallothioneins are a family of ubiquitous, cysteine rich proteins, whose amino acidic and genomic sequences have been highly conserved during evolution. MT synthesis is induced by heavy metals, glucocorticoids and a bacterial lipopolysaccharide in vivo and in vitro. MT forms stable complexes with heavy metals. One MTIIA gene, four MTI class genes and five pseudogenes have been isolated in humans. The cluster of MT genes is located on chromosome 16. The cloned, transfected genes retain metal inducibility. The first 150 bp of the 5' flanking region of mouse and human MT genes are essential for transcription and metal regulation. Two control regions have been identified. The distal region, between -151 and -78 is essential for efficient transcription and binding of cellular factor(s) which regulates MT gene expression. In Menkes' disease, a lethal X-linked recessive disorder, copper accumulates intracellularly bound to MT. Low doses of copper induce MT synthesis in Menkes' fibroblasts, but not in normal controls. Transfection experiments using the mouse MTI promoter fused to CAT show that the effect of copper in MT transcription is in trans. Menkes' cells are more sensitive to copper than normal controls and respond to copper poisoning by synthesizing two heat-shock like proteins. A mutation affecting copper transport or metabolism is discussed.

Topics: Animals; Brain Diseases, Metabolic; Cadmium; Cells, Cultured; Copper; Disease Models, Animal; Gene Expression Regulation; Humans; Menkes Kinky Hair Syndrome; Metalloproteins; Metallothionein; Promoter Regions, Genetic; Structure-Activity Relationship

A hypothesis is proposed admitting the participation of apometallothioneines (AMT) as a common link in the etiopathogenesis of hypertonic disease (HD) and some diseases with polygene heredity. The preconditions of the hypothesis are discussed (role of genetic disposition, external factors as salt, stress, tobacco smoking, alcohol, microelements--V and Cd and glucocorticoids in the origination of arterial hypertension). AMT homeostasis is discussed as well as the possible connection with the metabolism of Zn and Cu and Cu in organism. The chelating capacity of AMT makes it a potential regulatory protein, associated with the activity of Zn- and Cu-dependent enzymes and metalloenzymes. The mosaicism of pathology is explained with the genetic polymorphism of those enzymes (D beta H, MAO, etc), regardless of the common etiopathogenetic link. Some schemes are presented illustrating the hypothesis. The tendencies of the future studies are outlined in searching of direct proofs of the hypothesis.

Topics: Adrenal Cortex; Animals; Apoproteins; Blood Pressure; Copper; Disease Models, Animal; Disease Susceptibility; Genes; Genetic Diseases, Inborn; Glucocorticoids; Homeostasis; Humans; Hypertension; Menkes Kinky Hair Syndrome; Metallothionein; Terminology as Topic; Zinc

Various inherited disorders of copper metabolism in man and animals are reviewed. Emphasis is placed on the use of cultured cells from mutants to determine the primary molecular defects and to acquire basic knowledge of normal copper metabolism. This allows better diagnostic tests and possible treatment of the disorders. Menkes' disease in humans and mottled mouse mutants are discussed in detail, as they illustrate these approaches.

Topics: Animals; Brain Diseases, Metabolic; Cells, Cultured; Copper; Disease Models, Animal; Dogs; Electron Transport Complex IV; Female; Fibroblasts; Hepatolenticular Degeneration; Humans; Menkes Kinky Hair Syndrome; Metalloproteins; Metallothionein; Mice; Mice, Mutant Strains; Mice, Quaking; Pregnancy; Protein-Lysine 6-Oxidase

Neuroinflammation has a major role in several brain disorders including Alzheimer's disease (AD), yet at present there are no effective anti-neuroinflammatory therapeutics available. Copper(II) complexes of bis(thiosemicarbazones) (Cu

Topics: Alzheimer Disease; Animals; Chemotactic Factors; Coordination Complexes; Copper; Disease Models, Animal; Membrane Glycoproteins; Metallothionein; Mice; Microglia; Receptors, Immunologic; Thiosemicarbazones; Tumor Necrosis Factor-alpha

Non-alcoholic fatty liver disease (NAFLD), a metabolic disorder that develops from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH), has become an epidemic of chronic liver dysfunction worldwide. However, mechanisms that govern the transition from NAFL to NASH have not been fully elucidated.. Gene expression profile data of NAFLD liver tissues were obtained from Gene Expression Omnibus (GEO), including three microarray datasets with 60 NAFL and 44 NASH patients. Integrative differentially expressed genes (DEGs) between NAFL and NASH patients were identified using robust rank aggregation (RRA) analysis. Hub genes were identified combined with gene ontology functional annotation and protein-protein interaction network construction and validated using a sequencing dataset. Huh-7 cells with palmitate-induced lipid overload and NAFLD-diet mouse model of different stages were used to verify our findings.. RRA analysis determined 70 robust DEGs between NAFL and NASH. The most robustly upregulated genes were. Our integrated analysis identified novel gene signatures and provided comprehensive molecular mechanisms underlying the transition from NAFL to NASH. Metallothionein might be a potential intervention target for NAFLD progression.

Topics: Animals; Diet; Disease Models, Animal; Metallothionein; Mice; Non-alcoholic Fatty Liver Disease; Protein Interaction Maps

Radiotherapy or accidental exposure to ionizing radiation causes severe damage of healthy intestinal tissues. Intestinal barrier function is highly sensitive to ionizing radiation, and loss of epithelial integrity results in mucosal inflammation, bacterial translocation, and endotoxemia. Few studies have of epithelial integrity as a therapeutic target to treat radiation toxicity. Here, we examined the effects of pravastatin (PS) and the molecular mechanisms underlying epithelial integrity on radiation-induced enteropathy.. The radio-mitigative effects of PS were evaluated in a minipig model by quantifying clinical symptoms, and performing histological and serological analyses and mRNA sequencing in intestinal tissues. To evaluate the role of intercellular junctions on radiation damage, we used tight junction regulator and metallothionein 2 (MT2) as treatments in a mouse model of radiation-induced enteropathy. Caco-2 monolayers were used to examine functional epithelial integrityand intercellular junction expression.. Using a minipig model of pharmaceutical oral bioavailability, we found that PS mitigated acute radiation-induced enteropathy. PS-treated irradiated minipigs had mild clinical symptoms, lower intestinal inflammation and endotoxin levels, and improved gastrointestinal integrity, compared with control group animals. The results of mRNA sequencing analysis indicated that PS treatment markedly influenced intercellular junctions by inhibiting p38 MAPK signaling in the irradiated intestinal epithelium. The PS-regulated gene MT2 improved the epithelial barrier via enhancement of intercellular junctions in radiation-induced enteropathy.. PS regulated epithelial integrity by modulating MT2 in radiation-damaged epithelial cells. These findings suggested that maintenance of epithelial integrity is a novel therapeutic target for treatment of radiation-induced gastrointestinal damage.. As stated in the Acknowledgments.

Topics: Animals; Biopsy; Caco-2 Cells; Computational Biology; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation; Gene Ontology; Humans; Intestinal Diseases; Intestinal Mucosa; Male; Metallothionein; Mice; Pravastatin; Radiation Injuries; Radiation, Ionizing; Swine; Swine, Miniature; Tight Junctions

Mitochondrial diseases (MD), such as Leigh syndrome (LS), present with severe neurological and muscular phenotypes in patients, but have no known cure and limited treatment options. Based on their neuroprotective effects against other neurodegenerative diseases in vivo and their positive impact as an antioxidant against complex I deficiency in vitro, we investigated the potential protective effect of metallothioneins (MTs) in an Ndufs4 knockout mouse model (with a very similar phenotype to LS) crossed with an Mt1 overexpressing mouse model (TgMt1). Despite subtle reductions in the expression of neuroinflammatory markers GFAP and IBA1 in the vestibular nucleus and hippocampus, we found no improvement in survival, growth, locomotor activity, balance, or motor coordination in the Mt1 overexpressing Ndufs4

Topics: Animals; Ataxia; beta 2-Microglobulin; Biomarkers; Body Weight; Disease Models, Animal; Electron Transport Complex I; Female; Hippocampus; Inflammation; Male; Metabolome; Metallothionein; Mice, Knockout; Mitochondrial Diseases; Motor Activity; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; RNA, Messenger; Survival Analysis

Recent data suggest that rats with neonatal ventral hippocampal lesion (NVHL) show changes related to inflammatory processes and oxidative stress at the prefrontal cortex (PFC) level at post-pubertal age. The NVHL model is considered an animal model in schizophrenia. Here we analyzed the levels of nitrite, zinc, and metallothionein (MT) in cortical and subcortical regions of NVHL rats at pre-pubertal and post-pubertal ages. Nitric oxide (NO) levels were evaluated through measurement of nitrite levels. The locomotor activity was also evaluated in a novel environment. Animals with NVHL showed an increase in locomotor activity only at post-pubertal age. Furthermore, at pre-pubertal age, NVHL rats showed an increase in NO levels in ventral and dorsal hippocampus, thalamus, Caudate-putamen (CPu) and brainstem, in zinc levels in ventral and dorsal hippocampus, and CPu, and the MT level also in the ventral hippocampus and occipital cortex. In addition, at pre-pubertal age, a reduction in MT levels was also found in the PFC, parietal and temporal cortices, the CPu and the cerebellum. However, after puberty, NVHL caused an increase in NO levels in the PFC, and also zinc levels in the PFC and occipital and parietal cortices, with a reduction in MT levels in the thalamus and NAcc. Our results show the changes of these three molecules over time, among lesion (PD7), pre-pubertal and post-pubertal ages. This suggests changes at pre-pubertal age directly related to the site of the lesion, while at post-pubertal age, our data highlight changes in the PFC, a region mainly involved in schizophrenia.

Topics: Aging; Animals; Disease Models, Animal; Limbic System; Male; Metallothionein; Motor Activity; Neurons; Nitric Oxide; Rats; Rats, Sprague-Dawley; Schizophrenia; Zinc

Zinc is well known for its anti-inflammatory effects, including regulation of migration and activity of polymorphonuclear neutrophils (PMN). Zinc deficiency is associated with inflammatory diseases such as acute lung injury (ALI). As deregulated neutrophil recruitment and their hyper-activation are hallmarks of ALI, benefits of zinc supplementation on the development of lipopolysaccharides (LPS)-induced ALI were tested.. 64 C57Bl/6 mice, split into eight groups, were injected with 30 µg zinc 24 hours before exposure to aerosolised LPS for 4 hours. Zinc homoeostasis was characterised measuring serum and lung zinc concentrations as well as metallothionein-1 expression. Recruitment of neutrophils to alveolar, interstitial and intravascular space was assessed using flow cytometry. To determine the extent of lung damage, permeability and histological changes and the influx of protein into the bronchoalveolar lavage fluid were measured. Inflammatory status and PMN activity were evaluated via tumour necrosis factor α levels and formation of neutrophil extracellular traps. The effects of zinc supplementation prior to LPS stimulation on activation of primary human granulocytes and integrity of human lung cell monolayers were assessed as well.. Injecting zinc 24 hours prior to LPS-induced ALI indeed significantly decreased the recruitment of neutrophils to the lungs and prevented their hyperactivity and thus lung damage was decreased. Results from in vitro investigations using human cells suggest the transferability of the finding to human disease, which remains to be tested in more detail.. Zinc supplementation attenuated LPS-induced lung injury in a murine ALI model. Thus, the usage of zinc-based strategies should be considered to prevent detrimental consequences of respiratory infection and lung damage in risk groups.

Topics: Acute Lung Injury; Animals; Bronchoalveolar Lavage Fluid; Cation Transport Proteins; Cell Line; Cell Survival; Chemokine CXCL1; Disease Models, Animal; Gene Expression; Granulocyte Colony-Stimulating Factor; Homeostasis; Humans; L-Selectin; Lipopolysaccharides; Male; Metallothionein; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Neutrophils; Receptors, Complement 3b; RNA, Messenger; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha; Zinc

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate-intensity treadmill exercise and/or treatment with metallothionein-2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1

Topics: Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; Female; Metallothionein; Mice; Mice, Transgenic; Physical Conditioning, Animal; Quality of Life; Superoxide Dismutase-1

Alcohol-associated liver disease is a spectrum of liver disorders with histopathological changes ranging from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recent data suggest that chronic-plus-binge ethanol intake induces steatohepatitis by promoting release by hepatocytes of proinflammatory mitochondrial DNA-enriched (mtDNA-enriched) extracellular vesicles (EVs). The aim of the present study was to investigate the role of the stress kinase apoptosis signal-regulating kinase 1 (ASK1) and p38 mitogen-activated protein kinase (p38) in chronic-plus-binge ethanol-induced steatohepatitis and mtDNA-enriched EV release. Microarray analysis revealed the greatest hepatic upregulation of metallothionein 1 and 2 (Mt1/2), which encode 2 of the most potent antioxidant proteins. Genetic deletion of the Mt1 and Mt2 genes aggravated ethanol-induced liver injury, as evidenced by elevation of serum ALT, neutrophil infiltration, oxidative stress, and ASK1/p38 activation in the liver. Inhibition or genetic deletion of Ask1 or p38 ameliorated ethanol-induced liver injury, inflammation, ROS levels, and expression of phagocytic oxidase and ER stress markers in the liver. In addition, inhibition of ASK1 or p38 also attenuated ethanol-induced mtDNA-enriched EV secretion from hepatocytes. Taken together, these findings indicate that induction of hepatic mtDNA-enriched EVs by ethanol is dependent on ASK1 and p38, thereby promoting alcoholic steatohepatitis.

Topics: Alcoholism; Alcohols; Animals; Binge Drinking; Disease Models, Animal; DNA, Mitochondrial; Extracellular Vesicles; Fatty Liver, Alcoholic; Hepatocytes; Humans; Inflammation; Liver; MAP Kinase Kinase Kinase 5; Matrix Metalloproteinase 14; Metallothionein; Mice; p38 Mitogen-Activated Protein Kinases; Signal Transduction

Neurodegenerative disorders (ND) like Alzheimer's (AD), Parkinson's (PD), Huntington's or Prion diseases share similar pathological features. They are all age dependent and are often associated with disruptions in analogous metabolic processes such as protein aggregation and oxidative stress, both of which involve metal ions like copper, manganese and iron. Bush and Tanzi proposed 2008 in the 'metal hypothesis of Alzheimer's disease' that a breakdown in metal homeostasis is the main cause of NDs, and drugs restoring metal homeostasis are promising novel therapeutic strategies. We report here that metallothionein (MT), an endogenous metal detoxifying protein, is increased in young amyloid ß (Aß) expressing Caenorhabditis elegans, whereas it is not in wild type strains. Further MT induction collapsed in 8 days old transgenic worms, indicating the age dependency of disease outbreak, and sharing intriguing parallels to diminished MT levels in human brains of AD. A medium throughput screening assay method was established to search for compounds increasing the MT level. Compounds known to induce MT release like progesterone, ZnSO

Topics: Aging; alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Animals; Animals, Genetically Modified; Benzothiazoles; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Clioquinol; Disease Models, Animal; Emodin; Gene Knockdown Techniques; Homeostasis; Metallothionein; Metals; Neuroprotective Agents; Parkinson Disease; Quercetin; Signal Transduction

Intestinal epithelial cells (IECs) regulate intestinal immune cells, particularly development of T-helper 17 (Th17) cells. Deregulation of this process leads to intestinal inflammation and tumorigenesis, via unknown mechanisms. TANK-binding kinase 1 (TBK1) is expressed by IECs and cells in the innate immune system. We studied the functions of TBK1 in the intestinal immune response and tumorigenesis in mice.. We performed studies of wild-type mice, mice with conditional disruption of Tbk1 (Tbk1. Compared to Apc. Expression of TBK1 by IECs suppresses expression of MT1 and prevents expression of IL1β by macrophages and differentiation of Th17 cells, to prevent inflammation and tumorigenesis. Strategies to block this pathway might be developed for colorectal tumorigenesis.

Topics: Adenomatous Polyposis Coli; Animals; Cell Differentiation; Cell Transformation, Neoplastic; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Epithelial Cells; Genes, APC; Humans; Immunity, Innate; Immunity, Mucosal; Interleukin-17; Interleukin-1beta; Intestinal Mucosa; Intestinal Neoplasms; Macrophages; Metallothionein; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Protein Serine-Threonine Kinases; Th17 Cells

Doxorubicin (DOX) induced cardiotoxicity is a life-threatening side effect of chemotherapy and decreased cardiac function can present years after treatment. Despite the investigation of a broad range of pharmacologic interventions, to date the only drug shown to reduce DOX-related cardiotoxicity in preclinical studies and limited clinical trials is the iron chelating agent, dexrazoxane (DRZ), although the mechanisms responsible for DRZ mediated protection from DOX related cardiotoxicity remain unclear. Engineered cardiac tissues (ECTs) can be used for tissue repair strategies and as in vitro surrogate models to test cardiac toxicities and preventative countermeasures. Neonatal murine ECTs display cardiotoxicity in response to the environmental toxin, cadmium, and reduced cadmium toxicity with Zinc co-treatment, in part via the induction of the anti-oxidant Metallothionein (MT). We adapted our in vitro ECT model to determine the feasibility of using the ECT approach to investigate DOX-related cardiac injury and DRZ prevention. We found: (1) DOX induced dose and time dependent cell death in ECTs; (2) Zinc did not show protection from DOX cardiotoxicity; (3) MT overexpression induced by Zinc, low dose Cd pretreatment, or MT-overexpression (MT-TG) did not reduce ECT DOX cardiotoxicity; (4) DRZ reduced ECT DOX induced cell death; and (5) The mechanism of DRZ ECT protection from DOX cardiotoxicity was topoisomerase 2B (TOP2B) inhibition rather than reduced reactive oxygen species. Our data support the feasibility of ECTs as an in vitro platform technology for the investigation of drug induced cardiotoxicities including the role of TOP2B in DOX toxicity and DRZ mediated DOX toxicity prevention.

Topics: Animals; Animals, Newborn; Cardiotoxicity; Dexrazoxane; Disease Models, Animal; DNA Topoisomerases, Type II; Doxorubicin; Iron Chelating Agents; Metallothionein; Mice; Mice, Transgenic; Myocytes, Cardiac; Reactive Oxygen Species; Tissue Engineering

Platelet hyperaggregation and hypercoagulation are associated with increase of thrombogenic risk, especially in patients with type 2 diabetes (T2D). High activity of P2Y12 receptor is found in T2D patients, exposing such patients to a prothrombotic condition. P2Y12 is a promising target for antiplatelet, but due to P2Y12 receptor constitutive activation, the clinical practical phenomena such as "clopidogrel resistance" are commonly occurring. In this study, we investigate the role of lncRNA on platelet activation. By lncRNA array, we screened thousands of differentially expressed lncRNA in megakaryocytes from T2D patients and confirmed that lncRNA metallothionein 1 pseudogene 3 (MT1P3) was significantly upregulated in megakaryocytes from T2D patients than in healthy controls. And we further investigate the biofunction of MT1P3 on platelet activation and the regulatory mechanism on p2y12. MT1P3 was positively correlated with p2y12 mRNA levels and promoted p2y12 expression by sponging miR-126. Knockdown of MT1P3 by siRNA reduced p2y12 expression, inhibiting platelet activation and aggregation in diabetes animal model. In conclusion, our findings identify MT1P3 as a key regulator in platelet activation by increasing p2y12 expression through sponging miR-126 under T2D condition. These findings may provide a new insight for managing platelet hyperactivity-related diseases.

Topics: Animals; Diabetes Mellitus, Type 2; Disease Models, Animal; Female; Humans; Male; Metallothionein; Mice; MicroRNAs; Middle Aged; Pseudogenes; Rats; Rats, Wistar; Receptors, Purinergic P2Y12; RNA, Long Noncoding; Transfection

Topics: Adipokines; Animals; Apoptosis; Cell Movement; Cell Proliferation; Disease Models, Animal; Fibrosis; Heart; Hydrogen Peroxide; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Metallothionein; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardium; Signal Transduction; Superoxide Dismutase; Tumor Necrosis Factors; Ventricular Function, Left

Copper chelator cuprizone (CPZ) is neurotoxicant, which selectively disrupts oligodendroglial respiratory chain, leading to oxidative stress and subsequent apoptosis. Demyelination is, however, followed by spontaneous remyelination owing to the activation of intrinsic CNS repair mechanisms. To explore the participation of metallothioneins (MTs) in these processes, in this study we analyzed the expression profiles of MT-I/II and their receptor megalin (low-density lipoprotein receptor related protein-2) in the brain of mice subjected to different protocols of CPZ feeding. Experiments were performed in female C57BL/6 mice fed with 0.25% CPZ during 1, 3 and 5 weeks. They were sacrificed immediately after feeding with CPZ or 2 weeks after the withdrawal of CPZ. The data showed that CPZ-induced demyelination was followed by high astrogliosis and enhanced expression of MTs and megalin in white (corpus callosum and internal capsule) and gray matter of the brain (cortex, hippocampus, and cerebellum). Moreover, in numerous cortical neurons and progenitor cells the signs of MT/megalin interactions and Akt1 phosphorylation was found supporting the hypothesis that MTs secreted from the astrocytes might directly affect the neuronal differentiation and survival. Furthermore, in mice treated with CPZ for 5 weeks the prominent MTs and megalin immunoreactivities were found on several neural stem cells and oligodendrocyte progenitors in subgranular zone of dentate gyrus and subventricular zone of lateral ventricles pointing to high modulatory effect of MTs on adult neuro- and oligodendrogenesis. The data show that MT I/II perform important cytoprotective and growth-regulating functions in remyelinating processes activated after toxic demyelinating insults.

Topics: Animals; Brain; Cerebral Cortex; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Female; Gene Expression Profiling; Gene Expression Regulation; Low Density Lipoprotein Receptor-Related Protein-2; Metallothionein; Mice, Inbred C57BL; Neural Stem Cells; Neuroglia; Neurons; Remyelination

Atopic dermatitis (AD), one of the most common chronic eczematous skin disorders, is associated with cutaneous hyperactivity as a response to environmental triggers. Metallothionein (MT) plays a constitutive defensive role in the response to noxious stimuli. However, the role of MT in AD development is unclear. Using an AD-like murine model created by the topical application of 2.4-dinitrofluorobenzene, we studied the dynamic pattern of MT expression on AD development. AD-like lesions were evaluated based on the development of erythema, edema, exfoliation, scaling, increased thickness, and increased weight of lesional skin. These characteristics of AD-like lesions and thymic stromal lymphopoietin (TSLP) expression peaked at Day 1 of the establishment of our model and gradually alleviated over time. The MT expression in lesional skin was increased and peaked at Day 3. By immunostaining, increased expression of MT was translocated from the cytoplasm to the nucleus. MT-1/2 knockout (MT-/-) mice and wild type (MT+/+) mice were also used to evaluate the role of MT on AD. MT-/- mice had greater edema scores, thickness, lesional skin weight, as well as more CD4+ T cells, TSLP, superoxide dismutase, and NDUFAF1. These results suggest that MT may play a protective role against AD development, and that antioxidant and nuclear protective mechanisms may be involved.

Topics: Animals; Antioxidants; CD4-Positive T-Lymphocytes; Dermatitis, Atopic; Dinitrofluorobenzene; Disease Models, Animal; Edema; Gene Expression Regulation; Humans; Metallothionein; Mice; Mice, Knockout; NADH Dehydrogenase; Superoxide Dismutase

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.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acid Sequence; Animals; Cell Death; Cell Line, Tumor; Cell-Penetrating Peptides; Computer Simulation; Disease Models, Animal; Green Fluorescent Proteins; Humans; Metallothionein; Mice; Mitochondria; Neurons; Parkinson Disease; Protein Transport; Recombinant Fusion Proteins; Substantia Nigra; Tyrosine 3-Monooxygenase

Metallothioneins (MTs) play important roles in regulating oxidative stress, inflammation, and hormone signaling. These processes play a major role in labor at term and preterm. The aims of this study were to characterize (a) temporal- and labor-associated changes and (b) the effect of pro-inflammatory and pro-labor insults on the expression of MT1 isoforms, MT2A, MT3, and MT4 in fetal membranes and myometrium.. The expression of MTs was assessed in fetal membranes and myometrium from nonlaboring and laboring women at preterm and term by RT-qPCR. Tissue explants were used to assess the effect of pro-inflammatory cytokines and Toll-like receptor (TLR) ligands on the expression of MTs in fetal membranes and myometrium.. In fetal membranes, the expression of MT1A, MT1E, MT1F, MT1X, and MT2A was higher at term compared with preterm. Preterm labor and preterm histological chorioamnionitis were associated with increased expression of MT1A, MT1G, MT1M, MT1X, MT2A, and MT3. Term labor was associated with increased expression of MT1A, MT1F, MT1X, MT2A, and MT3 in fetal membranes and expression of MT1A, MT1E, MT1F, MT1G, MT1M, MT1X, MT2A, and MT3 in myometrium. Pro-inflammatory cytokines and TLR ligands increased the expression of MT1A, MT1E, MT1F, MT1G, MT1H, MT1X, and MT2A in fetal membranes and myometrium.. Temporal-, labor-, and infection-associated increases in MT1 isoforms, MT2A, and MT3 have been observed in fetal membranes and/or myometrium. Furthermore, pro-inflammatory cytokines and bacterial and viral products increased the expression of MT1 isoforms, MT2A, MT3, and MT4 mRNA expression in fetal membranes and myometrium.

Topics: Animals; Cells, Cultured; Cohort Studies; Disease Models, Animal; Extraembryonic Membranes; Female; Humans; Inflammation; Labor, Obstetric; Lipopolysaccharides; Metallothionein; Metallothionein 3; Mice; Mice, Inbred C57BL; Myometrium; Obstetric Labor, Premature; Pregnancy; Protein Isoforms; Signal Transduction

Wilson disease (WD) is an autosomal recessive disorder caused by mutation in the ATP7B gene that affects copper transport in the body. ATP7B mutation damages copper transporter function, ultimately resulting in excessive copper accumulation and subsequent toxicity in both the liver and brain. Mechanisms of copper toxicity, however, are not well defined. The Atp7b-/- mouse model is well-characterized and presents a hepatic phenotype consistent with WD. In this study, we found that the untreated Atp7b-/- mice accumulate approximately 2-fold excess hepatic zinc compared to the wild type. We used targeted transcriptomics and proteomics to analyze the molecular events associated with zinc and copper accumulation in the Atp7b-/- mouse liver. Altered gene expression of Zip5 and ZnT1 zinc transporters indicated a transcriptional homeostatic response, while increased copper/zinc ratios associated with high levels of metallothioneins 1 and 2, indicated altered Zn availability in cells. These data suggest that copper toxicity in Wilson disease includes effects on zinc-dependent proteins. Transcriptional network analysis of RNA-seq data reveals an interconnected network of transcriptional activators with over-representation of zinc-dependent and zinc-responsive transcription factors. In the context of previous research, these observations support the hypothesis that mechanisms of copper toxicity include disruption of intracellular zinc distribution in liver cells. The translational significance of this work lies in oral zinc supplementation in treatment for WD, which is thought to mediate protective effects through the induction of metallothionein synthesis in the intestine. This work indicates broader impacts of altered zinc-copper balance in WD, including global transcriptional responses and altered zinc balance in the liver.

Topics: Animals; Copper; Copper-Transporting ATPases; Disease Models, Animal; Gene Regulatory Networks; Hepatolenticular Degeneration; Liver; Metallothionein; Mice; Mice, Knockout; Zinc

To investigate the impact of bone mesenchymal stem cells (BMSCs) intervention on the viscoelasticity of sciatic nerve in rats with chronic alcohol intoxication (CAI).. The CAI rat models were prepared, divided into model groups, and treated with either BMSCs or basic fibroblast growth factor (bFGF). Then the rats underwent electrophysiological test and the serum levels of malondialdehyde (MDA), superoxide dismutase (SOD), and metallothionein (MT) were measured. Histological observation, stress relaxation test, and creep test were performed for the sciatic nerve of the CAI model in each group.. The MDA level of group BMSC was significantly lower (p<0.05) than that of groups MOD (the CIA model) and bFGF. The SOD and MT levels were higher in group BMSC than in groups MOD and bFGF (p<0.05). The motor nerve conduction velocity and amplitude were higher in group BMSC than in groups MOD and bFGF (p<0.05). The amounts of 7200s stress reduction and 7200 s strain increase of the sciatic nerve in group BMSC were greater than those in groups bFGF and MOD (p<0.05).. Bone mesenchymal stem cells can improve the metabolism of free radicals, restore the tissue morphology and viscoelasticity of the chronic alcohol intoxication animal model, and positively affect the repairing of the injured sciatic nerve.

Topics: Alcoholic Intoxication; Animals; Bone Marrow Cells; Bone Marrow Transplantation; Disease Models, Animal; Elasticity; Fibroblast Growth Factor 2; Male; Malondialdehyde; Mesenchymal Stem Cell Transplantation; Metallothionein; Nerve Regeneration; Rats; Rats, Wistar; Sciatic Nerve; Stress, Physiological; Superoxide Dismutase; Viscosity

 To characterize the patterns of cell differentiation, proliferation, and tissue invasion in eutopic and ectopic endometrium of rabbits with induced endometriotic lesions via a well- known experimental model, 4 and 8 weeks after the endometrial implantation procedure..  Twenty-nine female New Zealand rabbits underwent laparotomy for endometriosis induction through the resection of one uterine horn, isolation of the endometrium, and fixation of tissue segment to the pelvic peritoneum. Two groups of animals (one with 14 animals, and the other with15) were sacrificed 4 and 8 weeks after endometriosis induction. The lesion was excised along with the opposite uterine horn for endometrial gland and stroma determination. Immunohistochemical reactions were performed in eutopic and ectopic endometrial tissues for analysis of the following markers: metalloprotease (MMP-9) and tissue inhibitor of metalloprotease (TIMP-2), which are involved in the invasive capacity of the endometrial tissue; and metallothionein (MT) and p63, which are involved in cell differentiation and proliferation..  The intensity of the immunostaining for MMP9, TIMP-2, MT, and p63 was higher in ectopic endometria than in eutopic endometria. However, when the ectopic lesions were compared at 4 and 8 weeks, no significant difference was observed, with the exception of the marker p63, which was more evident after 8 weeks of evolution of the ectopic endometrial tissue..  Ectopic endometrial lesions seem to express greater power for cell differentiation and tissue invasion, compared with eutopic endometria, demonstrating a potentially invasive, progressive, and heterogeneous presentation of endometriosis..  Caracterizar o padrão de diferenciação celular, proliferação e invasão tecidual em endométrio eutópico e ectópico de coelhas com lesões de endometriose induzidas por um modelo experimental 4 e 8 semanas após o procedimento de implantação endometrial. MéTODOS:  Vinte e nove coelhas fêmeas Nova Zelândia foram submetidas a laparotomia para indução de endometriose através da ressecção de um dos cornos uterinos, isolamento do endométrio e fixação do tecido no peritônio pélvico. Dois grupos de animais (14 animais em um grupo e 15 animais no outro) foram sacrificados 4 e 8 semanas após a indução da endometriose. A lesão foi excisada junto com o corno uterino contralateral para determinação da presença de glândulas e de estroma endometrial. Reações de imunohistoquímica foram realizadas no tecido endometrial eutópico e ectópico para análise dos seguintes marcadores: metaloprotease (MMP9) e inibidor tecidual da metaloprotease 2 (TIMP-2), os quais estão envolvidos na capacidade de invasão do tecido endometrial; e metalotioneina (MT) e p63, os quais estão envolvidos na diferenciação e proliferação celular..  A intensidade da imunomarcação para MMP9, TIMP-2, MT e p63 foi mais alta nos endométrios ectópicos do que nos endométrios eutópicos. Contudo, quando as lesões foram comparadas entre 4 e 8 semanas, nenhuma diferença foi observada, com exceção do marcador p63, o qual foi mais evidente depois de 8 semanas de evolução do tecido endometrial ectópico. CONCLUSãO:  Lesões endometriais ectópicas parecem expressar maior poder de diferenciação celular e de invasão tecidual comparadas com endométrios eutópicos, demonstrando o potencial de invasão, de progressão e de apresentação heterogênea da endometriose.

Topics: Animals; Cell Differentiation; Cell Proliferation; Choristoma; Disease Models, Animal; Endometriosis; Endometrium; Female; Matrix Metalloproteinase 9; Membrane Proteins; Metallothionein; Rabbits; Tissue Inhibitor of Metalloproteinase-2

Osteopontin (OPN), an extracellular matrix (ECM) glyco-phosphoprotein, plays an important role in autoimmune-mediated demyelinating diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). As an integrin and CD44 binding protein it participates in bidirectional communication between the ECM and target cells and affects transduction pathways that maintain neuronal and immune cell homeostasis. Its biological activity is also heavily influenced by microenvironment, which stimulates the cleavage of OPN and changes its functions. In this study we estimated the expression profile of OPN in neural tissues of DA rats during the first relapse of chronic relapsing EAE and investigated the relationship of OPN to metallothionein I+II (MTs), which play pivotal role in zinc-related cell homeostasis and in protection of CNS against cytokine-induced injury. The data showed that in EAE rats OPN mRNA and protein levels increased concurrently with the transcription of MTs and that within the spinal cord (SC) lysates EAE-afflicted rats had a higher content of OPN fragments of low molecular weight than untreated and CFA-treated rats. The expression of OPN and MTs was upregulated on ependymal, lymphoid and astroglial cells and on multiple αvβ3+ neurons in SC and in the brain (cortex, white matter, hippocampus, and cerebellum). Besides, multiple cells co-expressed OPN and MTs. Granular OPN signals were detected in secretory vesicles of Golgy (αvβ3 neurons) and in patches adjacent to the plasma membrane (subventricular zone). The findings imply that in demyelinating lesions are generated proteolytic OPN fragments and that OPN/MT interactions contribute to tissue remodeling during an autoimmune attack.

Topics: Animals; Astrocytes; Brain; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Male; Metallothionein; Neurons; Osteopontin; Rats; Up-Regulation

Mammalian cardiomyocytes substantially lose proliferative capacity immediately after birth, limiting adult heart regeneration after injury. However, clinical myocarditis appears to be self-limiting with tissue-reparative properties. Here, we investigated the molecular mechanisms underlying the recovery from myocarditis with regard to cardiomyocyte proliferation using an experimental autoimmune myocarditis (EAM) model. Three weeks after EAM induction (EAM3w), cardiac tissue displayed infiltration of inflammatory cells with cardiomyocyte apoptosis. However, by EAM5w, the myocardial damage was remarkably attenuated, associated with an increase in cardiomyocytes that were positively stained with cell cycle markers at EAM3w. Cardiomyocyte fate mapping study revealed that the proliferating cardiomyocytes primarily derived from pre-existing cardiomyocytes. Signal transducer and activator of transcription 3 (STAT3) was robustly activated in cardiomyocytes during inflammation, accompanied by induction of interleukin-6 family cytokines. Cardiomyocyte-specific ablation of STAT3 gene suppressed the frequency of cycling cardiomyocytes in the recovery period without influencing inflammatory status, resulting in impaired tissue repair and cardiac dysfunction. Finally, microarray analysis revealed that the expression of regeneration-related genes, metallothioneins and clusterin, in cardiomyocytes was decreased by STAT3 gene deletion. These data show that adult mammalian cardiomyocytes restore regenerative capacity with cell cycle reentry through STAT3 as the heart recovers from myocarditis-induced cardiac damage.

Topics: Animals; Autoimmune Diseases; Cell Cycle; Cell Proliferation; Clusterin; Disease Models, Animal; Male; Metallothionein; Mice; Mice, Knockout; Myocarditis; Myocytes, Cardiac; Regeneration; Signal Transduction; STAT3 Transcription Factor

Topics: alpha-Linolenic Acid; Animals; Body Composition; Brain; Caloric Restriction; Copper; Corn Oil; Corticosterone; Disease Models, Animal; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Gene Expression Regulation; Liver; Male; Malnutrition; Manganese; Metallothionein; Monocarboxylic Acid Transporters; Rats; Rats, Sprague-Dawley; Receptors, Glucocorticoid; RNA, Messenger; Soybean Oil; Triiodothyronine; Zinc

Inflammatory demyelinating diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are often followed by cognitive deficits associated with the neuronal injury, synaptic loss and altered neurogenesis within the hippocampus. Changes depend on the genetic and epigenetic factors that ensure the cellular and environmental homeostasis and regulate the interactions of immunocompetent, glial and neural cells. Owing to high impact of stress proteins on these processes, in this study we compared the protein content of interleukin-6, transforming growth factor-β1, metallothioneins I/II (MTs) and glycoprotein 96 (gp96) in the hippocampus of DA and AO rats that differ in the susceptibility to the induction of EAE, and tested the relationship of MTs and gp96 to granule neurons, glial cells and neural progenitors in different subfields of dentate gyrus. Rats were immunized with bovine brain homogenate emulsified in complete Freund's adjuvant or only with CFA. The data showed that acute attack of EAE in DA rats was followed by accumulation of IL-6, TGF-β1 and MTs proteins, by increased expression of MTs in molecular and granular cell layer, by reduced expression of gp96/granular cell, by apoptosis and by microgliosis with appearance of Iba-1+ cells, co-expressing MT I/II and gp96. Furthermore, in subgranular zone (SGZ) of DA rats an augmented number of GFAP+ precursors, but decreased number of doublecortin (DCX)+ neuroblasts and immature NeuN+ neurons were found, implying that in DA rats the neurogenesis was delayed or reduced. Besides, in SGZ of both strains several DCX+ and NeuN+ cells co-expressing gp96 and MT I/II were found.

Topics: Animals; Apoptosis; Disease Models, Animal; Disease Susceptibility; Doublecortin Domain Proteins; Doublecortin Protein; Encephalomyelitis, Autoimmune, Experimental; Hippocampus; Interleukin-6; Male; Membrane Glycoproteins; Metallothionein; Microtubule-Associated Proteins; Neurons; Neuropeptides; Rats; Transforming Growth Factor beta1

Zinc transporters (ZnTs) and metallothioneins (MT) are important in maintaining Zn homeostasis in the brain. The present study was designed to find out whether alterations in ZnTs and MTs are associated with the pathophysiology of depression and the mechanism of antidepressant action.. Messenger RNA and proteins of ZnT1, ZnT3, ZnT4, ZnT5, ZnT6 and MT1/2 were measured in the prefrontal cortex (PFC) and hippocampus (Hp) of rats subjected to olfactory bulbectomy (OB) (a model of depression) and chronic amitriptyline (AMI) treatment by Real Time PCR and Western Blot/Immunohistochemistry (IHP).. Results in the OB rats showed: increases in the protein levels of ZnT1 in the PFC and Hp and MT1/2 in the PFC; a decrease in ZnT3 protein level in the PFC; no changes in ZnT4, ZnT5 and ZnT6 in the PFC and Hp. IHP labeling revealed increases in the optical densities of ZnT1-IR in the PFC and Hp and decreases in ZnT3 and ZnT4-IR in the PFC of OB rats. Although OB had no effects on gene expression of ZnTs, mRNAs for MT1/2 were increased. Chronic AMI treatment did not influence protein levels of ZnTs and MT1/2 in Sham and OB rats; however decreased mRNA levels of ZnT4 and ZnT5 in PFC and ZnT1, ZnT3, ZnT4 and ZnT6 in Hp of Sham rats and normalized OB induced increase in MT1/2 gene expression.. Changes in ZnTs and MT1/2 suggest altered cortical distribution of Zn in the OB model which further supports the hypothesis that Zn dyshomeostasis may be involved in the pathophysiology of depression.

Topics: Amitriptyline; Animals; Antidepressive Agents, Tricyclic; Brain; Carrier Proteins; Depression; Disease Models, Animal; Exploratory Behavior; Gene Expression Regulation; Homeostasis; Male; Metallothionein; Olfactory Bulb; Rats; Rats, Sprague-Dawley; RNA, Messenger; Zinc

We previously demonstrated that OVE transgenic diabetic mice are susceptible to chronic complications of diabetic nephropathy (DN) including substantial oxidative damage to the renal glomerular filtration barrier (GFB). Importantly, the damage was mitigated significantly by overexpression of the powerful antioxidant, metallothionein (MT) in podocytes. To test our hypothesis that GFB damage in OVE mice is the result of endothelial oxidative insult, a new JTMT transgenic mouse was designed in which MT overexpression was targeted specifically to endothelial cells. At 60 days of age, JTMT mice were crossed with age-matched OVE diabetic mice to produce bi-transgenic OVE-JTMT diabetic progeny that carried the endothelial targeted JTMT transgene. Renal tissues from the OVE-JTMT progeny were examined by unbiased TEM stereometry for possible GFB damage and other alterations from chronic complications of DN. In 150 day-old OVE-JTMT mice, blood glucose and HbA1c were indistinguishable from age-matched OVE mice. However, endothelial-specific MT overexpression in OVE-JTMT mice mitigated several DN complications including significantly increased non-fenestrated glomerular endothelial area, and elimination of glomerular basement membrane thickening. Significant renoprotection was also observed outside of endothelial cells, including reduced podocyte effacement, and increased podocyte and total glomerular cell densities. Moreover, when compared to OVE diabetic animals, OVE-JTMT mice showed significant mitigation of nephromegaly, glomerular hypertrophy, increased mesangial cell numbers and increased total glomerular cell numbers. These results confirm the importance of oxidative stress to glomerular damage in DN, and show the central role of endothelial cell injury to the pathogenesis of chronic complications of diabetes. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:560-576, 2017. © 2016 Wiley Periodicals, Inc.

Topics: Animals; Blood Glucose; Diabetic Nephropathies; Disease Models, Animal; Endothelial Cells; Kidney Glomerulus; Metallothionein; Mice; Mice, Transgenic; Oxidative Stress; Podocytes

Metallothioneins (MTs) are a family of low molecular-weight and cysteine-rich metalloproteins that regulate metal metabolism and protect cells from oxygen free radicals. Recent studies suggested that MTs have some anti-inflammatory effects. However, the role of MTs in post-burn inflammation remains unclear. This study is designed to investigate the role of MTs in post-burn inflammation in a mouse burn model. MT-I/II null (-/-) and C57BL/6 wild-type (WT) mice were randomly divided into sham burn, burn, Zn treated, and Zn-MT-2 treated groups. The inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). Myeloperoxidase (MPO) activity was determined by spectrophotometry. In in vitro study, exogenous MT-2 was added to macrophages that were stimulated with burn serum in the presence or absence of a p38 MAPK inhibitor SB203580. The IL-6 and TNF-α messenger RNA (mRNA) expression were detected by quantitative real-time polymerase chain reaction. The levels of p38 expression were determined by Western blot. Burn induced increased inflammatory cytokines such as interleukin (IL)-1β, IL-6, tumor necrosis factors-α, and macrophage chemoattractant protein-1 production in burn wound and serum. The MPO activities in the lung and heart were also increased after burn. These effects were significantly more prominent in MT (-/-) mice than in WT mice. Furthermore, these effects were inhibited by administration of exogenous MT-2 to both WT and MT (-/-) mice. Exogenous MT-2 inhibited the p38 expression and abrogated the increase of IL-6 and TNF-α mRNA expression from macrophages that were stimulated with burn serum. The effect of MT-2 was not further strengthened in the presence of SB203580. MTs may have a protective role against post-burn inflammation and inflammatory organ damage, at least partly through inhibiting the p38 MAPK signaling.

Topics: Animals; Burns; Chemokine CCL2; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Imidazoles; Inflammation; Interleukin-1beta; Interleukin-6; Macrophages; Male; MAP Kinase Signaling System; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; p38 Mitogen-Activated Protein Kinases; Peroxidase; Pyridines; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha

Alzheimer's disease (AD) is the most commonly diagnosed dementia, where signs of neuroinflammation and oxidative stress are prominent. In this study we intend to further characterize the roles of the antioxidant, anti-inflammatory, and heavy metal binding protein, metallothionein-1 (MT-1), by crossing Mt1 overexpressing mice with a well-known mouse model of AD, Tg2576 mice, which express the human amyloid-β protein precursor (hAβPP) with the Swedish K670N/M671L mutations. Mt1 overexpression increased overall perinatal survival, but did not affect significantly hAβPP-induced mortality and weight loss in adult mice. Amyloid plaque burden in ∼14-month-old mice was increased by Mt1 overexpression in the hippocampus but not the cortex. Despite full length hAβPP levels and amyloid plaques being increased by Mt1 overexpression in the hippocampus of both sexes, oligomeric and monomeric forms of Aβ, which may contribute more to toxicity, were decreased in the hippocampus of females and increased in males. Several behavioral traits such as exploration, anxiety, and learning were altered in Tg2576 mice to various degrees depending on the age and the sex. Mt1 overexpression ameliorated the effects of hAβPP on exploration in young females, and potentiated those on anxiety in old males, and seemed to improve the rate of spatial learning (Morris water maze) and the learning elicited by a classical conditioning procedure (eye-blink test). These results clearly suggest that MT-1 may be involved in AD pathogenesis.

Topics: Age Factors; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Anxiety; Disease Models, Animal; Exploratory Behavior; Female; Gene Expression Regulation; Humans; Male; Matrix Metalloproteinase 16; Maze Learning; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Activity; Mutation; Phenotype; Psychomotor Disorders

Heat stress (HS) is commonly used to refer to the heat load that an individual is subjected to due to either metabolic heat, or environmental factors, including high temperatures and high humidity levels. HS has been reported to affect and even damage the functioning of various organs; overexposure to high temperatures and high humidity may lead to accidental deaths. It has been suggested that the cardiovascular system is primarily targeted by exposure to HS conditions; the HS-induced dysfunction of cardiomyocytes, which is characterized by mitochondrial dysfunction, may result in the development of cardiovascular diseases. The excessive production of reactive oxygen species (ROS) also participates in mitochondrial dysfunction. However, effective methods for the prevention and treatment of mitochondrial and cardiovascular dysfunction induced by exposure to HS are lacking. In the present study, we hypothesized that vitamin E (VE), an antioxidant, is capable of preventing oxidative stress and mitochondrial injury in cardiomyocytes induced by exposure to HS. The results revealed that pre‑treatment with VE increased the expression of metallothionein (MT), which has previously been reported to confer cytoprotective effects, particularly on the cardiovascular system. Pre-treatment with VE restored mitochondrial function in cardiomyocytes under conditions of HS by increasing the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), and by increasing adenosine triphosphate (ATP) levels. Furthermore, pre-treatment with VE decreased the production of ROS, which was induced by exposure to HS and thus exerted antioxidant effects. In addition, pre-treatment with VE attenuated oxidative stress induced by exposure to HS, as demonstrated by the increased levels of antioxidant enzymes [superoxide dismutase (SOD) and glutathione (GSH)], and by the decreased levels of markers of oxidative injury [malondialdehyde (MDA) and lactate dehydrogenase (LDH)]. Taken together, these findings suggest that pre-treatment with VE can prevent mitochondrial dysfunction and oxidative stress in cardiomyocytes induced by exposure to HS, by increasing the expression of MT.

Topics: Adenosine Triphosphate; Animals; Antioxidants; Disease Models, Animal; Gene Expression Regulation; Heat Stress Disorders; Heat-Shock Response; Metallothionein; Mice; Mitochondria, Heart; Myocytes, Cardiac; Oxidative Stress; Protective Agents; Reactive Oxygen Species; Vitamin E

Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)-small molecule ubiquitin-like modifier protein (SUMO)-metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 10(10) Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract.

Topics: Administration, Oral; Animals; Brain; Calorimetry; Disease Models, Animal; Gastrointestinal Tract; Genetic Vectors; Glutathione Transferase; Humans; Intestines; Kidney; Lactococcus lactis; Lead; Lead Poisoning; Liver; Male; Metallothionein; Muscle, Skeletal; Plasmids; Rats; Recombinant Proteins; Testis; Thermodynamics; Tissue Distribution

Chronic oxidative stress and subacute inflammation have been implicated as causes of age-related macular degeneration (AMD). In this study, we tested whether an orally available 5-OH-tryptamine (5HT) 1a receptor agonist, xaliproden, could protect against retinal pigment epithelium (RPE) cell damage in culture and in a mouse model of geographic atrophy.. Paraquat was used to create mitochondrial oxidative stress in ARPE-19 cells, and tumor necrosis factor-α (TNF-α) was used to stimulate the production of inflammatory cytokines in these cells. The production of antioxidant proteins, metallothionein, and inflammatory cytokines was assayed with quantitative real-time PCR. Cell survival was analyzed with microscopy and a cell titer assay. Integrity of the RPE monolayer was determined by measuring the transepithelial electrical resistance (TEER) and with immunocytochemistry with zona occludens protein 1 (ZO-1) antibody. RPE atrophy was studied in mice deleted for Sod2 (the gene for mitochondrial superoxide dismutase) specifically in the RPE. The mice were treated orally with daily doses of xaliproden at 0.5 and 3 mg/kg for 4 months. The retinal structure was analyzed with spectral domain optical coherence tomography (SD-OCT) and with light and electron microscopy. Retinal function was assessed with full-field electroretinography (ERG) and with optokinetic measurements.. Xaliproden led to a dose-dependent increase in cell survival following treatment with paraquat. Synthesis of the antioxidant response genes NqO1, GSTM1, CAT, HO-1, and Nrf2 was increased in response to the drug, as was the zinc chaperone metallothionein. Treatment of cells with TNF-α led to increased production of IL-1β, IL-6, chemokine (C-C motif) ligand 20 (CCL20), and vascular endothelial growth factor (VEGF) by ARPE-19 cells, and this response was attenuated by treatment with xaliproden. TNF-α also led to a decrease in the TEER that was prevented by treatment with the 5HT1a agonist. Daily gavage with xaliproden at either dose induced the production of protective enzymes in the mouse retina, and treatment of the Sod2-deleted mice with the drug showed improved thickness of the outer nuclear layer and improved visual acuity relative to the control-treated mice. There was no significant difference in full-field scotopic ERG among the treatment groups, however. Vacuolization of the RPE and disorganization of the photoreceptor outer segments were reduced at both dose levels of xaliproden.. Xaliproden protected RPE cells from oxidative and inflammatory insults and protected the mouse RPE and retina from RPE atrophy in the face of excess mitochondrial oxidative stress. These results suggest that this drug, which had a reasonable safety profile in clinical trials, may be used to prevent the progression of geographic atrophy in humans.

Topics: Administration, Oral; Animals; Cell Line; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Repositioning; Electric Impedance; Electroretinography; Enzyme-Linked Immunosorbent Assay; Geographic Atrophy; Humans; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Transgenic; Naphthalenes; Pyridines; Real-Time Polymerase Chain Reaction; Retina; Retinal Pigment Epithelium; Serotonin 5-HT1 Receptor Agonists; Tomography, Optical Coherence; Zonula Occludens-1 Protein

Aims. Repetitive brief ischemia and reperfusion (I/R) is associated with left ventricular dysfunction during development of ischemic cardiomyopathy. We investigated the role of zinc-donor proteins metallothionein MT1 and MT2 in a closed-chest murine model of I/R. Methods. Daily 15-minute LAD-occlusion was performed for 1, 3, and 7 days in SV129 (WT)- and MT1/2 knockout (MT(-/-))-mice (n = 8-10/group). Hearts were examined with M-mode echocardiography and processed for histological and mRNA studies. Results. Expression of MT1/2 mRNA was transiently induced during repetitive I/R in WT-mice, accompanied by a transient inflammation, leading to interstitial fibrosis with left ventricular dysfunction without infarction. In contrast, MT(-/-)-hearts presented with enhanced apoptosis and small infarctions leading to impaired global and regional pump function. Molecular analysis revealed maladaptation of myosin heavy chain isoforms and antioxidative enzymes in MT1/2(-/-)-hearts. Despite their postponed chemokine induction we found a higher total neutrophil density and macrophage infiltration in small infarctions in MT(-/-)-hearts. Subsequently, higher expression of osteopontin 1 and tenascin C was associated with increased myofibroblast density resulting in predominately nonreversible fibrosis and adverse remodeling in MT1/2(-/-)-hearts. Conclusion. Cardioprotective effects of MT1/2 seem to be exerted via modulation of contractile elements, antioxidative enzymes, inflammatory response, and myocardial remodeling.

Topics: Animals; Apoptosis; Cardiomyopathies; Disease Models, Animal; Echocardiography; Metallothionein; Mice; Mice, Knockout; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Osteopontin; Tenascin

Herein, we describe the preparation of liposomes with folate-targeting properties for the encapsulation of anti-sarcosine antibodies (antisarAbs@LIP) and sarcosine (sar@LIP). The competitive inhibitory effects of exogenously added folic acid supported the role of folate targeting in liposome internalization. We examined the effects of repeated administration on mice PC-3 xenografts. Sar@LIP treatment significantly increased tumor volume and weight compared to controls treated with empty liposomes. Moreover, antisarAbs@LIP administration exhibited a mild antitumor effect. We also identified differences in gene expression patterns post-treatment. Furthermore, Sar@LIP treatment resulted in decreased amounts of tumor zinc ions and total metallothioneins. Examination of the spatial distribution across the tumor sections revealed a sarcosine-related decline of the MT1X isoform within the marginal regions but an elevation after antisarAbs@LIP administration. Our exploratory results demonstrate the importance of sarcosine as an oncometabolite in PCa. Moreover, we have shown that sarcosine can be a potential target for anticancer strategies in management of PCa.

Topics: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Disease Models, Animal; Dose-Response Relationship, Drug; Folic Acid; Humans; Liposomes; Male; Metallothionein; Mice; Models, Biological; Phosphatidylethanolamines; Prostatic Neoplasms; Sarcosine; Tumor Burden; Xenograft Model Antitumor Assays; Zinc

Intracellular zinc overload causes neuronal injury during the course of neurological disorders, whereas mild levels of zinc are beneficial to neurons. Previous reports indicated that non-steroidal anti-inflammatory drugs, including indomethacin and aspirin, can reduce the risk of ischemic stroke. This study found that chronic pretreatment of rats with indomethacin, a non-selective cyclooxygenase inhibitor, provided tolerance to ischemic injuries in an animal model of stroke by eliciting moderate zinc elevation in neurons. Consecutive intraperitoneal injection of indomethacin (3mg/kg/day for 28 days) led to modest increases in intraneuronal zinc as well as synaptic zinc content, with no significant stimulation of neuronal death. Furthermore, indomethacin induced the expressions of intracellular zinc homeostatic and neuroprotective proteins, rendering the brain resistant against ischemic damages and improving neurological outcomes. However, administration of a zinc-chelator, N,N,N',N'-tetra(2-picolyl)ethylenediamine (TPEN; 15 mg/kg/day), immediately after indomethacin administration eliminated the beneficial actions of the drug. Therefore, indomethacin preconditioning can modulate intracellular zinc availability, contributing to ischemic tolerance in the brain after stroke.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Brain; Carrier Proteins; Cell Death; Chelating Agents; Disease Models, Animal; Drug Administration Schedule; Ethylenediamines; HSP70 Heat-Shock Proteins; Hypoxia-Inducible Factor 1, alpha Subunit; Indomethacin; Infarction, Middle Cerebral Artery; Male; Metallothionein; Neurons; Rats; Rats, Sprague-Dawley; Time Factors; Zinc

Obesity continues to rise as an alarming global epidemic. System level mechanisms, diagnostics, and therapeutics are sorely needed so as to identify at risk individuals and design appropriate population scale interventions. The present study evaluated the protective role of metallothioneins (MTs) against obesity and high-fat diet-induced effects such as insulin resistance in both male and female MT-1+2 knockout and MT-3 knockout mice. As the metabolome is closest to the functional phenotype, changes in metabolite levels were also evaluated, and the direct or indirect involvement of MTs in metabolism examined. MT-1+2-, MT-3 knockout, and wild-type mice were given a high-fat diet for 2 months. Variation in body weight gain, tissue weight, and response to oral glucose tolerance test and insulin tolerance test were determined and compared to mice that received the control diet. Effect of the high-fat diet on the knockout mice were investigated on the metabolome level in specific tissues using metabolomics. Both knockout mice strains were more susceptible to high-fat diet-induced effects, such as weight gain and moderate insulin resistance, with the MT-3 knockout mice most susceptible. Brain tissue of the knockout mice showed most metabolic variation and pointed to possible impairment of mitochondrial function. The protective effect of MTs against high-fat diet and obesity-induced effects such as insulin resistance was evident from our observations. The putative role MTs play in mitochondrial function is possibly the main contributor to the lack of these effects in wild-type mice. Considering the expression profiles of the MT isoforms and similarity in brain metabolic variation in the knockout strains, it appears that they promote mitochondrial function in the hypothalamus, thereby limiting weight gain and insulin resistance. Furthermore, metabolomics research in preclinical models of obesity and in the clinic is warranted in the near future.

Topics: Adipose Tissue; Animals; Blood Glucose; Body Weight; Cerebral Cortex; Diet, High-Fat; Disease Models, Animal; Female; Glucose Tolerance Test; Insulin; Insulin Resistance; Male; Metabolome; Metabolomics; Metallothionein; Metallothionein 3; Mice; Mice, Knockout; Obesity

Disorders of the prostate and lower urinary tract are common in elderly men. We investigated the role of metallothionein-1 (MT1) in prostate carcinogenesis by generating a prostate-specific, MT1-expressing mouse. Unexpectedly, genomic analyses revealed that a 12.1-kb genomic region harboring several conserved noncoding elements was unintentionally deleted, upstream of the transgene integration site in the mouse, which we named it 12.1ΔMT1. Male 12.1ΔMT1 mice chronically treated with testosterone (T) plus 17β-estradiol (E2) to induce prostate cancer exhibited no evidence of precancerous or cancerous lesions. Instead, most of them exhibited a bladder outlet obstruction (BOO) phenotype not observed in treated wild-type (WT) mice. Thus, we hypothesized that 12.1ΔMT1 is a novel model for studying the hormonal requirement for BOO induction. Adult male 12.1ΔMT1 and WT mice were treated with T, E2, bisphenol A (BPA), T+E2, or T+BPA for up to 6 months. Histologic and immunohistochemical analysis of the prostate, bladder, and urethra were performed. No significant prostate pathologies were observed in WT or 12.1ΔMT1 mice treated with any of the hormone regimens. As expected, prostatic regression occurred in all E2-treated animals (WT and 12.1ΔMT1). Of great interest, despite a small prostate, 100% of E2-treated 12.1ΔMT1 mice, but only 40% of E2-treated WT mice, developed severe BOO (P<0.01). In contrast, T+E2 treatment was less effective than E2 treatment in inducing severe BOO in 12.1ΔMT1 mice (68%, P<0.05) and was completely ineffective in WT animals. Similarly, T, BPA, and T+BPA treatments did not induce BOO in either WT or 12.1ΔMT1 mice. The BOO pathology includes a thinner detrusor wall, narrowing of bladder neck and urethral lumen, and basal cell hyperplasia in the bladder body and urethra. These findings indicate that 12.1ΔMT1 mice exhibit enhanced susceptibility to E2-induced BOO that is independent of prostate enlargement but that is attenuated by the conjoint treatment with T.

Topics: Animals; Disease Models, Animal; Estradiol; Female; Gene Deletion; Hyperplasia; Immunohistochemistry; Male; Metallothionein; Mice; Mice, Transgenic; Phenotype; Prostate; Receptors, N-Methyl-D-Aspartate; Urethra; Urinary Bladder; Urinary Bladder Neck Obstruction

Diabetic cardiomyopathy is a prominent cause of heart failure in patients with diabetes mellitus. Currently, there is no specific treatment for diabetic cardiomyopathy. This study aimed to investigate the effect and underlying mechanisms of Zinc (Zn) supplementation in the protection against diabetic cardiomyopathy in a rat model of type 2 diabetes mellitus (T2DM). T2DM-like lesions in male Wistar rats were induced by introducing the high-fat diet and by administration of streptozocin (STZ). After STZ induction, animals with fasting plasma glucose level ≥16.7 mM were considered as diabetic, and randomly assigned to the group receiving physiological saline (control) or ZnSO4 for 56 days. On days 0, 7, 28 and 56 of treatment, animals were weighed, and their blood samples were analyzed. On day 56, hemodynamic assessment was performed right before the sacrifice of animals. Cardiac tissue specimens were collected and subjected to pathologic assessment, metallothionein (MT) concentration measurement and Western blot analysis of microtubule-associated protein light chain 3 (LC3), the marker of autophagy, and glucose-regulated protein-78 (GRP78), an oxidative stress marker. High-fat diet feeding followed by STZ administration resulted in weight loss, hyperglycemia, polydipsia, polyphagia, hemodynamic anomalies and a significant increase in the myocardial content of LC3 and GRP78 proteins, but not in MT protein. Zn supplementation effectively attenuated all these aberrations induced by high-fat diet and STZ. These findings suggest that Zn might be a protective factor in diabetic cardiomyopathy, acting in two ways: at least partially, through inhibiting autophagy and by endoplasmic reticulum stress.

Topics: Animals; Autophagy; Biomarkers; Blood Glucose; Diabetes Mellitus, Type 2; Diabetic Cardiomyopathies; Diet, High-Fat; Dietary Supplements; Disease Models, Animal; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Male; Metallothionein; Microtubule-Associated Proteins; Rats; Rats, Wistar; Streptozocin; Treatment Outcome; Zinc

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), an incurable motor neuron disease. The pathogenesis of the disease is poorly understood, but intracellular copper dyshomeostasis has been implicated as a key process in the disease. We recently observed that metallothioneins (MTs) are an excellent target for the modification of copper dyshomeostasis in a mouse model of ALS (SOD1(G93A)). Here, we offer a therapeutic strategy designed to increase the level of endogenous MTs. The upregulation of endogenous MTs by dexamethasone, a synthetic glucocorticoid, significantly improved the disease course and rescued motor neurons in SOD1(G93A) mice, even if the induction was initiated when peak body weight had decreased by 10%. Neuroprotection was associated with the normalization of copper dyshomeostasis, as well as with decreased levels of SOD1(G93A) aggregates. Importantly, these benefits were clearly mediated in a MT-dependent manner, as dexamethasone did not provide any protection when endogenous MTs were abolished from SOD1(G93A) mice. In conclusion, the upregulation of endogenous MTs represents a promising strategy for the treatment of ALS linked to mutant SOD1.

Topics: Age Factors; Amyotrophic Lateral Sclerosis; Animals; Anti-Inflammatory Agents; CD11b Antigen; Copper; Dexamethasone; Disease Models, Animal; Glial Fibrillary Acidic Protein; Humans; Lipid Peroxides; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphopyruvate Hydratase; Protein Isoforms; Spinal Cord; Superoxide Dismutase; Time Factors; Up-Regulation

Some dietary factors could inhibit lead toxicity. The aim of this study was to evaluate the effect of dietary compounds rich in unsaturated fatty acids (FA) on blood lead level, lipid metabolism, and vascular reactivity in rats. Serum metallothionein and organs' lead level were evaluated with the aim of assessing the possible mechanism of unsaturated FA impact on blood lead level. For three months, male Wistar rats that were receiving drinking water with (100 ppm Pb) or without lead acetate were supplemented per os daily with virgin olive oil or linseed oil (0.2 mL/kg b.w.) or egg derived lecithin fraction: "super lecithin" (50 g/kg b.w.). Mesenteric artery was stimulated ex vivo by norepinephrine (NE) administered at six different doses. Lecithin supplementation slightly reduced pressor responses of artery to NE. Lead administered to rats attenuated the beneficial effect of unsaturated FA on lipid metabolism and vascular reactivity to adrenergic stimulation. On the other hand, the super lecithin and linseed oil that were characterized by low omega-6 to omega-3 ratio (about 1) reduced the blood lead concentration. This effect was observed in lead poisoned rats (p < 0.0001) and also in rats nonpoisoned with lead (p < 0.05).

Topics: Animals; Carrier Proteins; Dietary Fats, Unsaturated; Dietary Supplements; Disease Models, Animal; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Fatty Acids, Unsaturated; In Vitro Techniques; Lead; Lead Poisoning; Lecithins; Linseed Oil; Lipids; Male; Mesenteric Arteries; Metallothionein; Norepinephrine; Olive Oil; Rats; Rats, Wistar; Vasoconstrictor Agents

Depleted uranium (DU) has been widely used in both civilian and military activities, and the kidney is the main target organ of DU during acute high-dose exposures. In this study, the nephrotoxicity caused by DU in metallothionein-1/2-null mice (MT-/-) and corresponding wild-type (MT+/+) mice was investigated to determine any associations with MT. Each MT-/- or MT+/+ mouse was pretreated with a single dose of DU (10mg/kg, intraperitoneal injection) or an equivalent volume of saline. After 4days of DU administration, kidney changes were assessed. After DU exposure, serum creatinine and serum urea nitrogen in MT-/- mice significantly increased than in MT+/+ mice, with more severe kidney pathological damage. Moreover, catalase and superoxide dismutase (SOD) decreased, and generation of reactive oxygen species and malondialdehyde increased in MT-/- mice. The apoptosis rate in MT-/- mice significantly increased, with a significant increase in both Bax and caspase 3 and a decrease in Bcl-2. Furthermore, sodium-glucose cotransporter (SGLT) and sodium-phosphate cotransporter (NaPi-II) were significantly reduced after DU exposure, and the change of SGLT was more evident in MT-/- mice. Finally, exogenous MT was used to evaluate the correlation between kidney changes induced by DU and MT doses in MT-/- mice. The results showed that, the pathological damage and cell apoptosis decreased, and SOD and SGLT levels increased with increasing dose of MT. In conclusion, MT deficiency aggravated DU-induced nephrotoxicity, and the molecular mechanisms appeared to be related to the increased oxidative stress and apoptosis, and decreased SGLT expression.

Topics: Animals; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Biomarkers; Blood Urea Nitrogen; Creatinine; Disease Models, Animal; Kidney; Kidney Diseases; Male; Metallothionein; Mice, Knockout; Oxidative Stress; Reactive Oxygen Species; Sodium-Glucose Transport Proteins; Time Factors; Uranyl Nitrate

Epilepsy is a neurological disorder that has been associated with oxidative stress therefore epilepsy models have been develop such as kainic acid and pentylenetetrazol are usually used to understanding of the molecular mechanisms of this disease. We examined the metallothionein expression in rat brains of treated with kainic acid and pentylenetetrazol. Increase in metallothionein and nitrotirosyne immunoreactivity of both seizures epilepsy models was observed. Moreover, we show a significant increase on levels of MT expression. These results suggest that the increase of metallothionein expression is related with kainic acid and pentylenetetrazol treatments as response to damage mediated by oxidative stress.

Topics: Animals; Brain; Disease Models, Animal; Epilepsy; Kainic Acid; Male; Metallothionein; Oxidative Stress; Pentylenetetrazole; Rats; Rats, Wistar; Tyrosine

Many studies have shown that the pathogenesis of liver injury includes oxidative stress. MicroRNA-122 may be a marker for the early diagnosis of drug-induced liver injury. However, the relationship between microRNA-122 and oxidative stress in anti-tuberculosis drug-induced liver injury remains unknown. We measured changes in tissue microRNA-122 levels and indices of oxidative stress during liver injury in mice after administration of isoniazid, a first-line anti-tuberculosis drug. We quantified microRNA-122 expression and indices of oxidative stress at 7 time points, including 1, 3, and 5 days and 1, 2, 3, and 4 weeks. The tissue microRNA-122 levels and oxidative stress significantly changed at 3 and 5 days, suggesting that isoniazid-induced liver injury reduces oxidative stress and microRNA-122 expression compared to in the control group (P < 0.05). Notably, over the time course of isoniazid-induced liver injury, mitochondrial ribosome protein S11 gene, the target of microRNA-122, began to change at 5 days (P < 0.05). The tissue microRNA-122 profile may affect oxidative stress by regulating mitochondrial ribosome protein S11 gene during isoniazid-induced liver injury, which may contribute to the response mechanisms of microRNA-122 and oxidative stress.

Topics: Animals; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Female; Gene Expression; Isoniazid; Liver Function Tests; Male; Metallothionein; Mice; MicroRNAs; Oxidation-Reduction; Oxidative Stress; Time Factors

Over 170 mutations in superoxide dismutase-1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS), a lethal motor neuron disease. Although the molecular properties of SOD1 mutants differ considerably, we have recently shown that intracellular copper dyshomeostasis is a common pathogenic feature of different SOD1 mutants. Thus, the potentiation of endogenous copper regulation could be a therapeutic strategy. In this study, we investigated the effects of the overexpression of metallothionein-I (MT-I), a major copper-regulating protein, on the disease course of a mouse model of ALS (SOD1(G93A)). Using double transgenic techniques, we found that the overexpression of MT-I in SOD1(G93A) mice significantly extended the lifespan and slowed disease progression, but the effects on disease onset were modest. Genetically induced MT-I normalized copper dyshomeostasis in the spinal cord without influencing SOD1 enzymatic activity. The overexpression of MT-I in SOD1(G93A) mice markedly attenuated the pathological features of the mice, including the death of motor neurons, the degeneration of ventral root axons, the atrophy of skeletal muscles, and the activation of glial cells. Double transgenic mice also showed a decreased level of SOD1 aggregates within the glial cells of the spinal cord. Furthermore, the overexpression of MT-I in SOD1(G93A) mice reduced the number of spheroid-shaped astrocytes cleaved by active caspase-3. We concluded that therapeutic strategies aimed at the potentiation of copper regulation by MT-I could be of benefit in cases of ALS caused by SOD1 mutations.

Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Caspase 3; Copper; Disease Models, Animal; Disease Progression; Female; Gene Expression; Humans; Intracellular Space; Longevity; Male; Metallothionein; Mice; Motor Neurons; Mutation; Neuroglia; Phenotype; Proteolysis; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1

To facilitate development of novel disease-modifying therapies for lysosomal storage disorder (LSDs) characterized by nervous system involvement such as metachromatic leukodystrophy (MLD), molecular markers for monitoring disease progression and therapeutic response are needed. To this end, we sought to identify blood transcripts associated with the progression of MLD.. Genome-wide expression analysis was performed in primary T lymphocytes of 24 patients with MLD compared to 24 age- and sex-matched healthy controls. Genes associated with MLD were identified, confirmed on a quantitative polymerase chain reaction platform, and replicated in an independent patient cohort. mRNA and protein expression of the prioritized gene family of metallothioneins was evaluated in postmortem patient brains and in mouse models representing 6 other LSDs. Metallothionein expression during disease progression and in response to specific treatment was evaluated in 1 of the tested LSD mouse models. Finally, a set of in vitro studies was planned to dissect the biological functions exerted by this class of molecules.. Metallothionein genes were significantly overexpressed in T lymphocytes and brain of patients with MLD and generally marked nervous tissue damage in the LSDs here evaluated. Overexpression of metallothioneins correlated with measures of disease progression in mice and patients, whereas their levels decreased in mice upon therapeutic treatment. In vitro studies indicated that metallothionein expression is regulated in response to oxidative stress and inflammation, which are biochemical hallmarks of lysosomal storage diseases.. Metallothioneins are potential markers of neurologic disease processes and treatment response in LSDs.

Topics: Animals; Biomarkers; Coculture Techniques; Disease Models, Animal; Humans; Leukocytes, Mononuclear; Leukodystrophy, Metachromatic; Lysosomal Storage Diseases; Metallothionein; Mice; Mice, Inbred C57BL; Molecular Dynamics Simulation; Primary Cell Culture

Human epidemiological and animal studies have shown the beneficial effect of zinc supplementation on mitigating diabetic nephropathy. However, the mechanism by which zinc protects the kidney from diabetes remains unknown. Here we demonstrate the therapeutic effects of zinc on diabetes-induced renal pathological and functional changes. These abnormalities were found in both transgenic OVE26 and Akt2-KO diabetic mouse models, accompanied by significant changes in glucose-metabolism-related regulators. The changes included significantly decreased phosphorylation of Akt and GSK-3β, increased phosphorylation of renal glycogen synthase, decreased expression of hexokinase II and PGC-1α, and increased expression of the Akt negative regulators PTEN, PTP1B, and TRB3. All of these were significantly prevented by zinc treatment for 3 months. Furthermore, zinc-stimulated changes in glucose metabolism mediated by Akt were actually found to be metallothionein dependent, but not Akt2 dependent. These results suggest that the therapeutic effects of zinc in diabetic nephropathy are mediated, in part, by the preservation of glucose-metabolism-related pathways via the prevention of diabetes-induced upregulation of Akt negative regulators. Given that zinc deficiency is very common in diabetics, this finding implies that regularly monitoring zinc levels in diabetic patients, as well as supplementing if low, is important in mitigating the development of diabetic nephropathy.

Topics: Animals; Diabetic Nephropathies; Disease Models, Animal; Glucose; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Metallothionein; Mice; Mice, Inbred NOD; Mice, Transgenic; Proto-Oncogene Proteins c-akt; Signal Transduction; Zinc

Inflammatory bowel diseases (IBDs) are recurrent intestinal pathologies characterized by a compromised epithelial barrier and an exaggerated immune activation. Mediators of immune cell infiltration may represent new therapeutic opportunities. Metallothioneins (MTs) are stress-responsive proteins with immune-modulating functions. Metallothioneins have been linked to IBDs, but their role in intestinal inflammation is inconclusive. We investigated MT expression in colonic biopsies from IBDs and acute infectious colitis patients and healthy controls and evaluated MT's role in experimental colitis using MT knockout mice and anti-MT antibodies. Antibody potential to target extracellular MT and its mechanism was tested in vitro. Biopsies of patients with active colitis showed infiltration of MT-positive cells in a pattern that correlated with the grade of inflammation. MT knockout mice displayed less severe acute dextran sulphate sodium (DSS)-induced colitis compared to congenic wild-type mice based on survival, weight loss, colon length, histological inflammation and leukocyte infiltration. Chronic DSS-colitis confirmed that Mt1 and Mt2 gene disruption enhances clinical outcome. Blockade of extracellular MT with antibodies reduced F4/80-positive macrophage infiltration in DSS- and trinitrobenzene sulphonic acid-colitis, with a tendency towards a better outcome. Whole-body single-photon emission computer tomography of mice injected with radioactive anti-MT antibodies showed antibody accumulation in the colon during colitis and clearance during recovery. Necrotic and not apoptotic cell death resulted in western blot MT detection in HT29 cell supernatant. In a Boyden chamber migration assay, leukocyte attraction towards the necrotic cell supernatant could be abolished with anti-MT antibody, indicating the chemotactic potential of endogenous released MT. Our results show that human colitis is associated with infiltration of MT-positive inflammatory cells. Since antibody blockade of extracellular MT can reduce colitis in mice, MT may act as a danger signal and may represent a novel target for reducing leukocyte infiltration and inflammation in IBD patients.

Topics: Acute Disease; Adolescent; Adult; Aged; Aged, 80 and over; Animals; Antibodies; Apoptosis; Biopsy; Case-Control Studies; Chemotaxis, Leukocyte; Chronic Disease; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Female; HT29 Cells; Humans; Macrophages; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Necrosis; Severity of Illness Index; Signal Transduction; Time Factors; Trinitrobenzenesulfonic Acid; Young Adult

To study the acute and subacute pulmonary toxicity of colloidal silver nanoparticles (Ag-NPs), 0 or 100 ppm of Ag-NPs were instilled intratracheally in mice. Cellular and biochemical parameters in bronchoalveolar lavage fluid (BALF) and histological alterations were determined 1, 3, 7, 15, and 30 days after instillation. Ag-NPs induced moderate pulmonary inflammation and injury on BALF indices during the acute period; however, these changes gradually regressed in a time-dependent manner. Concomitant histopathological and laminin immunohistochemical findings generally correlated to BALF data. Superoxide dismutase and metallothionein expression occurred in particle-laden macrophages and alveolar epithelial cells, which correlated to lung lesions in mice treated with Ag-NPs. These findings suggest that instillation of Ag-NPs causes transient moderate acute lung inflammation and tissue damage. Oxidative stress may underlie the induction of injury to lung tissue. Moreover, the expression of metallothionein in tissues indicated the protective response to exposure to Ag-NPs.

Topics: Acute Disease; Administration, Inhalation; Animals; Colloids; Disease Models, Animal; Epithelial Cells; Lung; Lung Diseases; Macrophages; Male; Metal Nanoparticles; Metallothionein; Mice; Mice, Inbred ICR; Silver; Time Factors

Metallothioneins (MT) are a family of low molecular weight proteins that are silenced during colorectal cancer progression, mainly through epigenetic mechanisms, and this loss is associated with poor survival. In this article, we show that overexpression of the MT1G isoform sensitizes colorectal cell lines to the chemotherapeutic agents oxaliplatin (OXA) and 5-fluorouracil (5-FU), in part through enhancing p53 and repressing NF-κB activity. Despite being silenced, MTs can be reinduced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate. In fact, this induction contributes to the cytotoxicity of these agents, given that silencing of MTs by siRNAs reduces their growth-inhibitory activities. Zinc ions also potently enhance MT expression and are cytotoxic to cancer cells. We show for the first time that OXA and 5-FU induce higher levels of intracellular labile zinc, as measured using the fluorescent probe FLUOZIN-3, and that such zinc contributes to the activation of p53 and repression of NF-κB. Addition of zinc enhanced growth inhibition by OXA and 5-FU, and was also capable of resensitizing 5-FU-resistant cell lines to levels comparable with sensitive cell lines. This effect was MT independent because silencing MTs did not affect zinc cytotoxicity. In conclusion, we show that MT induction and zinc administration are novel strategies to sensitize colorectal cancer cells to presently utilized chemotherapeutic agents.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Colorectal Neoplasms; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histone Deacetylase Inhibitors; HT29 Cells; Humans; Male; Metallothionein; NF-kappa B; Protein Transport; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays; Zinc

Chagas disease, caused by Trypanosoma cruzi, represents an endemic among Latin America countries. The participation of free radicals, especially nitric oxide (NO), has been demonstrated in the pathophysiology of seropositive individuals with T. cruzi. In Chagas disease, increased NO contributes to the development of cardiomyopathy and megacolon. Metallothioneins (MTs) are efficient free radicals scavengers of NO in vitro and in vivo. Here, we developed a murine model of the chronic phase of Chagas disease using endemic T. cruzi RyCH1 in BALB/c mice, which were divided into four groups: infected non-treated (Inf), infected N-monomethyl-L-arginine treated (Inf L-NAME), non-infected L-NAME treated and non-infected vehicle-treated. We determined blood parasitaemia and NO levels, the extent of parasite nests in tissues and liver MT-I expression levels. It was observed that NO levels were increasing in Inf mice in a time-dependent manner. Inf L-NAME mice had fewer T. cruzi nests in cardiac and skeletal muscle with decreased blood NO levels at day 135 post infection. This affect was negatively correlated with an increase of MT-I expression (r = -0.8462, p < 0.0001). In conclusion, we determined that in Chagas disease, an unknown inhibitory mechanism reduces MT-I expression, allowing augmented NO levels.

Topics: Animals; Antioxidants; Chagas Disease; Disease Models, Animal; Enzyme Inhibitors; Female; Heart; Metallothionein; Mice, Inbred BALB C; Muscle, Skeletal; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Oxidative Stress; Parasitemia; Real-Time Polymerase Chain Reaction; Statistics, Nonparametric; Time Factors; Trypanosoma cruzi

Neuronal ceroid lipofuscinoses (NCLs), a group of genetically distinct fatal neurodegenerative disorders with no treatment or cure, are clinically characterised by progressive motor and visual decline leading to premature death. While the underlying pathological mechanisms are yet to be precisely determined, the diseases share several common features including inflammation, lysosomal lipofuscin deposits and lipid abnormalities. An important hallmark of most common neurodegenerative disorders including Alzheimer's, Parkinson's and motor neuron diseases is deregulation of biologically active metal homeostasis. Metals such as zinc, copper and iron are critical enzyme cofactors and are important for synaptic transmission in the brain, but can mediate oxidative neurotoxicity when homeostatic regulatory mechanisms fail. We previously demonstrated biometal accumulation and altered biometal transporter expression in 3 animal models of CLN6 NCL disease. In this study we investigated the hypothesis that altered biometal homeostasis may be a feature of NCLs in general using 3 additional animal models of CLN1, CLN3 and CLN5 disease. We demonstrated significant accumulation of the biometals zinc, copper, manganese, iron and cobalt in these mice. Patterns of biometal accumulation in each model preceded significant neurodegeneration, and paralleled the relative severity of disease previously described for each model. Additionally, we observed deregulation of transcripts encoding the anti-oxidant protein, metallothionein (Mt), indicative of disruptions to biometal homeostasis. These results demonstrate that altered biometal homeostasis is a key feature of at least 4 genetically distinct forms of NCL disease.

Topics: Animals; Brain; Cobalt; Copper; Disease Models, Animal; Homeostasis; Iron; Lysosomal Membrane Proteins; Manganese; Membrane Glycoproteins; Metallothionein; Metals, Heavy; Mice; Mice, Knockout; Molecular Chaperones; Neuronal Ceroid-Lipofuscinoses; Thiolester Hydrolases; Transcription, Genetic; Zinc

Copper deficiency had been suggested to link between fructose-enriched diet (FED) and the development of non-alcoholic fatty liver disease (NAFLD). In this study, we characterized changes in hepatic copper concentrations and hepatic oxidative milieu, in rats with the metabolic syndrome and NAFLD as a result of FED with pharmacological manipulations to reduce blood pressure or plasma triglycerides. Changes in plasma and hepatic copper concentrations were correlated with changes observed in the immunohistochemical hepatic expression of copper-zinc-superoxide dismutase (CuZnSOD; SOD1), metallothionein (MT) and nitrotyrosine (NITT). FED administration was associated with a 2.2-fold reduction in hepatic copper concentrations, a decrease in the hepatic SOD1 expression, disappearance of the hepatic MT expression and increase in the hepatic NITT expression. Bezafibrate administration restored the hepatic copper concentrations and the hepatic SOD1 expression to levels that were observed in the control rats. A significant positive correlation between hepatic copper concentrations and the values of hepatic SOD1 expression of each animal included in this study was found. Administration of either captopril or bezafibrate increased hepatic MT expression, however, to levels that were lower than those observed in the control group. Administration of either amlodipine, or captopril or bezafibrate to the FED rats, had no effect on hepatic NITT expression. NAFLD development in FED rats is associated with a decrease in hepatic copper concentrations that is associated with a decrease in the hepatic SOD1 expression. Bezafibrate administration increases hepatic copper concentrations and restores the hepatic SOD1 expression.

Topics: Animals; Antihypertensive Agents; Bezafibrate; Captopril; Copper; Disease Models, Animal; Fatty Liver; Hypolipidemic Agents; Liver; Male; Metallothionein; Rats, Sprague-Dawley; Superoxide Dismutase

Muscular dystrophy (MD) is a disease characterized by skeletal muscle necrosis and the progressive accumulation of fibrotic tissue. While transforming growth factor (TGF)-β has emerged as central effector of MD and fibrotic disease, the cell types in diseased muscle that underlie TGFβ-dependent pathology have not been segregated. Here, we generated transgenic mice with myofiber-specific inhibition of TGFβ signaling owing to expression of a TGFβ type II receptor dominant-negative (dnTGFβRII) truncation mutant. Expression of dnTGFβRII in myofibers mitigated the dystrophic phenotype observed in δ-sarcoglycan-null (Sgcd(-/-)) mice through a mechanism involving reduced myofiber membrane fragility. The dnTGFβRII transgene also reduced muscle injury and improved muscle regeneration after cardiotoxin injury, as well as increased satellite cell numbers and activity. An unbiased global expression analysis revealed a number of potential mechanisms for dnTGFβRII-mediated protection, one of which was induction of the antioxidant protein metallothionein (Mt). Indeed, TGFβ directly inhibited Mt gene expression in vitro, the dnTGFβRII transgene conferred protection against reactive oxygen species accumulation in dystrophic muscle and treatment with Mt mimetics protected skeletal muscle upon injury in vivo and improved the membrane stability of dystrophic myofibers. Hence, our results show that the myofibers are central mediators of the deleterious effects associated with TGFβ signaling in MD.

Topics: Animals; Cell Membrane; Cobra Cardiotoxin Proteins; Crotoxin; Disease Models, Animal; Drug Combinations; Gene Expression Profiling; Gene Expression Regulation; Humans; Metallothionein; Mice; Mice, Transgenic; Muscular Dystrophies; Mutation; Myofibrils; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sarcoglycans; Satellite Cells, Skeletal Muscle; Signal Transduction; Transforming Growth Factor beta; Transgenes

The Polycomb group (PcG) protein Bmi1 is an essential epigenetic regulator of stem cell function during normal development and in adult organ systems. We show that mild up-regulation of Bmi1 expression in the adult stem cells of the skeletal muscle leads to a remarkable improvement of muscle function in a mouse model of Duchenne muscular dystrophy. The molecular mechanism underlying enhanced physiological function of Bmi1 depends on the injury context and it is mediated by metallothionein 1 (MT1)-driven modulation of resistance to oxidative stress in the satellite cell population. These results lay the basis for developing Bmi1 pharmacological activators, which either alone or in combination with MT1 agonists could be a powerful novel therapeutic approach to improve regeneration in muscle wasting conditions.

Topics: Animals; Cell Differentiation; Chronic Disease; Disease Models, Animal; DNA Damage; Gene Expression Profiling; Gene Expression Regulation; Humans; Macular Degeneration; Metallothionein; Mice, Inbred mdx; Mice, Transgenic; Muscle Development; Muscle Strength; Muscle, Skeletal; Oxidative Stress; PAX7 Transcription Factor; Polycomb Repressive Complex 1; Proto-Oncogene Proteins; Reactive Oxygen Species; Regeneration; Reproducibility of Results; Satellite Cells, Skeletal Muscle; Systems Biology

We previously demonstrated that metallothionein (MT)-mediated protection from diabetes-induced pathological changes in cardiac tissues is related to suppression of superoxide generation and protein nitration. The present study investigated which diabetes-nitrated protein(s) mediate the development of these pathological changes by identifying the panel of nitrated proteins present in diabetic hearts of wild-type (WT) mice and not in those of cardiac-specific MT-overexpressing transgenic (MT-TG) mice. At 2, 4, 8, and 16 wk after streptozotocin induction of diabetes, histopathological examination of the WT and MT-TG diabetic hearts revealed cardiac structure derangement and remodeling, significantly increased superoxide generation, and 3-nitrotyrosine accumulation. A nitrated protein of 58 kDa, succinyl-CoA:3-ketoacid CoA transferase-1 (SCOT), was identified by mass spectrometry. Although total SCOT expression was not significantly different between the two types of mice, the diabetic WT hearts showed significantly increased nitration content and dramatically decreased catalyzing activity of SCOT. Although SCOT nitration sites were identified at Tyr(76), Tyr(117), Tyr(135), Tyr(226), Tyr(368), and Trp(374), only Tyr(76) and Trp(374) were found to be located in the active site by three-dimensional structure modeling. However, only Trp(374) showed a significantly different nitration level between the WT and MT-TG diabetic hearts. These results suggest that MT prevention of diabetes-induced pathological changes in cardiac tissues is most likely mediated by suppression of SCOT nitration at Trp(374).

Topics: Animals; Coenzyme A-Transferases; Diabetes Mellitus, Experimental; Disease Models, Animal; Energy Metabolism; Heart Diseases; Male; Metallothionein; Mice; Mice, Inbred Strains; Mice, Transgenic; Myocardium; Nitrogen; Protein Structure, Tertiary; Superoxides; Tryptophan; Tyrosine

Compared to the Dark Agouti (DA), the Albino Oxford (AO) rat strain exhibits lower susceptibility to the induction of experimental autoimmune encephalomyelitis (EAE). Here, we investigated the potential contribution of the heavy metal-binding proteins metallothioneins (MTs) I/II to these effects.. Rats were immunized with bovine brain homogenate emulsified in complete Freund's adjuvant or only with complete Freund's adjuvant. The expression patterns of MTs mRNA and proteins and tissue concentrations of Zn2+ and Cu2+ were estimated in the brain and in the liver on days 7 and 12 after immunization, by real-time PCR, immunohistochemistry and inductively coupled plasma spectrometry, respectively. Additionally, the hepatic transforming growth factor beta and nuclear factor kappa B immunoreactivities were tested.. Clinical signs of EAE were not induced in AO rats, but they upregulated the expression of MT I/II proteins in the brain (hippocampus and cerebellum) and in the liver, similarly as DA rats. The transcriptional activation of MT-I occurred, however, only in DA rats, which accumulated also more zinc in the brain and in the liver. In contrast, intact AO rats had greater hepatic MT-I mRNA immunoreactivity and more Cu2+ in the hippocampus. Besides, in immunized AO rats a high upregulation of transforming growth factor beta and nuclear factor kappa B immunoreactivities was found in several hepatic structures (vascular endothelium, Kupffer cells and hepatocytes).. Our data show that AO and DA rats differ in constitutive and inductive MT-I gene expression in the brain and in the liver, as well as in the hepatic cytokine profile, suggesting that these mechanisms may contribute to the discrepancy in the susceptibility to EAE.

Topics: Animals; Brain; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Freund's Adjuvant; Gene Expression Regulation; Genetic Predisposition to Disease; Male; Metallothionein; Rats; RNA, Messenger; Species Specificity; Statistics, Nonparametric; T-Lymphocytes; Time Factors

Mutations in the CLN6 gene cause a variant late infantile form of neuronal ceroid lipofuscinosis (NCL; Batten disease). CLN6 loss leads to disease clinically characterized by vision impairment, motor and cognitive dysfunction, and seizures. Accumulating evidence suggests that alterations in metal homeostasis and cellular signaling pathways are implicated in several neurodegenerative and developmental disorders, yet little is known about their role in the NCLs. To explore the disease mechanisms of CLN6 NCL, metal concentrations and expression of proteins implicated in cellular signaling pathways were assessed in brain tissue from South Hampshire and Merino CLN6 sheep. Analyses revealed increased zinc and manganese concentrations in affected sheep brain in those regions where neuroinflammation and neurodegeneration first occur. Synaptic proteins, the metal-binding protein metallothionein, and the Akt/GSK3 and ERK/MAPK cellular signaling pathways were also altered. These results demonstrate that altered metal concentrations, synaptic protein changes, and aberrant modulation of cellular signaling pathways are characteristic features in the CLN6 ovine form of NCL.

Topics: Animals; Brain; Disease Models, Animal; Glycogen Synthase Kinase 3; Manganese; Membrane Proteins; Metallothionein; Mutation; Neuronal Ceroid-Lipofuscinoses; Phosphorylation; Proto-Oncogene Proteins c-akt; Sheep; Signal Transduction; Synapses; Zinc

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.. 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.. 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.. 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.

Topics: Animals; Aorta, Thoracic; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Dietary Supplements; Disease Models, Animal; Female; Male; Metallothionein; Mice; Mice, Transgenic; NF-E2-Related Factor 2; Up-Regulation; Zinc

Gene expression profiles in the amygdala of juvenile rats were compared between the two autistic rat models for mechanistic insights into impaired social behavior and enhanced anxiety in autism. The rats exposed to VPA by intraperitoneal administration to their dams at embryonic day (E) 12 were used as a model for autism (E2IP), and those by subcutaneous administration at postnatal day (P) 14 (P14SC) were used as a model for regressive autism; both of the models show impaired social behavior and enhanced anxiety as symptoms. Gene expression profiles in the amygdala of the rats (E12IP and P14SC) were analyzed by microarray and compared to each other. Only two genes, Neu2 and Mt2a, showed significant changes in the same direction in both of the rat models, and there were little similarities in the overall gene expression profiles between them. It was considered that gene expression changes per se in the amygdala might be an important cause for impaired social behavior and enhanced anxiety, rather than expression changes of particular genes.

Topics: Amygdala; Animals; Animals, Newborn; Anxiety; Autistic Disorder; Behavior, Animal; Disease Models, Animal; Female; Injections, Intraperitoneal; Injections, Subcutaneous; Maternal Exposure; Maternal-Fetal Exchange; Metallothionein; Neuraminidase; Pregnancy; Rats; Rats, Wistar; Social Behavior; Transcriptome; Valproic Acid

Androgen signalling through the androgen receptor (AR) plays a critical role in prostate cancer (PCa) initiation and progression. Estrogen in synergy with androgen is essential for cell growth of the normal and malignant prostate. However, the exact role that estrogen and the estrogen receptor play in prostate carcinogenesis remains unclear. We have previously demonstrated the metastasis-promoting effect of an estrogen receptor beta (ERβ) agonist (genistein) in a patient-derived PCa xenograft model mimicking localized and metastatic disease.. To test the hypothesis that the tumor-promoting activity of genistein was due to its estrogenic properties, we treated the xenograft-bearing mice with genistein and an anti-estrogen compound (ICI 182, 780) and compared the differential gene expression using microarrays.. Using a second xenograft model which was derived from another patient, we showed that genistein promoted disease progression in vivo and ICI 182, 780 inhibited metastatic spread. The microarray analysis revealed that the metallothionein (MT) gene family was differentially expressed in tumors treated by these compounds. Using qRT-PCR, the differences in expression levels were validated in the metastatic and non-metastatic LTL313 PCa xenograft tumor lines, both of which were originally derived from the same PCa patient.. Together our data provide evidence that genistein stimulates and ICI 182, 780 inhibits metastatic progression, suggesting that these effects may be mediated by ERβ signalling.

Topics: Animals; Antineoplastic Agents; Disease Models, Animal; Disease Progression; Estradiol; Estrogen Antagonists; Estrogen Receptor beta; Fulvestrant; Gene Expression Regulation, Neoplastic; Genistein; Humans; Male; Metallothionein; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Prostatic Neoplasms; RNA, Small Interfering; Treatment Outcome; Xenograft Model Antitumor Assays

Diabetic cardiomyopathy is a clinically distinct disease characterized by impaired cardiac function as a result of reduced contractility and hypertension-induced athero- or arteriosclerosis. This may be due either to generalized vascular disease, tissue-based injury such as focal cardiomyocyte dysmorphia, or microvascular damage manifested by myocardial capillary basement membrane (CBM) thickening. Hyperglycemia-driven increases in reactive oxygen species (ROS) have been proposed to contribute to such damage. To address this hypothesis, we utilized light (LM) and transmission electron microscopy (TEM) to demonstrate cardiomyocyte morphology and myocardial CBM thickness in the left ventricles of four mouse genotypes: FVB (background Friend virus B controls), OVE (transgenic diabetics), Mt [transgenics with targeted overexpression of the antioxidant protein metallothionein (MT) in cardiomyocytes], and OVEMt (bi-transgenic cross of OVE and Mt) animals. Mice were prepared for morphometric analysis by vascular perfusion. Focal myocardial disorganization was identified in OVE mice but not in the remaining genotypes. Not unexpectedly, myocardial CBM thickness was increased significantly in OVE relative to FVB (P < 0.05) and Mt (P < 0.05) animals (+28% and +39.5%, respectively). Remarkably, however, OVEMt myocardial CBMs showed no increase in width; rather they were ~3% thinner than FVB controls. Although the molecular mechanisms regulating CBM width remain elusive, it seems possible that despite a significant hyperglycemic environment, MT antioxidant activity may mitigate local oxidative stress and reduce downstream excess microvascular extracellular matrix (ECM) formation. In addition, the reduction of intra- and perivascular ROS may protect against incipient endothelial damage and the CBM thickening that results from such injury.

Topics: Animals; Basement Membrane; Blood Glucose; Capillaries; Coronary Vessels; Diabetic Cardiomyopathies; Disease Models, Animal; Genotype; Glycated Hemoglobin; Humans; Male; Metallothionein; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Myocytes, Cardiac; Oxidative Stress; Phenotype; Reactive Oxygen Species; Up-Regulation

Oxidative stress is considered to be one of the main causes of neural damage after injury. However, little is known about the changes in mRNA expression of antioxidant molecules that occur after injury and regeneration of the peripheral nerve. In the present study, the rat median nerve was transected, and transcriptional changes were studied at day 6 and day 12 after injury in both the proximal and the distal stumps, in the absence or presence of microsurgical repair. The expression profiles of the following genes were investigated: three metallothionein isoforms (MT-1, MT-2, and MT-3), the main antioxidant enzymes (catalase, superoxide dismutase, and glutathione-S-transferase), and the marker of cellular damage poly(ADP-ribose) polymerase-1 (PARP-1). The results showed that, in the proximal nerve stump, MT-3 mRNA expression was significantly and markedly up-regulated in the absence of surgical repair, whereas MT-1 and MT-2 showed significant down-regulation. In the distal nerve portion, mRNA expression of all MT isoforms decreased significantly in the absence of microsurgical reconstruction, whereas, after repair, MT-3 mRNA expression alone was up-regulated. Expression of all the antioxidant enzymes decreased significantly after repair in the proximal nerve portion, but a significant general increase in their mRNA expression was revealed in the distal nerve stump. PARP-1 expression was significantly up-regulated in the proximal nerve portion without repair but dramatically reduced after reconstruction. In contrast, PARP-1 expression increased markedly in the distal stump after surgical repair. Taken together, these findings indicate that antioxidant molecules are differentially modulated and might, therefore, play an important role in peripheral nerve injury and regeneration.

Topics: Animals; Antioxidants; Catalase; Disease Models, Animal; Female; Gene Expression Regulation; Glutathione Transferase; Median Neuropathy; Metallothionein; Nerve Regeneration; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Isoforms; Rats; Rats, Wistar; RNA, Messenger; Superoxide Dismutase; Time Factors

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognition. One of the hallmarks of AD is the accumulation of beta-amyloid (Aβ). Although endoplasmic reticulum stress, mitochondrial dysfunction, and oxidative stress have been implicated in Aβ toxicity, the molecular mechanism(s) of Aβ-induced neurotoxicity are not fully understood. In this study, we present evidence that the glia-derived stress protein metallothionein (MT) attenuates Aβ-induced neurotoxicity by unique mechanisms. MT expression was increased in brain astrocytes of a NSE-APPsw transgenic mouse model of AD. Astrocyte-derived MT protected N2a neuroblastoma cells and primary cortical neurons against Aβ toxicity with concurrent reduction of reactive oxygen species levels. MT reversed Aβ-induced down-regulation of Bcl-2 and survival signaling in neuroblastoma cells. Moreover, MT inhibited Aβ-induced proinflammatory cytokine production from microglia. The neurotoxicity of Aβ-stimulated microglia was significantly attenuated by MT-I. The results indicate that MT released from reactive astrocytes may antagonize Aβ neurotoxicity by direct inhibition of Aβ neurotoxicity and indirect suppression of neurotoxic microglial activation. These findings broaden the understanding of neurotoxic mechanisms of Aβ and the crosstalk between Aβ and MT in AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Astrocytes; Disease Models, Animal; DNA Fragmentation; Enzyme-Linked Immunosorbent Assay; Immunoblotting; Immunohistochemistry; Metallothionein; Mice; Mice, Transgenic; Microglia; Oxidative Stress; Reverse Transcriptase Polymerase Chain Reaction

Activation of cardiac STAT3 by IL-6 cytokine family contributes to cardioprotection. Previously, we demonstrated that IL-11, an IL-6 cytokine family, has the therapeutic potential to prevent adverse cardiac remodeling after myocardial infarction; however, it remains to be elucidated whether IL-11 exhibits postconditioning effects. To address the possibility that IL-11 treatment improves clinical outcome of recanalization therapy against acute myocardial infarction, we examined its postconditioning effects on ischemia/reperfusion (I/R) injury. C57BL/6 mice were exposed to ischemia (30 min) and reperfusion (24 h), and IL-11 was intravenously administered at the start of reperfusion. I/R injury mediated the activation of STAT3, which was enhanced by IL-11 administration. IL-11 treatment reduced I/R injury, analyzed by triphenyl tetrazolium chloride staining [PBS, 46.7 ± 14.4%; IL-11 (20 μg/kg), 28.6 ± 7.5% in the ratio of infarct to risk area]. Moreover, echocardiographic and hemodynamic analyses clarified that IL-11 treatment preserved cardiac function after I/R. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining revealed that IL-11 reduced the frequency of apoptotic cardiomyocytes after I/R. Interestingly, IL-11 reduced superoxide production assessed by in situ dihydroethidium fluorescence analysis, accompanied by the increased expression of metallothionein 1 and 2, reactive oxygen species (ROS) scavengers. Importantly, with the use of cardiac-specific STAT3 conditional knockout (STAT3 CKO) mice, it was revealed that cardiac-specific ablation of STAT3 abrogated IL-11-mediated attenuation of I/R injury. Finally, IL-11 failed to suppress the ROS production after I/R in STAT3 CKO mice. IL-11 administration exhibits the postconditioning effects through cardiac STAT3 activation, suggesting that IL-11 has the clinical therapeutic potential to prevent I/R injury in heart.

Topics: Animals; Animals, Newborn; Apoptosis; Cardiotonic Agents; Cells, Cultured; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; Hemodynamics; Humans; Injections, Intravenous; Interleukin-11; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Oxidative Stress; Phosphorylation; Rats; Rats, Wistar; Reactive Oxygen Species; RNA Interference; Signal Transduction; STAT3 Transcription Factor; Time Factors; Transfection; Ventricular Function, Left; Ventricular Pressure

Alzheimer's disease (AD) is by far the most commonly diagnosed dementia, and despite multiple efforts, there are still no effective drugs available for its treatment. One strategy that deserves to be pursued is to alter the expression and/or physiological action of endogenous proteins instead of administering exogenous factors. In this study, we intend to characterize the roles of the antioxidant, anti-inflammatory, and heavy-metal binding proteins, metallothionein-1 + 2 (MT1 + 2), in a mouse model of Alzheimer's disease, Tg2576 mice. Contrary to expectations, MT1 + 2-deficiency rescued partially the human amyloid precursor protein-induced changes in mortality and body weight in a gender-dependent manner. On the other hand, amyloid plaque burden was decreased in the cortex and hippocampus in both sexes, while the amyloid cascade, neuroinflammation, and behavior were affected in the absence of MT1 + 2 in a complex manner. These results highlight that the control of the endogenous production and/or action of MT1 + 2 could represent a powerful therapeutic target in AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Behavior, Animal; Body Weight; Disease Models, Animal; Female; Hippocampus; Humans; Male; Metallothionein; Mice; Mice, Knockout; Mice, Transgenic; Peptide Fragments

Multiple sclerosis (MS) is the most common neurodegenerative disease in the Western world affecting younger, otherwise healthy individuals. Today no curative treatment exists. Patients suffer from recurring attacks caused by demyelination and underlying neuroinflammation, ultimately leading to loss of neurons. Recent research shows that bio-liberation of gold ions from metallic gold implants can ameliorate inflammation, reduce apoptosis and promote proliferation of neuronal stem cells (NSCs) in a mouse model of focal brain injury. Based on these findings, the present study investigates whether metallic gold implants affect the clinical signs of disease progression and the pathological findings in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. Gold particles 20-45 μm suspended in hyaluronic acid were bilaterally injected into the lateral ventricles (LV) of young Lewis rats prior to EAE induction. Comparing gold-treated animals to untreated and vehicle-treated ones, a statistically significant slowing of disease progression in terms of reduced weight loss was seen. Despite massive inflammatory infiltration, terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed reduced apoptotic cell death in disease foci in the brain stem of gold-treated animals, alongside an up-regulation of glial fibrillary acidic protein-positive reactive astrocytes near the LV and in the brain stem. Cell counting of frizzled-9 and nestin-stained cells showed statistically significant up-regulation of NSCs migrating from the subventricular zone. Additionally, the neuroprotective proteins Metallothionein-1 and -2 were up-regulated in the corpus callosum. In conclusion, this study is the first to show that the presence of small gold implants affect disease progression in a rat model of MS, increasing the neurogenic response and reducing the loss of cells in disease foci. Gold implants might thus improve clinical outcome for MS patients and further research into the long-term effects of such localized gold treatment is warranted.

Topics: Animals; Brain Stem; Cell Movement; Corpus Callosum; Disease Models, Animal; Disease Progression; Encephalomyelitis, Autoimmune, Experimental; Female; Glial Fibrillary Acidic Protein; Gliosis; Gold; Intermediate Filament Proteins; Lateral Ventricles; Metallothionein; Multiple Sclerosis; Nerve Tissue Proteins; Nestin; Neural Stem Cells; Rats; Rats, Inbred Lew; Receptors, Neurotransmitter; Up-Regulation

Retinal photoreceptor damage is a common feature of ophthalmic disorders, such as age-related macular degeneration and retinitis pigmentosa. Oxidative stress has a key role in these diseases. Metallothioneins (MTs) are a family of cysteine-rich proteins, and various physiologic functions have been reported, including protection against metal toxicity and antioxidative potency. We investigated the functional role of MT-III in light-induced retinal damage.. The expression of retinal MT-I, -II, and -III mRNA was evaluated by real-time reverse-transcription PCR in retina exposed to light. Retinal damage in MT-deficient mice was induced by exposure to white light at 16,000 lux for 3 hours after dark adaptation. Photoreceptor damage was evaluated histologically by measuring the thickness of the outer nuclear layer (ONL) 5 days after light exposure and by electroretinogram recording. In an in vitro experiment, the MT-III siRNAs were tested for their effects on light-induced mouse photoreceptor cell (661W) damage.. The mRNAs of the MTs were increased significantly in murine retina after light exposure. The ONL in the MT-III-deficient mice was remarkably thinner compared to light-exposed wild-type (WT) mice, and a- and b-wave amplitudes were decreased; the damage induced in MT-I/-II-deficient mice was comparable to that observed in WT mice. MT-III knockdown by siRNA in 661W exacerbated the cell damage and increased the production of reactive oxygen species in response to light exposure.. These findings suggested that MT-III can help protect against light-induced retinal damage compared to MT-I/II. Some of these effects may be exerted by its antioxidative potency.

Topics: Animals; Cell Culture Techniques; Disease Models, Animal; Disease Progression; Electroretinography; Female; Gene Expression Regulation; Light; Male; Metallothionein; Metallothionein 3; Mice; Nerve Tissue Proteins; Oxidative Stress; Radiation Injuries, Experimental; Reactive Oxygen Species; Retina; Retinal Degeneration; RNA, Messenger

Consumption of beverages that contain fructose favors the increasing prevalence of metabolic syndrome alterations in humans, including non-alcoholic fatty liver disease (NAFLD). Although the only effective treatment for NAFLD is caloric restriction and weight loss, existing data show that atorvastatin, a hydroxymethyl-glutaryl-CoA reductase inhibitor, can be used safely in patients with NAFLD and improves hepatic histology. To gain further insight into the molecular mechanisms of atorvastatin's therapeutic effect on NAFLD, we used an experimental model that mimics human consumption of fructose-sweetened beverages. Control, fructose (10% w/v solution) and fructose+atorvastatin (30 mg/kg/day) Sprague-Dawley rats were sacrificed after 14 days. Plasma and liver tissue samples were obtained to determine plasma analytes, liver histology, and the expression of liver proteins that are related to fatty acid synthesis and catabolism, and inflammatory processes. Fructose supplementation induced hypertriglyceridemia and hyperleptinemia, hepatic steatosis and necroinflammation, increased the expression of genes related to fatty acid synthesis and decreased fatty acid β-oxidation activity. Atorvastatin treatment completely abolished histological signs of necroinflammation, reducing the hepatic expression of metallothionein-1 and nuclear factor kappa B binding. Furthermore, atorvastatin reduced plasma (x 0.74) and liver triglyceride (x 0.62) concentrations, decreased the liver expression of carbohydrate response element binding protein transcription factor (x 0.45) and its target genes, and increased the hepatic activity of the fatty acid β-oxidation system (x 1.15). These effects may be related to the fact that atorvastatin decreased the expression of fructokinase (x 0.6) in livers of fructose-supplemented rats, reducing the metabolic burden on the liver that is imposed by continuous fructose ingestion.

Topics: Animals; Atorvastatin; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Cyclic AMP-Dependent Protein Kinases; Dietary Carbohydrates; Disease Models, Animal; Down-Regulation; Fatty Acids; Fatty Liver; Fructokinases; Fructose; Gene Expression Regulation; Hepatitis; Heptanoic Acids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypertriglyceridemia; I-kappa B Proteins; Inflammation Mediators; Lipid Metabolism; Liver; Male; Metallothionein; Necrosis; NF-kappa B; Non-alcoholic Fatty Liver Disease; Oxidation-Reduction; Phosphorylation; Pyrroles; Rats; Rats, Sprague-Dawley; Triglycerides

The aggregation of α-synuclein (α-Syn), the major component of intracellular Lewy body inclusions in dopaminergic neurons of the substantia nigra, plays a critical role in the etiology of Parkinson disease (PD). Long-term effects of redox-active transition metals (Cu, Fe) and oxidative chemical imbalance underlie the disease progression and neuronal death. In this work, we provide evidence that a brain metalloprotein, Zn₇-metallothionein-3 (Zn₇MT-3), possesses a dynamic role in controlling aberrant protein-copper interactions in PD. We examined the properties of the α-Syn-Cu(II) complex with regard to molecular oxygen, the biological reducing agent ascorbate, and the neurotransmitter dopamine. The results revealed that under aerobic conditions α-Syn-Cu(II) possesses catalytic oxidase activity. The observed metal-centered redox chemistry significantly promotes the production of hydroxyl radicals and α-Syn oxidation and oligomerization, processes considered critical for cellular toxicity. Moreover, we show that Zn₇MT-3, through Cu(II) removal from the α-Syn-Cu(II) complex, efficiently prevents its deleterious redox activity. We demonstrate that the Cu(II) reduction by thiolate ligands of Zn₇MT-3 and the formation of Cu(I)₄Zn₄MT-3, in which an unusual oxygen-stable Cu(I)₄-thiolate cluster is present, comprise the underlying molecular mechanism by which α-Syn and dopamine oxidation, α-Syn oligomerization, and ROS production are abolished. These studies provide new insights into the bioinorganic chemistry of PD.

Topics: alpha-Synuclein; Animals; Cells, Cultured; Copper; Disease Models, Animal; Disease Progression; Dopamine; Homeostasis; Humans; Male; Metallothionein; Neurons; Oxidation-Reduction; Parkinson Disease; Rats; Substantia Nigra

Tumors affect myelopoeisis and induce the expansion of myeloid cells with immunosuppressive activity. In the MT/ret model of spontaneous metastatic melanoma, myeloid cells are the most abundant tumor infiltrating hematopoietic population and their proportion is highest in the most aggressive cutaneous metastasis. Our data suggest that the tumor microenvironment favors polarization of myeloid cells into type 2 cells characterized by F4/80 expression, a weak capacity to secrete IL-12 and a high production of arginase. Myeloid cells from tumor and spleen of MT/ret mice inhibit T cell proliferation and IFNγ secretion. Interestingly, T cells play a role in type 2 polarization of myeloid cells. Indeed, intra-tumoral myeloid cells from MT/ret mice lacking T cells are not only less suppressive towards T cells than corresponding cells from wild-type MT/ret mice, but they also inhibit more efficiently melanoma cell proliferation. Thus, our data support the existence of a vicious circle, in which T cells may favor cancer development by establishing an environment that is likely to skew myeloid cell immunity toward a tumor promoting response that, in turn, suppresses immune effector cell functions.

Topics: Animals; Antigens, Differentiation; CD11b Antigen; Cell Proliferation; Disease Models, Animal; Disease Progression; Female; Flow Cytometry; Humans; Interferon-gamma; Interleukin-10; Interleukin-12; Male; Melanoma; Metallothionein; Mice; Mice, Knockout; Mice, Transgenic; Myeloid Cells; Proto-Oncogene Proteins c-ret; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Tumor Microenvironment

Metallothioneins (MTs) are intracellular low-molecular-weight, cysteine-rich proteins with potent metal-binding and redox functions, but with limited membrane permeativity. The aim of this study was to investigate whether we could enhance delivery of MT-1 to pancreatic islets or β cells in vitro and in vivo. The second goal was to determine whether increased MT-1 could prevent cellular toxicity induced by high glucose and free fatty acids in vitro (glucolipotoxicity) and ameliorate the development of diabetes induced by streptozotocin in mice or delay the development of diabetes by improving insulin secretion and resistance in the OLETF rat model of type 2 diabetes. Expression of HIV-1 Tat-MT-1 enabled efficient delivery of MT into both INS-1 cells and rat islets. Intracellular MT activity increased in parallel with the amount of protein delivered to cells. The formation of reactive oxygen species, glucolipotoxicity, and DNA fragmentation due to streptozotocin decreased after treating pancreatic β cells with Tat-MT in vitro. Importantly, in vivo, intraperitoneal injection resulted in delivery of the Tat-MT protein to the pancreas as well as liver, muscle, and white adipose tissues. Multiple injections increased radical-scavenging activity, decreased apoptosis, and reduced endoplasmic reticulum stress in the pancreas. Treatment with Tat-MT fusion protein delayed the development of diabetes in streptozotocin-induced mice and improved insulin secretion and resistance in OLETF rats. These results suggest that in vivo transduction of Tat-MT may offer a new strategy to protect pancreatic β cells from glucolipotoxicity, may improve insulin resistance in type 2 diabetes, and may have a protective effect in preventing islet destruction in type 1 diabetes.

Topics: Animals; Cells, Cultured; Diabetes Mellitus, Type 2; Disease Models, Animal; Gene Products, tat; Gene Transfer Techniques; HIV-1; Insulin; Insulin Resistance; Insulin Secretion; Male; Metallothionein; Mice; Mice, Inbred ICR; Rats; Rats, Inbred OLETF; Rats, Sprague-Dawley; Reactive Oxygen Species; Recombinant Fusion Proteins; Streptozocin

The discovery of neural stem cells (NSCs) provides new therapeutic strategies for brain injury by means of endogenous cell renewal. In the injured mouse brain, bio-liberated gold ions from gold implants mediate anti-inflammatory and antiapoptotic effects and activation of NSCs. This paper investigates the neuroprotective effects of gold following brain injury in mice. We show for the first time that endogenous NSCs express macrophage colony-stimulating factor (M-CSF) as part of their post-injury activation and that gold implants increase this response. Also, gold increases expression of neurotrophin (NT)-4, transforming growth factor-beta 3 (TGF-beta 3), leukemia inhibitory factor (LIF) and metallothionein I+II (MT-I+II) post-injury. This paper shows that gold ions modulate neurotrophic factors after injury and that hematopoietic factor M-CSF is expressed in activated NSCs.

Topics: Adult Stem Cells; Animals; Brain Injuries; Disease Models, Animal; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Gold; Lateral Ventricles; Macrophage Colony-Stimulating Factor; Metallothionein; Mice; Mice, Inbred C57BL; Nerve Growth Factors; Time Factors

Cadmium (Cd) inhalation can result in emphysema. Cd exposure of rat lung fibroblasts (RFL6) enhanced levels of metal scavenging thiols, e.g., metallothionein (MT) and glutathione (GSH), and the heavy chain of gamma-glutamylcysteine synthetase (gamma-GCS), a key enzyme for GSH biosynthesis, concomitant with downregulation of lysyl oxidase (LO), a copper-dependent enzyme for crosslinking collagen and elastin in the extracellular matrix (ECM). Cd downregulation of LO in treated cells was closely accompanied by suppression of synthesis of collagen, a major structure component of the lung ECM. Using rats intratracheally instilled with cadmium chloride (30 microg, once a week) as an animal model, we further demonstrated that although 2-week Cd instillation induced a non-significant change in the lung LO activity and collagen synthesis, 4- and 6-week Cd instillation resulted in a steady decrease in the lung LO and collagen expression. The lung MT and total GSH levels were both upregulated upon the long-term Cd exposure. Emphysematous lesions were generated in lungs of 6-week Cd-dosed rats. Increases of cellular thiols by transfection of cells with MT-II expression vectors or treatment of cells with GSH monoethyl ester, a GSH delivery system, markedly inhibited LO mRNA levels and catalytic activities in the cell model. Thus, Cd upregulation of cellular thiols may be a critical cellular event facilitating downregulation of LO, a potential mechanism for Cd-induced emphysema.

Topics: Animals; Cadmium; Cell Line; Collagen; Disease Models, Animal; Emphysema; Extracellular Matrix; Gene Expression Regulation, Enzymologic; Glutamate-Cysteine Ligase; Glutathione; Homeostasis; Lung; Metallothionein; Protein-Lysine 6-Oxidase; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sulfhydryl Compounds

Metallothioneins I and II (MTI/II) are metal-binding proteins overexpressed in response to brain injury. Recently, we have designed a peptide, termed EmtinB, which is modeled after the beta-domain of MT-II and mimics the biological effects of MTI/II in vitro. Here, we demonstrate the neuroprotective effect of EmtinB in the in vitro and in vivo models of kainic acid (KA)-induced neurotoxicity. We show that EmtinB passes the blood-brain barrier and is detectable in plasma for up to 24 hr. Treatment with EmtinB significantly attenuates seizures in C57BL/6J mice exposed to moderate (20 mg/kg) and high (30 mg/kg) KA doses and tends to decrease mortality induced by the high KA dose. Histopathological evaluation of hippocampal (CA3 and CA1) and cortical areas of mice treated with 20 mg/kg KA shows that EmtinB treatment reduces KA-induced neurodegeneration in the CA1 region. These findings establish EmtinB as a promising target for therapeutic development.

Topics: Animals; Blood-Brain Barrier; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Epilepsy, Temporal Lobe; Hippocampus; Intercellular Signaling Peptides and Proteins; Kainic Acid; Male; Metallothionein; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neuroprotective Agents; Neurotoxins; Peptides; Rats; Rats, Wistar; Seizures

To elucidate the role of metal-related molecules in hepatocarcinogenesis, we examined immunolocalization of transferrin receptor (Tfrc), ceruloplasmin (Cp) and metallothionein (MT)-1/2 in relation to liver cell foci positive for glutathione-S-transferase placental form (GST-P) in the early stage of tumor promotion by fenbendazole (FB), phenobarbital, piperonyl butoxide or thioacetamide in a rat two-stage hepatocarcinogenesis model. To estimate the involvement of oxidative stress responses to the promotion, immunolocalization of 4-hydroxy-2-nonenal, malondialdehyde and acrolein was similarly examined. Our findings showed that MT-1/2 immunoreactivity was not associated with the cellular distribution of GST-P and proliferating cell nuclear antigen, suggesting no role of MT-1/2 in hepatocarcinogenesis. We also found enhanced expression of Tfrc after treatment with strong tumor-promoting chemicals. With regard to Cp, the population showing down-regulation was increased in the GST-P-positive foci in relation to tumor promotion. Up-regulation of Tfrc and down-regulation of Cp was maintained in GST-P-positive neoplastic lesions induced after long-term promotion with FB, suggesting the expression changes occurring downstream of the signaling pathway involved in the formation of GST-P-positive lesions. Furthermore, enhanced accumulation of lipid peroxidation end products was observed in the GST-P-positive foci by promotion. Post-initiation treatment with peroxisome proliferator-activated receptor alpha agonists did not enhance any such distribution changes in GST-P-negative foci. The results thus suggest that facilitation of lipid peroxidation is involved in the induction of GST-P-positive lesions by tumor promotion from an early stage, and up-regulation of Tfrc and down-regulation of Cp may be a signature of enhanced oxidative cellular stress in these lesions.

Topics: Animals; Carcinogenicity Tests; Carcinogens; Ceruloplasmin; Copper; Disease Models, Animal; Down-Regulation; Gene Expression Regulation, Neoplastic; Glutathione S-Transferase pi; Iron; Lipid Peroxidation; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; Oxidative Stress; PPAR alpha; Rats; Rats, Inbred F344; Receptors, Transferrin; Up-Regulation

Traumatic injury to the brain is one of the leading causes of injury-related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock-out mice. We herewith report the results comparing full mouse MT-1 with the independent alpha and beta domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota-rod tests; all the proteins showed a modest effect in the former test, while only full MT-1 improved the performance of animals in the rota-rod, and the alpha domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host-response genes such as GFAP, Mac1 and ICAM, in some cases being the beta domain more effective than the alpha domain or even the full MT-1. A MT-1-to-MT-3 mutation blunted some but not all the effects caused by the normal MT-1, and in some cases increased its potency. Thus, splitting the two MT-1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions.

Topics: Animals; Body Weight; Brain Injuries; Disease Models, Animal; Gene Expression Regulation; Metallothionein; Metallothionein 3; Mice; Mice, Knockout; Motor Activity; Mutation; Nerve Tissue Proteins; Protein Structure, Tertiary; Psychomotor Performance

A major obstacle that hampers the design of drug therapy for traumatic brain injury is the incomplete understanding of the biochemical pathways that lead to secondary cellular injury and contribute to cell death. One such pathway involves reactive species that generate potentially cytotoxic zinc ion fluctuations as a major executor of neuronal, and possibly glial, cell death. Whether zinc ions released during traumatic brain injury are toxic or protective is controversial but can be approached by investigating the exact concentrations of free zinc ions, the thresholds of compromised zinc buffering capacity, and the mechanism of cellular homeostatic control of zinc. Rapidly stretch-injured rat pheochromocytoma (PC12) cells express cellular zinc ion fluctuations that depend on the production of nitric oxide. Chelation of cellular zinc ions after rapid stretch injury, however, increases cellular reactive oxygen species. In a rat model of traumatic brain injury, parasagittal fluid percussion, analysis of the metal load of metallothionein was used as an indicator of changes in cellular zinc ion concentrations. The combined results from the cellular and in vivo investigations caution against interpreting zinc ion fluctuations in the early phase (24h) after injury as a primarily cytotoxic event.

Topics: Animals; Brain; Brain Injuries; Cell Survival; Disease Models, Animal; Intracellular Space; Ions; Male; Metallothionein; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; PC12 Cells; Random Allocation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction; Time Factors; Zinc

To examine in rodent models of retinitis pigmentosa (RP) the expression of the neuroprotectants metallothionein-I and -II and of megalin, an endocytic receptor that mediates their transport into neurons.. Gene and protein expression were analyzed in retinas of rd1 and rds mice and in those of RCS (Royal College of Surgeons) rats of various ages. Glial cell markers (cellular retinaldehyde binding protein, CRALBP; glial fibrillary acidic protein, GFAP; CD11b; and isolectin B4) were used to establish the identity of the cells.. Metallothionein-I and -II gene expression increased with age in normal and degenerating retinas and was significantly greater in the latter. Protein expression, corresponding to metallothionein-I+II, was first observed in rd1 mice in Müller cells at postnatal day (P)12 and in rds mice at P16, coinciding with the onset of GFAP expression in these cells. In RCS rats, the same distribution was observed, but not until P32, long after the onset of GFAP expression. Metallothionein-I+II was observed also in a small number of microglial cells. Megalin was expressed in the nerve fiber layer and in the region of the inner and outer segments in normal animals, but expression in the outer retina was lost with age in degenerating retinas.. Induction of metallothionein-I and -II occurs in the RP models studied and correlates with glial activation. The progressive loss of megalin suggests that transport of metallothionein-I+II into the degenerating photoreceptors (from e.g., Müller cells), could be impaired, potentially limiting the actions of these metallothioneins.

Topics: Animals; Astrocytes; Avidin; Biomarkers; Cyclic Nucleotide Phosphodiesterases, Type 6; Disease Models, Animal; Glial Fibrillary Acidic Protein; In Situ Nick-End Labeling; Low Density Lipoprotein Receptor-Related Protein-2; Metallothionein; Mice; Mice, Inbred C3H; Mice, Mutant Strains; Microglia; Nerve Tissue Proteins; Oligonucleotide Array Sequence Analysis; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Ganglion Cells; Retinitis Pigmentosa

Training under hypoxia has several advantages over normoxic training in terms of enhancing the physical performance. Therefore, we tested the protective effect of hypoxia preconditioning by hypoxia mimetic cobalt chloride against exercise-induced oxidative damage in the skeletal muscles and improvement of physical performance.. Male Sprague-Dawley rats were randomly divided into four groups (n=8), namely control, cobalt-supplemented, training and cobalt with training. The red gastrocnemius muscle was examined for all measurements, viz. free radical generation, lipid peroxidation, muscle damage and antioxidative capacity.. Hypoxic preconditioning with cobalt along with training significantly increased physical performance (33%, P<0.01) in rats compared with training-only rats. Cobalt supplementation activated cellular oxygen sensing system in rat skeletal muscle. It also protected against training-induced oxidative damage as observed by an increase in the GSH/GSSG ratio (36%, P<0.001; 28%, P<0.01 respectively) and reduced lipid peroxidation (15%, P<0.01; 31%, P<0.01 respectively) in both trained and untrained rats compared with their respective controls. Cobalt supplementation along with training enhanced the expression of antioxidant proteins haem oxygenase-1 (HO-1; 1.2-fold, P<0.05) and metallothionein (MT; 4.8-fold, P<0.001) compared with training only. A marked reduction was observed in exercise-induced muscle fibre damage as indicated by decreased necrotic muscle fibre, decreased lipofuscin content of muscle and plasma creatine kinase level (16%, P<0.01) in rats preconditioned with cobalt.. Our study provides strong evidence that hypoxic preconditioning with cobalt chloride enhances physical performance and protects muscle from exercise-induced oxidative damage via GSH, HO-1 and MT-mediated antioxidative capacity.

Topics: Animals; Antioxidants; Cell Hypoxia; Cobalt; Creatine Kinase, MM Form; Cytoprotection; Disease Models, Animal; Dose-Response Relationship, Drug; Glutathione; Heme Oxygenase (Decyclizing); Lactic Acid; Lipid Peroxidation; Lipofuscin; Male; Metallothionein; Mitochondria, Muscle; Mitochondrial Proteins; Muscle, Skeletal; Muscular Diseases; Necrosis; Oxidative Stress; Physical Endurance; Physical Exertion; Rats; Rats, Wistar; Reactive Oxygen Species

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.. 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.. 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.. 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.

Topics: Animals; Blood Cell Count; Bone Marrow; Colitis; Colon; Disease Models, Animal; Drug Therapy, Combination; Enzyme Inhibitors; Intestinal Mucosa; Male; Metallothionein; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Trinitrobenzenesulfonic Acid

Spinal cord injury (SCI) is a world-wide health problem. After traumatic injury, spinal cord tissue starts a series of self-destructive mechanisms, known as the secondary lesion. The leading mechanisms of damage after SCI are excitotoxicity, free radicals' overproduction, inflammation and apoptosis. Metallothionein (MT) and reduced glutathione (GSH) are low-molecular-weight, cysteine-rich peptides able to scavenge free radicals. MT and GSH participation as neuroprotective molecules after SCI is unknown. The aim of the present study is to describe the changes of MT and GSH contents and GSH peroxidase (GPx) activity in the acute phase after SCI in rats. Female Wistar rats weighing 200-250g were submitted to spinal cord contusion model, by means of a computer-controlled device (NYU impactor). Rats receiving laminectomy were used as a control group. Animals were killed 2, 4, 12 and 24h after surgery. MT was quantified by the silver-saturation method, using atomic absorption spectrophotometry. GSH and GPx were assayed by spectrophotometry. Results indicate an increased MT content by effect of SCI, only at 4 and 24h, as compared to sham group values. Meanwhile, GSH was found decreased at 4, 12 and 24h after SCI. Interestingly, GPx activity was raised at all time points, indicating that this enzymatic defense is activated soon after SCI. Results suggest that thiol-based defenses, MT and GSH, are differentially expressed by spinal cord tissue to cope with the various processes of damage after lesion.

Topics: Animals; Antioxidants; Disease Models, Animal; Female; Gene Expression Regulation; Glutathione; Metallothionein; Oxidoreductases; Rats; Rats, Wistar; Spinal Cord Injuries; Time Factors

We have previously demonstrated that ethanol teratogenicity is associated with metallothionein-induced fetal zinc (Zn) deficiency, and that maternal subcutaneous Zn treatment given with ethanol in early pregnancy prevents fetal abnormalities and spatial memory impairments in mice. Here we investigated whether dietary Zn supplementation throughout pregnancy can also prevent ethanol-related dysmorphology.. Pregnant mice were injected with saline or 25% ethanol (0.015 ml/g intraperitoneally at 0 and 4 hours) on gestational day (GD) 8 and fed either a control (35 mg Zn/kg) or a Zn-supplemented diet (200 mg Zn/kg) from GD 0 to 18. Fetuses from the saline, saline + Zn, ethanol and ethanol + Zn groups were assessed for external birth abnormalities on GD 18. In a separate cohort of mice, postnatal growth and survival of offspring from these treatment groups were examined from birth until postnatal day 60.. Fetuses from dams treated with ethanol alone in early pregnancy had a significantly greater incidence of physical abnormalities (26%) compared to those from the saline (10%), saline + Zn (9%), or ethanol + Zn (12%) groups. The incidence of abnormalities in ethanol + Zn-supplemented fetuses was not different from saline-treated fetuses. While ethanol exposure did not affect the number of fetal resorptions or pre- or postnatal weight, there were more stillbirths with ethanol alone, and cumulative postnatal mortality was significantly higher in offspring exposed to ethanol alone (35% deaths) compared to all other treatment groups (13.5 to 20.5% deaths). Mice supplemented with Zn throughout pregnancy had higher plasma Zn concentrations than those in un-supplemented groups.. These findings demonstrate that dietary Zn supplementation throughout pregnancy ameliorates dysmorphology and postnatal mortality caused by ethanol exposure in early pregnancy.

Topics: Abnormalities, Drug-Induced; Alcoholism; Animals; Central Nervous System Depressants; Dietary Supplements; Disease Models, Animal; Embryonic Development; Ethanol; Female; Liver; Male; Metallothionein; Mice; Mice, Inbred C57BL; Pregnancy; Pregnancy Outcome; Pregnancy, Animal; Prenatal Exposure Delayed Effects; Sodium Chloride; Zinc

Fetal zinc (Zn) deficiency arising from ethanol-induction of the Zn-binding protein metallothionein (MT) in the mother's liver has been proposed as a mechanism of teratogenicity. Here, we determine the ontogeny of MT and Zn homeostasis in rats and mice and then examine the effect of acute ethanol exposure in early embryonic development on this relationship. The protective effect of Zn against ethanol-mediated fetal dysmorphology is also examined.. Study 1: Maternal liver MT and Zn homeostasis was determined in Sprague-Dawley rats and C57BL/6J mice throughout gestation. Study 2: Rats were administered ethanol (25% in saline, intraperitoneal 0.015 ml/g) or vehicle alone on gestational day (GD) 9. Maternal liver MT and Zn, and plasma Zn was determined over the ensuing 24 hours. Study 3: Pregnant rats were treated with ethanol and Zn (s.c. 2.5 microg Zn/g) on GD9 and fetal dysmorphology was assessed on GD 19.. Study 1: Maternal liver MT began to rise around GD 9 peaking on GD 15 before falling to nonpregnant levels around term. The pregnancy-related increase in MT was associated with a fall in plasma Zn which was significantly lower on GD 15 thereafter returning to nonpregnant levels by parturition. Study 2: Ethanol administered to pregnant rats on GD 9 resulted in a 10-fold induction of MT in the maternal liver and was associated with a 33% rise in liver Zn and a 30% fall in plasma Zn, 16 hours after treatment. Study 3: Ethanol treatment on GD 9 resulted in a significant increase in craniofacial malformations which were prevented by concurrent Zn treatment.. The findings indicate that maternal liver MT levels are lowest in early gestation (before GD 10) making this a sensitive period where ethanol-induction of MT can affect fetal Zn homeostasis and cause fetal dysmorphology. The study further provides evidence of a protective role for Zn against ethanol-mediated teratogenicity.

Topics: Animals; Central Nervous System Depressants; Craniofacial Abnormalities; Disease Models, Animal; Dose-Response Relationship, Drug; Ethanol; Female; Fetal Development; Fetus; Homeostasis; Liver; Male; Metallothionein; Mice; Mice, Inbred C57BL; Pregnancy; Pregnancy, Animal; Rats; Rats, Sprague-Dawley; Zinc

A dyshomeostasis of extra- and intracellular Ca(2+) and Zn(2+) occurs in rats receiving chronic aldosterone/salt treatment (ALDOST). Herein, we hypothesized that the dyshomeostasis of intracellular Ca(2+) and Zn(2+) is intrinsically coupled that alters the redox state of cardiac myocytes and mitochondria, with Ca(2+) serving as a pro-oxidant and Zn(2+) as an antioxidant. Toward this end, we harvested hearts from rats receiving 4 weeks of ALDOST alone or cotreatment with either spironolactone (Spiro), an aldosterone receptor antagonist, or amlodipine (Amlod), an L-type Ca(2+) channel blocker, and from age/sex-matched untreated controls. In each group, we monitored cardiomyocyte [Ca(2+)]i and [Zn(2+)]i and mitochondrial [Ca(2+)]m and [Zn(2+)]m; biomarkers of oxidative stress and antioxidant defenses; expression of Zn transporters, Zip1 and ZnT-1; metallothionein-1, a Zn(2+)-binding protein; and metal response element transcription factor-1, a [Zn(2+)]i sensor and regulator of antioxidant defenses. Compared with controls, at 4-week ALDOST, we found the following: (a) increased [Ca(2+)]i and [Zn(2+)]i, together with increased [Ca(2+)]m and [Zn(2+)]m, each of which could be prevented by Spiro and attenuated with Amlod; (b) increased levels of 3-nitrotyrosine and 4-hydroxy-2-nonenal in cardiomyocytes, together with increased H(2)O(2) production, malondialdehyde, and oxidized glutathione in mitochondria that were coincident with increased activities of Cu/Zn superoxide dismutase and glutathione peroxidase; and (c) increased expression of metallothionein-1, Zip1 and ZnT-1, and metal response element transcription factor-1, attenuated by Spiro. Thus, an intrinsically coupled dyshomeostasis of intracellular Ca(2+) and Zn(2+) occurs in cardiac myocytes and mitochondria in rats receiving ALDOST, where it serves to alter their redox state through a respective induction of oxidative stress and generation of antioxidant defenses. The importance of therapeutic strategies that can uncouple these two divalent cations and modulate their ratio in favor of sustained antioxidant defenses is therefore suggested.

Topics: Aldehydes; Aldosterone; Amlodipine; Animals; Calcium; Calcium Channel Blockers; Chronic Disease; Disease Models, Animal; Glutathione Peroxidase; Homeostasis; Hydrogen Peroxide; Hyperaldosteronism; Male; Metallothionein; Mineralocorticoid Receptor Antagonists; Mitochondria, Heart; Myocytes, Cardiac; Oxidative Stress; Rats; Rats, Sprague-Dawley; Spironolactone; Superoxide Dismutase; Tyrosine; Zinc

One key mechanism for endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O(2)(*-) rather than NO because of deficient eNOS cofactor tetrahydrobiopterin (BH4). This study was designed to examine the effect of BH4 deficiency on cardiac morphology and function, as well as the impact of metallothionein (MT) on BH4 deficiency-induced abnormalities, if any. Friend virus B (FVB) and cardiac-specific MT transgenic mice were exposed to 2,4-diamino-6-hydroxy-pyrimidine (DAHP; 10 mmol/L, 3 weeks), an inhibitor of the BH4 synthetic enzyme GTP cyclohydrolase I. DAHP reduced plasma BH4 levels by 85% and elevated blood pressure in both FVB and MT mice. Echocardiography found decreased fractional shortening and increased end-systolic diameter in DAHP-treated FVB mice. Cardiomyocytes from DAHP-treated FVB mice displayed enhanced O(2)(*-) production, contractile and intracellular Ca(2+) defects including depressed peak shortening and maximal velocity of shortening/relengthening, prolonged duration of relengthening, reduced intracellular Ca(2+) rise, and clearance. DAHP triggered mitochondrial swelling/myocardial filament aberrations and mitochondrial O(2)(*-) accumulation, assessed by transmission electron microscopy and MitoSOX Red fluorescence, respectively. DAHP also promoted the N(G)-nitro-l-arginine methyl ester-inhibitable O(2)(*-) production and eNOS phosphorylation at Thr497. Although MT had little effect on cardiac mechanics and ultrastructure, it attenuated DAHP-induced defects in cardiac function, morphology, O(2)(*-) production, and eNOS phosphorylation (Thr497). The DAHP-induced cardiomyocyte mechanical responses were alleviated by in vitro BH4 treatment. DAHP inhibited mitochondrial biogenesis, mitochondrial uncoupling protein 2, and chaperone heat shock protein 90, and all but uncoupling protein 2 were rescued by MT. Our data suggest a role for BH4 deficiency in cardiac dysfunction and the therapeutic potential of antioxidants against eNOS uncoupling in the heart.

Topics: Analysis of Variance; Animals; Biopterins; Blotting, Western; Chromatography, High Pressure Liquid; Disease Models, Animal; Echocardiography; GTP Cyclohydrolase; Male; Metallothionein; Mice; Mice, Transgenic; Mitochondria, Heart; Myocardial Contraction; Myocytes, Cardiac; Nitric Oxide; Oxygen Consumption; Probability; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; RNA; Sensitivity and Specificity; Sugar Acids

Chemotherapy-induced small intestinal mucositis is a debilitating side effect of cancer chemotherapy and currently there are no effective therapies. Zinc (Zn), an essential trace element required for normal growth and development and tissue repair processes, may be a potential treatment strategy. Zn induces metallothionein (MT) which has been shown to sequester free radicals. The aim of this study was to determine the capacity for dietary Zn supplementation to ameliorate methotrexate (MTX)-induced intestinal mucositis.. The duodenum and jejunum were significantly (p < 0.05) damaged at the 500 mg/kg MTX dose compared to 300 and 400 mg/kg MTX doses. Dietary Zn supplementation did not induce gut MT in MT(+/+) mice nor ameliorate MTX-induced gut damage in either MT(+/+) or MT(-/-) mice. However, MT(-/-) mice had markedly (p < 0.05) higher histological severity scores and MPO activity compared to MT(+/+) mice, irrespective of dietary Zn. methods: MT-knockout (MT(-/-)) and wild-type (MT(+/+)) mice were fed either a 10 mg/kg (control) or 400 mg/kg diet (high Zn) for 7 d and intestinal mucositis was induced by a single injection of MTX (500 mg/kg) subcutaneously. Mice were sacrificed at 24 and 72 h (n = 8/timepoint/genotype) after the MTX injection while continuing their respective diets. Daily weights were recorded and gut tissues were collected for histology, MT levels and myeloperoxidase (MPO) activity.. Dietary Zn supplementation did not ameliorate MTX-induced small bowel damage, possibly signifying a deficiency in induction of MT by Zn. However, the presence of MT was able to reduce histological damage and neutrophil infiltration caused by MTX in the gut.

Topics: Animals; Dietary Supplements; Disease Models, Animal; Intestine, Small; Male; Metallothionein; Methotrexate; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucositis; Zinc

Dopamine agonists have neuroprotective properties in addition to their original pharmacologic function. We examined the effects of pergolide mesilate (PM) on the levels of metallothionein mRNA expression and lipid peroxidation in the corpus striata of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonian mice. Mice were administered normal saline (vehicle as a control), PM, or MPTP. A consecutive 7-d administration of MPTP via a gastric tube at a dose of 30 mg/kg significantly decreased metallothionein (MT)-I mRNA expression but did not influence MT-III mRNA expression. Lipid peroxidation, measured as the production of malondialdehyde reactive substances, did not increase after MPTP treatment. Although PM administration alone did not effect MT-I expression, an additional consecutive 7-d administration of PM (30 mug/kg) following MPTP treatment recovered the decreased MT-I level and increased MT-III expression. Lipid peroxidation was significantly suppressed. These results suggest that PM exerts an antioxidative property through the induction of MT-I and MT-III mRNAs simultaneously in response to cellular and/or tissue injury.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Antioxidants; Brain; Disease Models, Animal; Dopamine Agonists; Gene Expression; Lipid Peroxidation; Malondialdehyde; Metallothionein; Mice; Mice, Inbred C57BL; Parkinson Disease; Pergolide; RNA, Messenger

Metallothionein (MT) is a small molecular weight protein possessing metal binding and free radical scavenging properties. We hypothesized that MT-1/MT-2 null (MT(-/-)) mice would display exacerbated soleus muscle atrophy, oxidative injury, and contractile dysfunction compared with the response of wild-type (WT) mice following acute spinal cord transection (SCT). Four groups of mice were studied: WT laminectomy, WT transection, MT(-/-) laminectomy (MT(-/-) lami), and MT(-/-) transection (MT(-/-) trans). Laminectomy animals served as surgical controls. Mice in SCT groups experienced similar percent body mass (BM) losses at 7 days postinjury. Soleus muscle mass (MM) and MM-to-BM ratio were lower at 7 days postinjury in SCT vs. laminectomy mice, with no differences observed between strains. However, soleus muscles from MT(-/-) trans mice showed reduced maximal specific tension compared with MT(-/-) lami animals. Mean cross-sectional area (microm(2)) of type I and type IIa fibers decreased similarly in SCT groups compared with laminectomy controls, and no difference in fiber distribution was observed. Lipid peroxidation (4-hydroxynoneal) was greater in MT(-/-) trans vs. MT(-/-) lami mice, but protein oxidation (protein carbonyls) was not altered by MT deficiency or SCT. Expression of key antioxidant proteins (catalase, manganese, and copper-zinc superoxide dismutase) was similar between the groups. In summary, MT deficiency did not impact soleus MM loss, but resulted in contractile dysfunction and increased lipid peroxidation following acute SCT. These findings suggest a role of MT in mediating protective adaptations in skeletal muscle following disuse mediated by spinal cord injury.

Topics: Acute Disease; Animals; Antioxidants; Catalase; Disease Models, Animal; Laminectomy; Lipid Peroxidation; Male; Metallothionein; Mice; Mice, Knockout; Muscle Contraction; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle Strength; Muscle, Skeletal; Muscular Atrophy; Oxidative Stress; Protein Carbonylation; Spinal Cord Injuries; Superoxide Dismutase; Thoracic Vertebrae

Iterations in Ca2+ and Mg2+ balance accompany aldosteronism (inappropriate for dietary Na+ intake). Increased Zn excretion and Zn translocation to injured tissues, including the heart, also occurs. Several causes and consequences of Zn dyshomeostasis in rats receiving aldosterone/salt treatment (ALDOST) were examined. (1) To study the role of urinary acidification in promoting hyperzincuria, acetazolamide (75 mg/kg), a carbonic anhydrase inhibitor, was used as cotreatment to raise urinary HCO3 excretion. (2) To assess Zn levels in the heart, including cardiomyocyte cytosolic free [Zn2+]i and mitochondrial Zn, the expression of metallothionein (MT-I), a Zn binding protein, and biomarkers of oxidative stress were examined. (3) Oxidative stress and cardiac pathology in response to ZnSO4 supplement (40 mg/d) were also studied. Comparison of controls and rats receiving 4 weeks ALDOST revealed the following: (1) an acidification of urine and metabolic alkalosis associated with increased urinary Zn excretion and hypozincemia, each of which were prevented by acetazolamide; (2) a rise in cardiac Zn, including increased [Zn2+]i and mitochondrial Zn, associated with increased tissue MT-I, 8-isoprostane, malondialdehyde, and gp91(phox), coupled with oxidative stress in plasma and urine; (3) ZnSO4 prevented hypozincemia, but not ionized hypocalcemia, and attenuated oxidative stress and microscopic scarring without preventing the vasculitis and perivascular fibrosis of intramural coronary arteries. Thus, the hyperzincuria seen with ALDOST is due to urinary acidification. The oxidative stress that appears in the heart is accompanied by increased tissue Zn serving as an antioxidant. Cotreatment with ZnSO4 attenuated cardiomyocyte necrosis; however, polynutrient supplement may be required to counteract the dyshomeostasis of all 3 cations that accompanies aldosteronism and contributes to cardiac pathology.

Topics: Acetazolamide; Aldosterone; Animals; Calcium; Chronic Disease; Disease Models, Animal; Homeostasis; Hydrogen-Ion Concentration; Hyperaldosteronism; Magnesium; Male; Metallothionein; Myocytes, Cardiac; Necrosis; Oxidative Stress; Rats; Rats, Sprague-Dawley; Urine; Zinc; Zinc Sulfate

Metallothioneins (MTs) are metal-binding proteins that can be upregulated in the brain after injury and are associated with neuroprotection. A recent genomics study has shown that brain MT-1 and MT-2 mRNA levels are upregulated following intracerebral hemorrhage (ICH) in rats. Our study examines whether brain MT-1 and MT-2 protein levels are increased after ICH. We also investigated the effect of exogenous MT-1 in perihematomal edema formation in vivo and iron-induced cell death in vitro. We found that MT-1/-2 immunoreactivity in ipsilateral basal ganglia was significantly increased after ICH and exogenous MT-1 attenuated perihematomal edema formation. In addition, MT-1 also reduced cell death induced by iron in cultured astrocytes. These results suggest a role for MT in ICH-induced brain injury, and MT could be a therapeutic target for ICH.

Topics: Analysis of Variance; Animals; Animals, Newborn; Astrocytes; Basal Ganglia; Brain Injuries; Cells, Cultured; Cerebral Cortex; Cerebral Hemorrhage; Disease Models, Animal; Dose-Response Relationship, Drug; L-Lactate Dehydrogenase; Male; Metallothionein; Rats; Rats, Sprague-Dawley; Up-Regulation

Infection with the gastric pathogen Helicobacter pylori (H. pylori) causes chronic gastritis, peptic ulcer, and gastric adenocarcinoma. These diseases are associated with production of reactive oxygen species (ROS) from infiltrated macrophages and neutrophiles in inflammatory sites. Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that can act not only as a metal-binding protein, but also as a ROS scavenger. In the present study, we examined the role of MT in the protection against H. pylori-induced gastric injury using MT-null mice. Female MT-null and wild-type mice were challenged with H. pylori SS1 strain, and then histological changes were evaluated with the updated Sydney grading system at 17 and 21 wk after challenge. Although the colonization efficiency of H. pylori was essentially the same for MT-null and wild-type mice, the scores of activity of inflammatory cells were significantly higher in MT-null mice than in wild-type mice at 17 wk after challenge. Histopathological examination revealed erosive lesions accompanied by infiltration of inflammatory cells in the infected MT-null mice but not in wild-type mice. Furthermore, activation of NF-kappaB and expression of NF-kappaB-mediated chemokines such as macrophage inflammatory protein-1alpha and monocytes chemoattractant protein-1 in gastric cells were markedly higher in MT-null mice than in wild-type mice. These results suggest that MT in the gastric mucosa might play an important role in the protection against H. pylori-induced gastric ulceration.

Topics: Animals; Chemokine CCL2; Chemokine CCL3; Chemotaxis, Leukocyte; Cytoprotection; Disease Models, Animal; Female; Gastric Mucosa; Helicobacter Infections; Helicobacter pylori; Leukocytes; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Reactive Oxygen Species; Stomach Ulcer; Time Factors

To investigate the protective effects of metallothionein (MT) induced by dexamethasone (DEX) against ischemia/reperfusion (I/R) injury of myocardium of isolated rat heart.. Thirty-two Sprague-Dawley (SD) rats were divided randomly into the DEX and control groups. In the former group, the rats were pretreated with DEX, and in latter group distilled water was given before their hearts were isolated for Langendorff perfusion and I/R. MT was assessed by Western blotting. The left ventricular developed pressure (LVDP), maximal change rate of intraventricular pressure rise/down (+/-dp/dt max), coronary artery flow (CF) and reperfusion arrhythmias were observed dynamically before ischemia and during 60-minute reperfusion following 30-minute ischemia, The hearts were perfused with triphenyltetrazolium (TTC) after 60-minute reperfusion. The myocardial infarct size was measured with Adobe Photoshop. The levels of MB isoenzyme of creatine kinase (CK-MB), malonaldehyde (MDA), total superoxide dismutase (T-SOD), CuZn-SOD, catalase (CAT), glutathione peroxidase (GSH-Px) and the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase were detected.. Compared with control group, the expression of MT was significantly increased (3.085+/-1.065 vs. 1.028+/-0.016, P<0.05), the LVDP, +/-dp/dt max and CF were greatly improved (all P<0.05), the accumulated point of ventricular arrhythmia and the infarct size were significantly reduced in DEX group [(2.00+/-1.41) scores vs. (6.63+/-4.24) scores and (28.38+/-11.22)% vs. (47.39+/-8.30)%, respectively, both P<0.01]. The level of CK-MB was significantly lowered in the DEX group compared with control group [(8.69+/-4.16)U/g vs. (18.15+/-5.59) U/g, P<0.01], and myocardium MDA content was decreased (P<0.05). Moreover, the levels of T-SOD, CuZn-SOD, CAT, GSH-Px, and the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were significantly increased (P<0.05 or/and P<0.01) in DEX group.. DEX induces upregulation of MT, which attenuates I/R injury in rat heart.

Topics: Animals; Ca(2+) Mg(2+)-ATPase; Catalase; Dexamethasone; Disease Models, Animal; Glutathione Peroxidase; Male; Metallothionein; Myocardial Reperfusion Injury; Myocardium; Random Allocation; Rats; Rats, Sprague-Dawley; Sodium-Hydrogen Exchangers; Superoxide Dismutase

Zinc, an essential micronutrient, is involved in wound healing. The hypozincemia seen with chronic aldosteronism is associated with enhanced fecal and urinary excretory Zn losses, and its tissue distribution is less certain. This study monitored tissue 65Zn distribution in uninephrectomized rats at weeks 1 and 4 of aldosterone/salt treatment (ALDOST). Plasma and tissue total radionucleotide uptake was determined by calculating its mean radioactivity at 1, 4, 8, 24, and 48 hours after intravenous 65Zn administration and where respective area under the concentration-time curves (AUC) were determined by the linear trapezoidal rule and expressed as a tissue:plasma AUC ratio. Examined tissues included: (1) injured heart and kidney in response to ALDOST and incised skin; (2) noninjured liver, skeletal muscle, and spleen sites of stress-linked Zn uptake; and (3) bone, a major storage and release site when Zn homeostasis is threatened. In comparison with age-matched and gender-matched controls, the following were found with week 1 and 4 ALDOST: (1) reduced plasma 65Zn; (2) an accumulation of 65Zn in heart and kidneys, where a well-known vasculopathy involves intramural vessels, and in incised skin at week 1; (3) an organ-specific increase in tissue 65Zn in liver, in keeping with upregulated metallothionein expression, skeletal muscle, and spleen; and (4) a fall in bone and healed skin Zn at week 4. Thus a wide-ranging disturbance in Zn homeostasis appears during ALDOST to include its translocation from plasma to injured heart, kidneys, and skin and noninjured liver, skeletal muscle, and spleen together with a resorption of stored Zn in bone at week 4. Zinc dyshomeostasis is an integral feature of chronic aldosteronism.

Topics: Animals; Bone Resorption; Chronic Disease; Disease Models, Animal; Feces; Hyperaldosteronism; Kidney; Liver; Male; Metallothionein; Muscle, Skeletal; Nephrectomy; Organ Specificity; Rats; Rats, Sprague-Dawley; Zinc; Zinc Radioisotopes

Topics: Animals; Blood Coagulation; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Disseminated Intravascular Coagulation; Fibrinogen; Galactosamine; Lipopolysaccharides; Liver; Liver Diseases; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Platelet Count; Time Factors

There is clinical evidence linking asthma with the trace element, zinc (Zn). Using a mouse model of allergic inflammation, we have previously shown that labile Zn decreases in inflamed airway epithelium (Truong-Tran AQ, Ruffin RE, Foster PS, Koskinen AM, Coyle P, Philcox JC, Rofe AM, Zalewski PD. Am J Respir Cell Mol Biol 27: 286-296, 2002). Moreover, mild nutritional Zn deficiency worsens lung function. Recently, a number of proteins belonging to the Solute Carrier Family 39 (ZIP) and Solute Carrier Family 30 (ZnT) have been identified that bind Zn and regulate Zn homeostasis. Mice were sensitized, and subsequently aerochallenged, with ovalbumin to induce acute and chronic airway inflammation. Mice received 0, 54, or 100 microg of Zn intraperitoneally. Tissues were analyzed for Zn content and histopathology. Inflammatory cells were counted in bronchoalveolar lavage fluid. Cytokine and Zn transporter mRNA levels were determined by cDNA gene array and/or real-time PCR. Zn supplementation decreased bronchoalveolar lavage fluid eosinophils by 40 and 80%, and lymphocytes by 55 and 66%, in the acute and chronic models, respectively. Alterations in Zn transporter expression were observed during acute inflammation, including increases in ZIP1 and ZIP14 and decreases in ZIP4 and ZnT4. Zn supplementation normalized ZIP1 and ZIP14, but it did not affect mRNA levels of cytokines or their receptors. Our results indicate that inflammation-induced alterations in Zn transporter gene expression are directed toward increasing Zn uptake. Increases in Zn uptake may be needed to counteract the local loss of Zn in the airway and to meet an increased demand for Zn-dependent proteins. The reduction of inflammatory cells by Zn in the airways provides support for Zn supplementation trials in human asthmatic individuals.

Topics: Animals; Anti-Inflammatory Agents; Body Weight; Bronchoalveolar Lavage Fluid; Cation Transport Proteins; Cytokines; Dietary Supplements; Disease Models, Animal; Female; Gene Expression Regulation; Inflammation; Inflammation Mediators; Lung; Metallothionein; Mice; Receptors, Cytokine; Respiratory Hypersensitivity; RNA, Messenger; Zinc

It has been hypothesized that copper-mediated oxidative stress contributes to the pathogenesis of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease in humans. To verify this hypothesis, we examined the copper and zinc concentrations and the amounts of lipid peroxides, together with that of the expression of metallothionein (MT) isoforms in a mouse model [superoxide dismutase1 transgenic (SOD1 Tg) mouse] of ALS. The expression of MT-I and MT-II (MT-I/II) isoforms were measured together with Western blotting, copper level, and lipid peroxides amounts increased in an age-dependent manner in the spinal cord, the region responsible for motor paralysis. A significant increase was already seen as early as 8-week-old SOD1 Tg mice, at which time the mice had not yet exhibited motor paralysis, and showed a further increase at 16 weeks of age, when paralysis was evident. Inversely, the spinal zinc level had significantly decreased at both 8 and 16 weeks of age. The third isoform, the MT-III level, remained at the same level as an 8-week-old wild-type mouse, finally increasing to a significant level at 16 weeks of age. It has been believed that a mutant SOD1 protein, encoded by a mutant SOD1, gains a novel cytotoxic function while maintaining its original enzymatic activity, and causes motor neuron death (gain-of-toxic function). Copper-mediated oxidative stress seems to be a probable underlying pathogenesis of gain-of-toxic function. Taking the above current concepts and the classic functions of MT into account, MTs could have a disease modifying property: the MT-I/II isoform for attenuating the gain-of-toxic function at the early stage of the disease, and the MT-III isoform at an advanced stage.

Topics: Age Factors; Amyotrophic Lateral Sclerosis; Animals; Blotting, Western; Cerebellum; Copper; Disease Models, Animal; Female; Humans; Lipid Peroxides; Male; Metallothionein; Mice; Mice, Transgenic; Mutation; Polymerase Chain Reaction; Protein Isoforms; Spinal Cord; Superoxide Dismutase; Time Factors; Zinc

Zinc (Zn) and its binding protein metallothionein (MT) have been proposed to suppress the disease activity in ulcerative colitis. To determine the role of Zn and MT in the dextran sulfate sodium (DSS)-induced model of colitis in mice, a DSS dose-response study was conducted in male C57BL/6 wild-type (MT+/+) and MT-null (MT-/-) mice by supplementing 2%, 3%, and 4% DSS in the drinking water for 6 days. In the intervention study, colitis was induced with 2% DSS, Zn (24 mg/ml as ZnO) was gavaged (0.1 ml) daily, concurrent with DSS administration, and the disease activity index (DAI) was scored daily. Histology, MT levels, and myeloperoxidase (MPO) activity were determined. DAI was increased (P<0.05) by 16% and 21% with 3% and 4% concentrations of DSS, respectively, compared to 2%, evident after 5 days of DSS administration. MPO activity was increased in MT+/+ compared to MT-/- mice and those receiving DSS. Zn administration had a 50% (P<0.05) lower DAI compared to DSS alone. Zn partially prevented the distal colon of MT+/+ by 47% from DSS-induced damage compared to MT-/- mice. MT did not prevent DSS-induced colitis and Zn was partially effective in amelioration of DSS-induced colitis.

Topics: Animals; Colitis, Ulcerative; Colon; Dextran Sulfate; Disease Models, Animal; Dose-Response Relationship, Drug; Follow-Up Studies; Male; Metallothionein; Mice; Mice, Inbred C57BL; Peroxidase; Plasma Substitutes; Severity of Illness Index; Spectrophotometry; Trace Elements; Treatment Outcome; Zinc

Zinc is a structural constituent of many proteins, including Cu/Zn superoxide dismutase (SOD), an endogenous antioxidant enzyme. Hypozincemia has been found in patients hospitalized with congestive heart failure, where neurohormonal activation, including the renin-angiotensin-aldosterone system (RAAS), is expected and oxidative stress is present. This study was undertaken to elucidate potential pathophysiological mechanisms involved in Zn dyshomeostasis in aldosteronism. In rats receiving aldosterone/salt treatment (ALDOST) alone for 1 and 4 wk or in combination with spironolactone (Spiro), an ALDO receptor antagonist, we monitored 24-h urinary and fecal Zn excretion and tissue Zn levels in heart, liver, and skeletal muscle, together with tissue metallothionein (MT)-I, a Zn(2+)-binding protein, and Cu/Zn-SOD activities in plasma and tissues. When compared with unoperated, untreated, age-/sex-matched controls, urinary and, in particular, fecal Zn losses were markedly increased (P < 0.05) at days 7 and 28 of ALDOST, leading to hypozincemia and a fall (P < 0.05) in plasma Cu/Zn-SOD activity. Microscopic scars and perivascular fibrosis of intramural coronary arteries first appeared in the right and left ventricles at week 4 of ALDOST and were accompanied by increased (P < 0.05) tissue Zn, MT-I, and Cu/Zn-SOD activity, which were not found in uninjured liver or skeletal muscle. Spiro cotreatment prevented cardiac injury and Zn redistribution to the heart. Thus increased urinary and fecal Zn losses, together with their preferential translocation to sites of cardiac injury, where MT-I overexpression and increased Cu/Zn-SOD activity appeared, contribute to Zn dyshomeostasis in rats with aldosteronism, which were each prevented by Spiro. These findings may shed light on Zn dyshomeostasis found in patients with decompensated heart failure.

Topics: Aldosterone; Animals; Coronary Vessels; Disease Models, Animal; Feces; Fibrosis; Homeostasis; Hyperaldosteronism; Liver; Male; Metabolic Diseases; Metallothionein; Mineralocorticoid Receptor Antagonists; Muscle, Skeletal; Myocardium; Nephrectomy; Oxidative Stress; Rats; Rats, Sprague-Dawley; Spironolactone; Superoxide Dismutase; Time Factors; Zinc

Metallothionein (MT)-I/II has been shown to be neuroprotective and neuroregenerative in a model of rat cortical brain injury. Here we examine expression patterns of MT-I/II and its putative receptor megalin in rat retina. At neonatal stages, MT-I/II was present in retinal ganglion cells (RGCs) but not glial or amacrine cells; megalin was present throughout the retina. Whilst MT-I/II was absent from adult RGC in normal animals and after optic nerve transection, the constitutive megalin expression in RGCs was lost following optic nerve transection. In vitro MT-IIA treatment stimulated neuritic growth: more RGCs grew neurites longer than 25 microm (P < 0.05) in dissociated retinal cultures and neurite extension increased in retinal explants (P < 0.05). MT-IIA treatment of mixed retinal cultures increased megalin expression in RGCs, and pre-treating cells with anti-megalin antibodies prevented MT-IIA-stimulated neurite extension. Our results indicate that MT-IIA stimulates neurite outgrowth in RGCs and may do so via the megalin receptor; we propose that neurite extension is triggered via signal transduction pathways activated by the NPxY motifs of megalin's cytoplasmic tail.

Topics: Animals; Animals, Newborn; Antibodies; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Low Density Lipoprotein Receptor-Related Protein-2; Male; Metallothionein; Neurites; Optic Nerve Injuries; Rats; Retinal Ganglion Cells; Signal Transduction; Up-Regulation

Topics: Animals; Antioxidants; Calpain; Disease Models, Animal; Humans; Immobilization; Metallothionein; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Oxidative Stress; Peptide Hydrolases; Proteasome Endopeptidase Complex; Spinal Cord Injuries; Time Factors; Ubiquitin; Ubiquitin-Protein Ligases

To investigate the chemopreventive efficacy of the Indian medicinal plant Acanthus ilicifolius L Acanthaceae in a transplantable Ehrlich ascites carcinoma (EAC)-bearing murine model.. Male Swiss albino mice were divided into four groups: Group A was the untreated normal control; Group B was the EAC control mice group that received serial, intraperitoneal (ip) inoculations of rapidly proliferating 2 x 10(5) viable EAC cells in 0.2 mL of sterile phosphate buffered saline; Group C was the plant extract-treated group that received the aqueous leaf extract (ALE) of the plant at a dose of 2.5 mg/kg body weight by single ip injections, once daily for 10, 20 and 30 consecutive days following tumour inoculation (ALE control); and Group D was the EAC + ALE-treatment group. The chemopreventive potential of the ALE was evaluated in a murine model by studying various biological parameters and genotoxic markers, such as tumour cell count, mean survival of the animals, haematological indices, hepatocellular histology, immunohistochemical expression of liver metallothionein (MT) protein, sister-chromatid exchanges (SCEs), and DNA alterations.. Treatment of the EAC-bearing mice with the ALE significantly (P < 0.001) reduced viable tumour cell count by 68.34% (228.7 x 10(6) +/- 0.53) when compared to EAC control mice (72.4 x 10(6) +/- 0.49), and restored body and organ weights almost to the normal values. ALE administration also increased (P < 0.001) mean survival of the hosts from 35 +/- 3.46 d in EAC control mice to 83 +/- 2.69 d in EAC + ALE-treated mice. Haematological indices also showed marked improvement with administration of ALE in EAC-bearing animals. There was a significant increase in RBC count (P < 0.001), hemoglobin percent (P < 0.001), and haematocrit value (P < 0.001) from 4.3 +/- 0.12, 6.4 +/- 0.93, and 17.63 +/- 0.72 respectively in EAC control mice to 7.1 +/- 0.13, 12.1 +/- 0.77, and 30.23 +/- 0.57 respectively in EAC + ALE-treated group, along with concurrent decrement (P < 0.001) in WBC count from 18.8 +/- 0.54 in EAC control to 8.4 +/- 0.71 in EAC + ALE. Furthermore, treatment with ALE substantially improved hepatocellular architecture and no noticeable neoplastic lesions or foci of cellular alteration were observed. Daily administration of the ALE was found to limit liver MT expression, an important marker of cell proliferation with concomitant reduction in MT immunoreactivity (62.25 +/- 2.58 vs 86.24 +/- 5.69, P < 0.01). ALE was also potentially effective in reducing (P < 0.001) the frequency of SCEs from 14.94 +/- 2.14 in EAC control to 5.12 +/- 1.16 in EAC + ALE-treated group. Finally, in comparison to the EAC control, ALE was able to suppress in vivo DNA damage by abating the generations of 'tailed' DNA by 53.59% (98.65 +/- 2.31 vs 45.06 +/- 1.14, P < 0.001), and DNA single-strand breaks (SSBs) by 38.53% (3.14 +/- 0.31 vs 1.93 +/- 0.23, P < 0.01) in EAC-bearing murine liver.. Our data indicate that, ALE is beneficial in restoring haematological and hepatic histological profiles and in lengthening the survival of the animals against the proliferation of ascites tumour in vivo. Finally, the chemopreventive efficacy of the ALE is manifested in limiting MT expression and in preventing DNA alterations in murine liver. The promising results of this study suggest further investigation into the chemopreventive mechanisms of the medicinal plant A. ilicifolius in vivo and in vitro.

Topics: Acanthaceae; Animals; Carcinoma, Ehrlich Tumor; Cell Proliferation; Cell Transplantation; Disease Models, Animal; DNA Breaks, Single-Stranded; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Drug; Liver; Male; Metallothionein; Mice; Plant Extracts; Sister Chromatid Exchange

After intracerebral hemorrhage (ICH), many changes of gene transcription occur that may be important because they will contribute to understanding mechanisms of injury and recovery. Therefore, gene expression was assessed using Affymetrix microarrays in the striatum and the overlying cortex at 24 h after intracranial infusions of blood into the striatum of adult rats. Intracerebral hemorrhage regulated 369 of 8,740 transcripts as compared with saline-injected controls, with 104 regulated genes shared by the striatum and cortex. There were 108 upregulated and 126 downregulated genes in striatum, and 170 upregulated and 69 downregulated genes in the cortex. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed upregulation of IL-1-beta, Lipcortin 1 (annexin) and metallothionein 1,2, and downregulation of potassium voltage-gated channel, shaker-related subfamily, beta member 2 (Kcnab2). Of the functional groups of genes modulated by ICH, many metabolism and signal-transduction-related genes decreased in striatum but increased in adjacent cortex. In contrast, most enzyme, cytokine, chemokine, and immune response genes were upregulated in both striatum and in the cortex after ICH, likely in response to foreign proteins from the blood. A number of these genes may contribute to brain edema and cellular apoptosis caused by ICH. In addition, downregulation of growth factor pathways and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway could also contribute to perihematoma cell death/apoptosis. Intracerebral hemorrhage-related downregulation of GABA-related genes and potassium channels might contribute to perihematoma cellular excitability and increased risk of post-ICH seizures. These genomic responses to ICH potentially provide new therapeutic targets for treatment.

Topics: Animals; Annexin A1; Brain; Cerebral Hemorrhage; Cluster Analysis; Disease Models, Animal; Down-Regulation; gamma-Aminobutyric Acid; Gene Expression Profiling; Genomics; Growth Substances; Interleukin-1; Male; Metallothionein; Oligonucleotide Array Sequence Analysis; Phosphatidylinositol 3-Kinases; Potassium Channels, Voltage-Gated; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Up-Regulation

Inflammation can play a key role in Cd-induced dysfunctions. Quercetin is a potent oxygen free radical scavenger and a metal chelator. Our aim was to study the effect of quercetin on Cd-induced kidney damage and metallothionein expression. The study was performed in Wistar rats that were administered during 9 weeks with either cadmium (1.2 mg Cd/kg/day, s.c.), quercetin (50 mg/kg/day, i.p.) or cadmium + quercetin. Renal toxicity was evaluated by measuring blood urea nitrogen concentration and urinary excretion of enzymes marker of tubular damage. Endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) renal expression were assessed by Western blot. Renal expression of metallothionein 1 and 2 (MT-1, MT-2) and eNOS mRNA was assessed by Northern blot. Our data demonstrated that Cd-induced renal toxicity was markedly reduced in rats that also received quercetin. MT-1 and MT-2 mRNA levels in kidney were substantially increased during treatment with Cd, being even higher when the animals received Cd and quercetin. Renal eNOS expression was significantly higher in rats receiving Cd and quercetin than in animals receiving Cd alone or in control rats. In the group that received Cd, COX-2 and iNOS expression was markedly higher than in control rats. In the group Cd+quercetin, no changes in COX-2 and iNOS expression were observed compared with the control group. Our results demonstrate that quercetin treatment prevents Cd-induced overexpression of iNOS and COX-2, and increases MT expression. These effects can explain the protection by quercetin of Cd-induced nephrotoxicity.

Topics: Animals; Cadmium Compounds; Chronic Disease; Cyclooxygenase 2; Disease Models, Animal; Enzyme Induction; Kidney Diseases; Male; Metallothionein; Nitric Oxide Synthase; Protective Agents; Quercetin; Rats; Rats, Wistar

Upper aerodigestive tract (UADT) cancer, including oral and esophageal cancer, is an important cause of cancer deaths worldwide. Patients with UADT cancer are frequently zinc deficient (ZD) and show a loss of function of the pivotal tumor suppressor gene p53. The present study examined whether zinc deficiency in collaboration with p53 insufficiency (p53+/-) promotes lingual and esophageal tumorigenesis in mice exposed to low doses of the carcinogen 4-nitroquinoline 1-oxide. In wild-type mice, ZD significantly increased the incidence of lingual and esophageal tumors from 0% in zinc sufficient (ZS) ZS:p53+/+ mice to approximately 40%. On the p53+/- background, ZD:p53+/- mice had significantly greater tumor incidence and multiplicity than ZS:p53+/- and ZD:p53+/+ mice, with a high frequency of progression to malignancy. Sixty-nine and 31% of ZD:p53+/- lingual and esophageal tumors, respectively, were squamous cell carcinoma versus 19 and 0% of ZS:p53+/- tumors (tongue, P = 0.003; esophagus, P = 0.005). Immunohistochemical analysis revealed that the increased cellular proliferation observed in preneoplastic lingual and esophageal lesions, as well as invasive carcinomas, was accompanied by overexpression of cytokeratin 14, cyclooxygenase-2 and metallothionein. In summary, a new UADT cancer model is developed in ZD:p53+/- mouse that recapitulates aspects of the human cancer and provides opportunities to probe the genetic changes intrinsic to UADT carcinogenesis and to test strategies for prevention and reversal of this deadly cancer.

Topics: 4-Nitroquinoline-1-oxide; Animals; Biomarkers, Tumor; Carcinogens; Cyclooxygenase 2; Disease Models, Animal; Esophageal Neoplasms; Female; Gene Expression; Genetic Predisposition to Disease; Immunohistochemistry; Keratin-14; Keratins; Male; Metallothionein; Mice; Mice, Mutant Strains; Precancerous Conditions; Tongue Neoplasms; Tumor Suppressor Protein p53; Zinc

Regional distribution of coenzyme Q10 and mitochondrial complex-1 activity were estimated in the brains of control-(C57BL/6), metallothionein knock out-, metallothionein transgenic-, and homozygous weaver mutant mice; and human dopaminergic (SK-N-SH) cells with a primary objective to determine the neuroprotective potential of coenzyme Q10 in Parkinson's disease. Complex-1 activity as well as coenzyme Q10 were significantly higher in the cerebral cortex as compared to the striatum in all the genotypes examined. Complex-1 activity and coenzyme Q10 were significantly reduced in weaver mutant mice and metallothionein knock out mice, but were significantly increased in metallothionein transgenic mice. The reduced complex-1 activity and 18F-DOPA uptake occurred concomitantly with negligible differences in the coenzyme Q10 between in the cerebral cortex and striatum of weaver mutant mice. Administration of coenzyme Q10 increased complex-1 activity and partially improved motoric performance in weaver mutant mice. Direct exposure of rotenone also reduced coenzyme Q10, complex-1 activity, and mitochondrial membrane potential in SK-N-SH cells. Rotenone-induced down-regulation of complex-1 activity was attenuated by coenzyme Q10 treatment, suggesting that complex-1 may be down regulated due to depletion of coenzyme Q10 in the brain. Therefore, metallothionein-induced coenzyme Q10 synthesis may provide neuroprotection by augmenting mitochondrial complex-1 activity in Parkinson's disease.

Topics: Analysis of Variance; Animals; Brain; Cell Line, Tumor; Chromatography, High Pressure Liquid; Coenzymes; Dihydroxyphenylalanine; Disease Models, Animal; Electron Transport Complex I; Fluorine Radioisotopes; Humans; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Mice, Transgenic; Neuroblastoma; Neuroprotective Agents; Parkinson Disease; Positron-Emission Tomography; Statistics as Topic; Tissue Distribution; Ubiquinone

In the present study, we investigated the antitumour efficacy of vanadium in a defined rodent model of experimental hepatocarcinogenesis. Hepatic preneoplasia was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal injection of diethylnitrosamine (DEN) (200 mg/kg body weight) followed by promotion with phenobarbital (PB). The levels of modified DNA bases 8-hydroxy-2'-deoxyguanosine (8-OHdG), a potential marker involved in the initiation of carcinogenesis, were measured by high-performance liquid chromatography, whereas tissue trace element status and expression of metallothionein (MT), a Cu-Zn metalloprotein associated with neoplastic cell growth and subsequent development of premalignant phenotype of the cell, were studied by energy-dispersive X-ray fluorescence spectrometry and enzyme-coupled immunohistochemistry, respectively. There was a significant and steady elevation of modified bases (8-OHdG) along with substantial increase in MT immunoexpression and disturbance in trace element homeostasis following DEN exposure. Supplementation of vanadium at a dose of 0.5 ppm for four consecutive weeks strictly abated the formation of 8-OHdG (P < 0.0001; 81.28%) in preneoplastic rat liver. In a long-term DEN plus PB regimen, vanadium was able to limit in situ MT expression with a concomitant decrease in MT immunoreactivity (P < 0.05). Furthermore, vanadium treatment throughout the study restored hepatic levels of essential trace elements and decreased nodular incidence (58.34%) and nodule multiplicity (P < 0.001; 66.89%) in rats treated with DEN plus PB. Taken together, the study provides evidence in support of the chemopreventive potential of vanadium in limiting neoplastic transformation during the preneoplastic stages of hepatocarcinogenesis in rats.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Cell Transformation, Neoplastic; Chemoprevention; Deoxyguanosine; Diethylnitrosamine; Disease Models, Animal; DNA Damage; Elements; Liver; Liver Neoplasms, Experimental; Male; Metallothionein; Phenobarbital; Rats; Rats, Sprague-Dawley; Spectrometry, X-Ray Emission; Vanadium

Trypanosoma cruzi infection and nonsteroidal anti-inflammatory drugs inhibit colorectal carcinogenesis by mechanisms not completely known and metallothionein proteins (MTs) may be involved in this process. Sixty-six male Wistar rats weighing 90 to 120 g were randomly divided into seven groups (GI to GVII). GI, GII and GIII animals were subcutaneously infected with 200,000 trypomastigote forms of the Y strain of T. cruzi. After 8 weeks, GI, GII, GIV, and GVI were injected with one weekly subcutaneous dose of 12 mg/kg dimethylhydrazine for 4 weeks. In sequence, GI, GIV and GV were treated with nimesulide (10 mg/kg per dose, five times per week for 8 weeks). Groups I, III, IV, and VI had 12 animals, and each of the other groups had 6 animals. All the animals were euthanized 8 weeks after the last dimethylhydrazine injection. The colons were fixed and processed for MT immunohistochemistry. The index of MT-overexpressing colonic crypts (MTEC) was estimated as the percentage of MT-stained crypts in relation to the total number of crypts scored. Five hundred crypts per animal were scored. Data were analyzed by the Kruskal-Wallis test followed by the Dunn test. There was an increase in MTEC index in the groups either infected with T. cruzi or treated with nimesulide or both infected and treated when compared to control (401, 809, and 1011%, respectively). We suggest that the increased formation of MTEC may be related to the protection against carcinogenesis provided both by T. cruzi infection and nimesulide.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carcinogens; Chagas Disease; Colorectal Neoplasms; Dimethylhydrazines; Disease Models, Animal; Immunohistochemistry; Male; Metallothionein; Random Allocation; Rats; Rats, Wistar; Sulfonamides; Trypanosoma cruzi

Previous studies have described altered expression of metallothioneins (MTs) in neurodegenerative diseases like multiple sclerosis (MS), Down syndrome, and Alzheimer's disease (AD). In order to gain insight into the possible role of MTs in neurodegenerative processes and especially in human diseases, the use of animal models is a valuable tool. Several transgenic mouse models of AD amyloid deposits are currently available. These models express human beta-amyloid precursor protein (AbetaPP) carrying different mutations that subsequently result in a varied pattern of beta-amyloid (Abeta) deposition within the brain. We have evaluated the expression of MT-I and MT-III mRNA by in situ hybridization in three different transgenic mice models of AD: Tg2576 (carrying AbetaPP harboring the Swedish K670N/M671L mutations), TgCRND8 (Swedish and the Indiana V717F mutations), and Tg-SwDI (Swedish and Dutch/Iowa E693Q/D694N mutations). MT-I mRNA levels were induced in all transgenic lines studied, although the pattern of induction differed between the models. In the Tg2576 mice MT-I was weakly upregulated in cells surrounding Congo Red-positive plaques in the cortex and hippocampus. A more potent induction of MT-I was observed in the cortex and hippocampus of the TgCRND8 mice, likely reflecting their higher amyloid plaques content. MT-I upregulation was also more significant in Tg-SwDI mice, especially in the subiculum and hippocampus CA1 area. Immunofluorescence stainings demonstrate that astrocytes and microglia/macrophages surrounding the plaques express MT-I&II. In general, MT-I regulation follows a similar but less potent response than glial fibrillary acidic protein (GFAP) expression. In contrast to MT-I, MT-III mRNA expression was not significantly altered in any of the models examined suggesting that the various MT isoforms may have different roles in these experimental systems, and perhaps also in human AD.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Astrocytes; Brain; Cerebral Cortex; Disease Models, Animal; Female; Gene Expression Regulation, Enzymologic; Glial Fibrillary Acidic Protein; Hippocampus; Male; Metallothionein; Metallothionein 3; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Mutation; Nerve Tissue Proteins; Plaque, Amyloid; RNA, Messenger

Metallothionein (MT) mRNA expression was investigated in a rodent model (G93A SOD1 transgenic mouse) for a lethal motor neuron disease, amyotrophic lateral sclerosis (ALS). In 8-wk-old mice that did not yet exhibit motor paralysis, MT-I mRNA expression was already significantly upregulated in the region of the spinal cord responsible for motor paralysis. The expression of another isoform, MT-III, was not changed. In the cerebellum, which is not responsible for motor paralysis in ALS, neither the expression profiles of MT-I nor MT-III were altered. In 16-wk-old mice exhibiting motor paralysis, the expression of MT-I mRNA remained upregulated and the MT-III level tended to be elevated. Although no significant differences were found in the levels of both isoforms in the liver or kidney of 8-wk-old mice, the MT-I mRNA expression level was significantly upregulated in the kidney and liver of 16-wk-old mice. These results indicated that the MT-I isoform, but not the MT-III isoform, is associated with motor neuron death in ALS and suggested that the disease might be a systemic disorder to which the spinal cord is particularly susceptible.

Topics: Amyotrophic Lateral Sclerosis; Animals; Cerebellum; Disease Models, Animal; Gene Expression Regulation; Kidney; Liver; Metallothionein; Metallothionein 3; Mice; Mice, Transgenic; Motor Activity; Nerve Tissue Proteins; RNA, Messenger; Spinal Cord; Spleen; Up-Regulation

Oxygen free radicals and nitric oxide (NO) participate in the pathogenesis of acute central nervous system (CNS) injury by forming peroxynitrite, which promotes oxidative damage and tyrosine nitration. Neuronal nitration is associated with cell death, but little is known of the characteristics and cell fate of nitrated astrocytes. In this study, we have used a postnatal excitotoxic lesion model (intracortical NMDA injection) and our aims were (i) to evaluate the temporal and spatial pattern of astroglial nitration in correlation with the neuropathological process and the sources of NO; and (ii) to establish, if any, the correlation among astrocyte nitration and other events such as expression of cytoskeletal proteins, antioxidant enzymes, and cell death markers to cope with nitration and/or undergo cell death. Our results show that after postnatal excitotoxic damage two distinct waves of nitration were observed in relation to astrocytes. At 24 h post-lesion, early-nitrated astrocytes were found within the neurodegenerating area, coinciding with the time of maximal cell death. These early-nitrated astrocytes are highly ramified protoplasmic cells, showing diffuse glial fibrillary acidic protein (GFAP) content and expressing inducible NOS. At later time-points, when astrogliosis is morphologically evident, nitrated hypertrophied reactive astrocytes are observed in the penumbra and the neurodegenerated area, displaying increased expression of GFAP and vimentin cytoskeletal proteins and of metallothionein I-II and Cu/Zn superoxide dismutase antioxidant proteins. Moreover, despite revealing activated caspase-3, they do not show TUNEL labeling. In summary, we show that nitrated astrocytes in vivo constitute a subpopulation of highly reactive astrocytes which display high resistance towards oxidative stress induced cell death.

Topics: Animals; Animals, Newborn; Astrocytes; Brain Injuries; Cytoskeletal Proteins; Disease Models, Animal; Female; Male; Metallothionein; Nerve Tissue Proteins; Neurotoxins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Rats, Long-Evans; Superoxide Dismutase; Tyrosine

We evaluated whether modulation of metallothionein (MT, and subsequent oxidative stress) would influence the development and progression of colitis.. MT-transgenic (MT-TG), MT-knockout (MT-KO), and wild-type (WT) mice were treated with Dextran Sodium Sulfate (DSS) to induce colitis compared to controls (no DSS).. The DSS treated MT-TG and MT-KO mice responded in a manner similar to that of DSS treated WT mice, with all groups developing severe colitis. The colonic MT content in DSS treated animals increased drastically when compared to controls. The colonic and blood levels of the antioxidant, glutathione (GSH), were significantly reduced in DSS treated MT-TG and MT-KO mice. Howevei there was no significant difference in the cysteine levels in these mice. The subeellular level of MT was highest in intestinal crypts loci of MT-TG mice and was not observed in MT-KO mice as well as in the inflammatory lesions.. We conclude that MT does not appear to influence the development or progression of intestinal pathology in the DSS animal model of IBD.

Topics: Animals; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Gene Expression; Humans; Immunohistochemistry; Inflammatory Bowel Diseases; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic

To monitor the systemic gene expression profile in a murine model of lipopolysaccharide-induced acute lung injury.. Acute lung injury was induced by intratracheal injection of lipopolysaccharide in 3 mice. Another 3 normal mice receiving same volume of normal saline were taken as the controls. The comprehensive gene expression profile was monitored by the recently modified long serial analysis of gene expression.. A total of 24,670 tags representing 12,168 transcripts in the control mice and 26,378 tags representing 13,397 transcripts in the mice with lung injury were identified respectively. There were 11 transcripts increasing and 7 transcripts decreasing more than 10 folds in the lipopolysaccharide-treated mice. The most overexpressed genes in the mice with lung injury included serum amyloid A3, metallothionein 2, lipocalin 2, cyclin-dependent kinase inhibitor 1A, lactate dehydrogenase 1, melatonin receptor, S100 calcium-binding protein A9, natriuretic peptide precursor, etc. Mitogen activated protein kinase 3, serum albumin, complement component 1 inhibitor, and ATP synthase were underexpressed in the lung injury mice.. Serial analysis of gene expression provides a molecular characteristic of acute lung injury.

Topics: Animals; Cyclin-Dependent Kinases; Disease Models, Animal; DNA-Binding Proteins; Gene Expression; Gene Expression Profiling; Lipopolysaccharides; Male; Metallothionein; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Nuclear Proteins; Nucleophosmin; Protein Folding; Reference Values; Respiratory Distress Syndrome; S100 Proteins; Serum Amyloid A Protein

Long-Evans Cinnamon (LEC) rats are characterized by an abnormal hepatic deposition of copper (Cu) due to a lack of the Cu-transporter P-type adenosine triphosphatase: accordingly, the strain is a good animal model of Wilson's disease. The effect of oral zinc (Zn) acetate treatment on the development of acute hepatitis and the biochemical parameters of Cu-induced liver damage was studied in 5-week-old LEC rats (n=52).. Rats receiving 50 or 80 mg/ml/day Zn acetate by gavage and control rats receiving a daily dose of glucose solution 0.02 g/ml by gastric intubation were killed at 1, 2 or 8 weeks after the start of treatment.. Treatment with Zn acetate resulted in the prevention of acute hepatitis: 10 of the 13 untreated rats developed signs and symptoms compatible with acute hepatitis between the 6th and 7th week of treatment. Tissue metallothionein (MT) significantly increased in the treated rats and positively correlated with Zn concentrations within the liver. Control rats had a significantly higher iron concentration in the liver and kidneys compared with supplemented rats, after both short- and long-term experiments. 8-hydroxy-2'-deoxyguanosine amounts were significantly lower in untreated rats.. Zn acetate prevents acute hepatitis, by increasing tissue MT concentrations, reducing Cu absorption and interfering with Fe metabolism.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Administration, Oral; Animals; Chemical and Drug Induced Liver Injury; Deoxyguanosine; Disease Models, Animal; Dose-Response Relationship, Drug; Hepatitis, Animal; Iron; Kidney; Liver; Male; Metallothionein; Protective Agents; Rats; Rats, Inbred LEC; Zinc; Zinc Acetate

Topics: Animals; Cardiomyopathy, Alcoholic; Disease Models, Animal; Ethanol; Fibrosis; Heart; Metallothionein; Mice; Mice, Knockout; Myocardium; Zinc

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis (MS). EAE and MS are characterized by CNS inflammation, demyelination and neurodegeneration. The inflammatory response occurring within the CNS leads to glial activation, dysfunction and death, as well as axonal damage and neurological deficit. Although the pathogenic mechanisms involved in EAE/MS are not well understood, accumulating data suggest that oxidative stress plays a major role in lesion development, and contributes to axonal dysfunction and degeneration. Metallothionein-I and -II are anti-inflammatory, neuroprotective, antioxidant proteins expressed during EAE and MS, in which they might play a protective role. The present study aimed to describe the expression profile of a group of inflammatory, neurodegenerative and tissue repair markers as well as metallothioneins during proteolipid protein-induced EAE, and to establish the time-relationships these molecules had during EAE. Interestingly, we found two marker expression profiles. In the first, marker expression increased as clinical signs worsened and reverted to baseline expression during recovery; in the second, marker expression increased at a later point during relapse, peaked at highest clinical score, and remained elevated throughout recovery. Of note, metallothionein expression was found to be related to the second profile, which would suggest that metallothionein proteins are implicated in the clinical recovery of EAE and perhaps these antioxidant proteins may provide therapeutic benefits in MS.

Topics: Animals; Apoptosis; Biomarkers; Central Nervous System; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Immunohistochemistry; In Situ Nick-End Labeling; Inflammation; Metallothionein; Mice; Multiple Sclerosis; Nerve Degeneration; Oxidative Stress; Time Factors

Gene expression analysis is a powerful tool that has been used to define the pathological processes underlying many diseases. Several laboratories, including our own, have used this approach to identify molecular abnormalities in the G93A/SOD1 mouse, an animal model of amyotrophic lateral sclerosis (ALS). Here, we report the results of analysis of an expanded panel of genes throughout the entire lifetime in the spinal cord of these animals. In addition to upregulation of microglia/neuroinflammatory genes identified previously, we observed upregulation of metallothionein-I and -II (MT-I, MT-II). MT-I and MT-II play an important role in disposition of zinc ion, and other studies have also indicated their levels are altered in development of motor neuron disease in these animals. We also analyzed the effect on these expression profiles of several candidate drugs that have been shown to have neuroprotective effects in vivo or in vitro. That is, we asked whether administration to the G93A/SOD1 mice of any of these drugs could reverse the alterations in gene expression patterns that occur as the animals develop. The mice were given daily doses of these drugs when they were 9-11 weeks old, at a stage early in development of motor neuron disease, continuing for 5 weeks, at which time they were sacrificed. Treatment of the mice with l-carnosine, a dipeptide that scavenges free radicals and chelates zinc, did not affect expression of any of the genes altered in these animals. However, it did upregulate 3 genes unaffected by the presence of the G93A/SOD1 mutation: glial fibrillary acidic protein (GFAP), stroma-derived factor-1 (SDF-1), and excitatory amino acid transporter-2 (EAAT2). In contrast, metallothionein-III (MT-III) was downregulated. Treatment of the animals with baicalein, an herbal extract with anti-inflammatory and numerous other effects, downregulated the microglia markers CD68, CD80, and CD86, all of which were upregulated in untreated mutant animals. Baicalein treatment also downregulated tumor necrosis factor receptor (TNFRp55) and upregulated noninducible nitric oxide synthase (nNOS) and glutamine synthase (GS). These 3 genes were unaffected by the presence of the G93A mutation. We discuss the implication of these results for testing the effects of these and other candidate drugs in mutant SOD1 mice.

Topics: Amyotrophic Lateral Sclerosis; Animals; Antioxidants; Apoptosis; Carnosine; Disease Models, Animal; Drug Evaluation, Preclinical; Flavanones; Gene Expression; Gene Expression Regulation; Genetic Markers; Inflammation; Metallothionein; Mice; Mice, Knockout; Neuroprotective Agents; Neurotoxins; Oxidative Stress; RNA; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1

The mouse model of experimentally induced systemic AA amyloidosis is long established, well validated, and closely analogous to the human form of this disease. However, the induction of amyloid by experimental inflammation is unpredictable, inconsistent, and difficult to modulate. We have previously shown that murine AA amyloid deposits can be imaged using iodine-123 labeled SAP scintigraphy and report here substantial refinements in both the imaging technology and the mouse model itself. In this regard, we have generated a novel prototype of AA amyloid in which mice expressing the human interleukin 6 gene, when given amyloid enhancing factor, develop extensive and progressive systemic AA deposition without an inflammatory stimulus, i.e., a transgenic rapidly inducible amyloid disease (TRIAD) mouse. Additionally, we have constructed high-resolution micro single photon emission computed tomography (SPECT)/computed tomography (CT) instrumentation that provides images revealing the precise anatomic location of amyloid deposits labeled by radioiodinated serum amyloid P component (SAP). Based on reconstructed microSPECT/CT images, as well as autoradiographic, isotope biodistribution, and quantitative histochemical analyses, the (125)I-labeled SAP tracer bound specifically to hepatic and splenic amyloid in the TRIAD animals. The ability to discern radiographically the extent of amyloid burden in the TRIAD model provides a unique opportunity to evaluate the therapeutic efficacy of pharmacologic compounds designed to inhibit fibril formation or effect amyloid resolution.

Topics: Amyloid; Amyloidosis; Animals; Autoradiography; Disease Models, Animal; Humans; Interleukin-6; Iodine Radioisotopes; Metallothionein; Mice; Mice, Transgenic; Promoter Regions, Genetic; Radiography; Tomography, Emission-Computed, Single-Photon

Spinal ischemia is a frequent cause of paralysis. Here we explore the biological basis of ischemic preconditioning (IPC), the phenomenon in which a brief period of ischemia can confer protection against subsequent longer and normally injurious ischemia, to identify mediators of endogenous neuroprotection. Using microarrays, we examined gene expression changes induced by brief spinal ischemia using a rat balloon occlusion model. Among the nearly 5000 genes assayed, relatively few showed two-fold changes, and three groups stood out prominently. The first group codes for heat shock protein 70, which is induced selectively and robustly at 30 min after brief ischemia, with increases up to 100-fold. A second group encodes metallothioneins 1 and 2. These mRNAs are increased at 6 and 12 h after ischemia, up to 12-fold. The third group codes for a group of immediate-early genes not previously associated with spinal ischemia: B-cell translocation gene 2 (BTG2), the transcription factors early growth response 1 (egr-1) and nerve growth factor inducible B (NGFI-B), and a mitogen-activated protein kinase phosphatase, ptpn16, an important cell signaling regulator. These mRNAs peak at 30 min and return to baseline or are decreased 6 h after ischemia. Several other potentially protective genes cluster with these induced mRNAs, including small heat shock proteins, and many have not been previously associated with IPC. These results provide both putative mediators of IPC and molecular targets for testing preconditioning therapies.

Topics: Animals; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation; Genes, Immediate-Early; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Male; Metallothionein; Oligonucleotide Array Sequence Analysis; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord; Spinal Cord Ischemia

Epidemiology investigation showed that no worker drunk Maotai liquor for nearly 30 years died of hepatic diseases, and no obvious hepatic fibrosis and cirrhosis were found in 99 workers who had drunk Maotai liquor for a long period by epidemiology investigation and needle biopsy. The same finding was detected in rats that were drunk by Maotai liquor continued for 56 days. This study was to investigate the effects of Maotai liquor on the liver and its mechanism of preventing hepatic fibrosis.. After ingestion of Maotai for 56 consecutive days, male SD rats were killed for detecting the levels of metallothionein and malondialdehyde (MDA) in liver tissues. Rat hepatic stellate cells (HSCs) and human HSCs were cultured in vitro to observe the effect of Maotai on HSCs proliferation and collagen synthesis. After ingestion of Maotai for 14 consecutive weeks, the livers of male SD rats were harvested for pathohistological examination.. The level of metallothionein in the liver of Maotai-induced rats increased by 22 folds, whereas the levels of hepatic lipid peroxide and MDA was decreased significantly (P<0.05) in Maotai-induced animals suffering from CCl4. Maotai demonstrated obvious inhibitory effect on proliferation of HSCs and the inhibition was concentration-dependent. Gene expression and protein secretion of collagens could also be inhibited by Maotai. In alcoholic group, typical liver cirrhosis was observed. In Maotai group, however, though fatty degeneration of hepatocytes and mild fibrosis of the interstitium were observed, no obvious hepatic fibrosis and cirrhosis were found.. It might be an important mechanism of interfering the progress of hepatic fibrosis that Maotai increases the level of metallothionein in the liver and inhibits the activation of HSCs and the synthesis of collagen proteins.

Topics: Alcoholic Beverages; Animals; Base Sequence; Biopsy, Needle; Cell Division; Cells, Cultured; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Hepatocytes; Immunohistochemistry; Liver Cirrhosis, Alcoholic; Male; Metallothionein; Molecular Sequence Data; Polymerase Chain Reaction; Probability; Rats; Rats, Sprague-Dawley; Reference Values; Risk Assessment; Sensitivity and Specificity

In experimental studies on the common human coxsackievirus B type 3 (CB3) infection, administered cadmium (Cd) is known to accumulate in the liver and kidneys. CB3 adapted to Balb/c mice was used to study whether infection affects the Cd-binding protein, metallothionein (MT) and if this alters the normal physiological trace element balance in the liver, kidney, spleen and brain. On day 3 of infection, degradation of liver proteins (44%, P<0.01) occurred, whereas in the spleen, protein increased (63%, P<0.05). The infection increased MT five-fold (P<0.01) in liver and kidneys, and in spleen by 34% (P<0.05). A redistribution of Cd and copper (Cu) from the liver to the kidney was associated with this increase in MT, resulting in an increased (P<0.01) kidney/liver ratio for both elements. The infection increased the zinc (Zn) concentration more in the kidney than in the liver, but the kidney/liver ratio was not significantly affected. Results show that MT is increased in several organs during the early phase of infection and is associated with redistribution of both essential and non-essential trace elements. This may be a normal response in common infections that could adversely influence the pathogenesis when the host is concomitantly exposed to potentially toxic trace elements, even at levels in the physiological range.

Topics: Acute-Phase Reaction; Animals; Brain; Cadmium; Copper; Disease Models, Animal; Enterovirus B, Human; Enterovirus Infections; Female; Kidney; Liver; Metallothionein; Mice; Mice, Inbred BALB C; Organ Size; Proteins; Spleen; Trace Elements; Zinc

Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-IL6 mice) induces significant inflammation and neurodegeneration but also affords neuroprotection against acute traumatic brain injury. This neuroprotection is likely mediated by the IL-6-induced protective factors metallothioneins-I and -II (MT-I+II). Here we evaluate the neuroprotective roles of IL-6 vs. MT-I+II during 6-aminonicotinamide (6-AN)-induced neurotoxicity, by using GFAP-IL6 mice and transgenic mice overexpressing MT-I (TgMT) as well as GFAP-IL6 mice crossed with TgMT mice (GFAP-IL6 x TgMT). 6-AN caused acute damage of brainstem gray matter areas identified by necrosis of astrocytes, followed by inflammatory responses. After 6-AN-induced toxicity, secondary damage was observed, consisting of oxidative stress, neurodegeneration, and apoptotic cell death. We hereby show that the primary injury caused by 6-AN was comparable in wild-type and GFAP-IL6 mice, but MT-I overexpression could significantly protect the brain tissue. As expected, GFAP-IL6 mice showed increased CNS inflammation with more gliosis, macrophages, and lymphocytes, including increased cytokine expression, relative to the other mice. However, GFAP-IL6 mice showed reduced oxidative stress (judged from nitrotyrosine, malondialdehyde, and 8-oxoguanine stainings), neurodegeneration (accumulation of neurofibrillary tangles), and apoptosis (determined from TUNEL and caspase-3). MT-I+II expression was significantly higher in GFAP-IL6 mice than in wild types, which may contribute to the IL-6-induced neuroprotection. In support of this, overexpression of MT-I in GFAP-IL6 x TgMT as well as TgMT mice protected the brainstem tissue significantly from 6-AN-induced toxicity and secondary brain tissue damage. Overall, the results demonstrate that brain MT-I+II proteins are fundamental neuroprotective factors, which in the future may become therapeutic agents.

Topics: 6-Aminonicotinamide; Animals; Apoptosis; Brain Stem; Caspase 3; Caspases; Crosses, Genetic; Disease Models, Animal; Down-Regulation; Encephalitis; Free Radicals; Glial Fibrillary Acidic Protein; Gliosis; Interleukin-6; Metallothionein; Mice; Mice, Transgenic; Nerve Degeneration; Neurofibrillary Tangles; Neuroglia; Neuroprotective Agents; Neurotoxins; Oxidative Stress; Promoter Regions, Genetic

Metallothionein (MT) is a small cysteine-rich protein that sequesters and distributes metal ions. Its overexpression stimulates cell proliferation and inhibits apoptosis. We investigated the effects of burn injury on MT expression and metal localization. We also sought to determine roles of MT in the pathophysiologic alterations in the liver after injury.. Mice (C57BLKS/J, MT-I/II knock-out, KO, and wild-type control mice) were subjected to an 18% burn. Liver tissues harvested after injury were analyzed for the MT expression and the levels of zinc, copper, manganese, and iron. Levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase were measured in serum samples from MT-I/MT-II KO mice and controls after injury.. Transient induction of MT-I and MT-II mRNAs was observed 3-6 h after injury, while MT-I/MT-II protein peaked on day 1. The induction was localized to hepatocytic nuclei. The intrahepatic levels of zinc, copper, and iron were transiently elevated on day 1, when a downregulation of manganese was evident. Interestingly, only the serum levels of aspartate aminotransferase were significantly augmented in MT-I/MT-II KO mice compared to controls after injury.. These data suggest that MT and metals may participate in the pathogenesis of the liver after burn injury.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Blotting, Western; Burns; Disease Models, Animal; Enzymes; Gene Expression; Immunoenzyme Techniques; Liver; Metallothionein; Metals, Heavy; Mice; Mice, Inbred C57BL; Mice, Knockout; RNA, Messenger

We report on the cochlea of a novel metallothionein-I (MT)/RFP-RET transgenic mouse model with severe systemic melanosis. Electron microscopy revealed that these transgenic mice possess abundant quantities of melanin in the intermediate cells of the stria vascularis. High performance liquid chromatography analysis indicated that cochleae of these transgenic mice contained about twice as much eumelanin as cochleae of control C57BL/6 mice and that the amount of pheomelanin was approximately equal in these two strains. Auditory brainstem responses at 2, 4, 8, and 16 kHz were not significantly different between transgenic and control mice. This is the first report on a mouse model of overproduction of cochlear eumelanin, and our results suggest that this transgenic mouse is an excellent model for investigating the effects of overexpression of cochlear eumelanin. In addition, we provide evidence that eumelanin overproduction in the cochlea does not affect normal hearing.

Topics: Animals; Chromatography, High Pressure Liquid; Cochlea; Disease Models, Animal; Evoked Potentials, Auditory, Brain Stem; Melanins; Melanosis; Metallothionein; Mice; Mice, Inbred Strains; Mice, Transgenic; Microscopy, Electron; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Receptor Protein-Tyrosine Kinases; Severity of Illness Index; Stria Vascularis

Both the immediate insult and delayed apoptosis contribute to functional deficits after brain injury. Secondary, delayed apoptotic death is more rapid in immature than in adult CNS neurons, suggesting the presence of age-dependent protective factors. To understand the molecular pathobiology of secondary injury in the context of brain development, we identified changes in expression of oxidative stress response genes during postnatal development and target deprivation-induced neurodegeneration. The antioxidants metallothionein I and II (MT I/II) were increased markedly in the thalamus of adult C57BL/6 mice compared to mice <15 days old. Target deprivation generates reactive oxygen species that mediate neuronal apoptosis in the central nervous system; thus the more rapid apoptosis observed in the immature brain might be due to lower levels of MT I/II. We tested this hypothesis by documenting neuronal loss after target-deprivation injury. MT I/II-deficient adult mice experienced greater thalamic neuron loss at 96 hr after cortical injury compared to that in controls (80 +/- 2% vs. 57 +/- 4%, P < 0.01), but not greater overall neuronal loss (84 +/- 4% vs. 79 +/- 3%, MT I/II-deficient vs. controls). Ten-day-old MT I/II-deficient mice, however, experienced both faster onset of secondary neuronal death (30 vs. 48 hr) and greater overall neuronal loss (88 +/- 2% vs. 69 +/- 4%, P = 0.02). MT I/II are thus inhibitors of age-dependent secondary brain injury, and the low levels of MT I/II in immature brains explains, in part, the enhanced susceptibility of the young brain to neuronal loss after injury. These findings have implications for the development of age-specific therapeutic strategies to enhance recovery after brain injury.

Topics: Age Factors; Analysis of Variance; Animals; Animals, Newborn; Apoptosis; Brain Injuries; Cell Count; Decerebrate State; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Functional Laterality; Gene Expression Profiling; Gene Expression Regulation, Developmental; Geniculate Bodies; Immunoblotting; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Degeneration; Neurons; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Thalamus; Time Factors

The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long-Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.

Topics: Animals; Catalase; Copper; Disease Models, Animal; Glutathione; Hepatolenticular Degeneration; Intestinal Mucosa; Intestines; Iron; Kidney; Liver; Male; Metallothionein; Rats; Rats, Inbred LEC; Superoxide Dismutase; Zinc

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age of six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats.

Topics: Animals; Cation Transport Proteins; Copper Transporter 1; Disease Models, Animal; Glutathione; Glutathione Disulfide; Hepatolenticular Degeneration; Liver; Membrane Proteins; Metallothionein; Rats; RNA, Messenger; Soybean Proteins

In an attempt to establish a primate model of chronic cadmium (Cd) toxicosis, we ovariectomized cynomolgus monkeys and treated with CdCl2 by repeated intravenous injections for 13 to 15 months. The animals showed an increase in blood glucose from Month 10 and a decrease in blood insulin at Month 11of the Cd-treatment. Histopathological examination of the Cd-treated animals revealed islet atrophy with reduction in islet number and vacuolation of the islet cells, whereas there was no remarkable change in the acinar cells of the exocrine pancreas. In histomorphometrical examination, insulin-positive areas in the islets were significantly decreased, accompanying a relative increase of glucagon-positive areas. Large amounts of Cd accumulated in the pancreas, and metallothionein (MT), a Cd binding protein, was localized in the islets of Cd-treated animals. The present study demonstrated that the chronic intravenous injection of Cd to cynomolgus monkeys induced the accumulation of the metal in the pancreas, degeneration of islet B cells and the diabetic clinical signs. Therefore the islet B cell is one of the major targets of the chronic Cd poisoning in monkeys.

Topics: Animals; Blood Glucose; Cadmium; Cadmium Poisoning; Disease Models, Animal; Female; Glucagon; Insulin; Islets of Langerhans; Macaca fascicularis; Metallothionein; Monkey Diseases; Ovariectomy; Random Allocation

Metallothioneins (MTs) are small, cysteine-rich, metal binding proteins. Their function has often been considered as stress-related proteins capable of protecting cells from heavy metal toxicity and oxidative free radicals. However, recent interest has focused on the brain-specific MT-III isoform, which has neurite-inhibitory properties. To investigate the effect of another MT isoform, human MT-IIA, on neurite growth, we used rat cortical neuron cultures. MT-IIA promoted a significant increase in the rate of initial neurite elongation of individually plated neurons. We also investigated the effect of MT-IIA on the neuronal response to axonal transection in vitro. MT-IIA promoted reactive axonal growth after injury, and, by 18 hr after transection, MT-IIA had promoted axonal growth across the injury tract. Exogenous application of MT-IIA after cortical brain injury promoted wound healing, as observed by a significant decrease in cellular degradation at 4 d after injury. Furthermore, MT-IIA-treated rats exhibited numerous SMI-312-immunoreactive axonal processes within the injury tract. This was in contrast to vehicle-treated animals, in which few axonal sprouts were observed. By 7 d after injury, MT-IIA treatment resulted in a total closing over of the injury tract by microglia, astrocytes, and reactive axonal processes. However, although some reactive axonal processes were observed within the injury tract of vehicle-treated rats, the tract itself was almost never entirely enclosed. These results are discussed in relation to a possible physiological role of metallothioneins in the brain, as well as in a therapeutic context.

Topics: Animals; Astrocytes; Axons; Brain Injuries; Cell Division; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Humans; Immunohistochemistry; Male; Metallothionein; Microglia; Neurites; Neurofilament Proteins; Neurons; Rats; Rats, Wistar; Wound Healing

Experimental autoimmune encephalomyelitis (EAE) is an animal model for the human demyelinating disease multiple sclerosis (MS). EAE and MS are characterized by significant inflammation, demyelination, neuroglial damage, and cell death. Metallothionein-I and -II (MT-I + II) are antiinflammatory and neuroprotective proteins that are expressed during EAE and MS. We have shown recently that exogenous administration of Zn-MT-II to Lewis rats with EAE significantly reduced clinical symptoms and the inflammatory response, oxidative stress, and apoptosis of the infiltrated central nervous system areas. We show for the first time that Zn-MT-II treatment during EAE significantly prevents demyelination and axonal damage and transection, and stimulates oligodendroglial regeneration from precursor cells, as well as the expression of the growth factors basic fibroblast growth factor (bFGF), transforming growth factor (TGF)beta, neurotrophin-3 (NT-3), NT-4/5, and nerve growth factor (NGF). These beneficial effects of Zn-MT-II treatment could not be attributable to its zinc content per se. The present results support further the use of Zn-MT-II as a safe and successful therapy for multiple sclerosis.

Topics: Animals; Antioxidants; Axons; Cell Division; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Growth Substances; Metallothionein; Multiple Sclerosis; Myelin Sheath; Nerve Regeneration; Oligodendroglia; Rats; Rats, Inbred Lew; Stem Cells; Up-Regulation; Wallerian Degeneration

6-aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray matter astrocytes mainly in the brainstem. We have examined the role of interleukin-6 (IL-6) in this degenerative process by using transgenic mice with astrocyte-targeted IL-6 expression (GFAP-IL6 mice). This study demonstrates that transgenic IL-6 expression significantly increases the 6-AN-induced inflammatory response of reactive astrocytes, microglia/macrophages, and lymphocytes in the brainstem. Also, IL-6 induced significant increases in proinflammatory cytokines IL-1, IL-12, and tumor necrosis factor-alpha as well as growth factors basic fibroblast growth factor (bFGF), transforming growth factor-beta, neurotrophin-3, angiopoietin, vascular endothelial growth factor, and the receptor for bFGF. In accordance, angiogenesis was increased in GFAP-IL6 mice relative to controls after 6-AN. Moreover, oxidative stress and apoptotic cell death were significantly reduced by transgenic IL-6 expression. IL-6 is also a major inducer in the CNS of metallothionein I and II (MT-I+II), which were significantly increased in the GFAP-IL6 mice. MT-I+II are antioxidants and neuroregenerative factors in the CNS, so increased MT-I+II levels in GFAP-IL6 mice could contribute to the reduction of oxidative stress and cell death in these mice.

Topics: 6-Aminonicotinamide; Angiogenesis Inducing Agents; Animals; Apoptosis; Astrocytes; Brain Stem; Cell Count; Central Nervous System; Cytokines; Disease Models, Animal; Gene Targeting; Glial Fibrillary Acidic Protein; Growth Substances; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-6; Lymphocytes; Macrophages; Malondialdehyde; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Nerve Degeneration; Oxidative Stress; Staining and Labeling; Stem Cells; Teratogens; Tyrosine

Rats injected with mercuric chloride develop an acute renal tubular necrosis with uremia, which is frequently lethal. Pretreatment for 3 or 7 days with a protein-free diet reduces the mortality, the clinical signs (tremor), and the severity of renal tubular necrosis, and ameliorates the uremic chemical findings in the serum. Similar results followed injection of a nephrotoxic amino acid, D-serine, after pretreatment with a protein-free diet. Indirect evidence suggests that induction of metallothionein may be involved, at least in the experiments with mercury. Acute uremia produced by nephrotoxic chemicals may be useful for further studies of the role of nutrition in uremia, while avoiding the surgical procedures and prolonged observations required for the "remnant kidney" models.

Topics: Acute Disease; Animals; Biomarkers; Body Weight; Diet, Protein-Restricted; Disease Models, Animal; Dose-Response Relationship, Drug; Injections, Subcutaneous; Kidney; Kidney Tubular Necrosis, Acute; Male; Mercuric Chloride; Metallothionein; Phosphates; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Sucrose; Time Factors; Treatment Outcome; Urea; Uremia

Transgenic mice overexpressing the human mutated form (G93A) of Cu,Zn-superoxide dismutase (mSOD1) develop motor neuron degeneration resembling amyotrophic lateral sclerosis. In vitro studies have shown that mSOD1-induced, reactive oxygen species-mediated apoptosis of motor neurons depends on the presence of copper and the relative absence of zinc. Oral intake of zinc sulphate induces the expression of metallothioneins, enzymes that decrease oxidative stress, and leads to higher serum zinc, and lower copper levels. We therefore tested the effect of chronic oral administration of zinc sulfate (0.075 and 0.375 g/kg) on disease onset and survival of mSOD1 transgenic mice. We observed that zinc sulfate, rather than prolonging survival, decreased survival. We conclude that zinc sulfate amplifies the mSOD1 transgenic mouse phenotype. This finding may shed more light on the role of zinc in mSOD1-mediated toxicity.

Topics: Amyotrophic Lateral Sclerosis; Animals; Disease Models, Animal; Female; Humans; Male; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Transgenic; Superoxide Dismutase; Zinc Sulfate

We applied serial analysis of gene expression (SAGE) to study differentially expressed genes in mouse brain 14 hr after the induction of focal cerebral ischemia. Analysis of >60,000 transcripts revealed 83 upregulated and 94 downregulated transcripts (more than or equal to eightfold). Reproducibility was demonstrated by performing SAGE in duplicate on the same starting material. Metallothionein-II (MT-II) was the most significantly upregulated transcript in the ischemic hemisphere. MT-I and MT-II are assumed to be induced by metals, glucocorticoids, and inflammatory signals in a coordinated manner, yet their function remains elusive. Upregulation of both MT-I and MT-II was confirmed by Northern blotting. MT-I and MT-II mRNA expression increased as early as 2 hr after 2 hr of transient ischemia, with a maximum after 16 hr. Western blotting and immunohistochemistry revealed MT-I/-II upregulation in the ischemic hemisphere, whereas double labeling demonstrated the colocalization of MT with markers for astrocytes as well as for monocytes/macrophages. MT-I- and MT-II-deficient mice developed approximately threefold larger infarcts than wild-type mice and a significantly worse neurological outcome. For the first time we make available a comprehensive data set on brain ischemic gene expression and underscore the important protective role of metallothioneins in ischemic damage of the brain. Our results demonstrate the usefulness of SAGE to screen functionally relevant genes and the power of knock-out models in linking function to expression data generated by high throughput techniques.

Topics: Animals; Blotting, Northern; Blotting, Western; Brain; Brain Ischemia; Disease Models, Animal; Gene Expression Profiling; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Microcirculation; Monte Carlo Method; Neuroprotective Agents; Reproducibility of Results; RNA, Messenger; Time Factors; Up-Regulation

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene cause one form of familial amyotrophic lateral sclerosis, a progressive disorder of motor neurons leading to weakness and death of affected individuals. Experiments using both transgenic mice expressing mutant SOD1 and SOD1 knock-out mice have demonstrated that disease is caused by a toxic gain of function and not by a loss of normal SOD1 activity. Precise mechanisms underlying mutant SOD1 toxicity are unclear but may involve abnormal interactions between zinc and SOD1. The metallothioneins (MTs) represent a family of zinc binding proteins that can function as zinc chaperones for apo-SOD1 in vitro. We hypothesized that manipulation of metallothioneins in vivo might alter the disease phenotype of transgenic mice expressing G93A SOD1 and therefore crossed this line with MT-I and MT-II or MT-III knock-out mice. G93A SOD1 mice deficient of either MT-I and MT-II or MT-III exhibited significant reductions in survival compared with G93A SOD1 mice. In addition, motor dysfunction was markedly accelerated in G93A SOD1 mice deficient in metallothioneins with regard to onset (MT-I and MT-II) or progression (MT-III). These results indicate that the disease course in G93A SOD1 mice is dependent on levels of metallothionein expression. Because MT-I and MT-II are expressed in glia whereas MT-III is found in neurons, these results also indicate that primary changes within non-neuronal cells can affect mutant SOD1-induced disease and do so in ways distinct from primary neuronal changes.

Topics: Amyotrophic Lateral Sclerosis; Animals; Blotting, Western; Cell Count; Crosses, Genetic; Disease Models, Animal; Disease Progression; Glial Fibrillary Acidic Protein; Humans; Metallothionein; Metallothionein 3; Mice; Mice, Transgenic; Motor Activity; Nerve Tissue Proteins; Neuroglia; Neurons; Phenotype; Spinal Cord; Superoxide Dismutase; Survival Analysis; Zinc

The response to injury is dependent on several factors, including the type and extent of the injury, genetics, and the environment. In the present study, the genetic contribution to sepsis was evaluated in a mouse model. Sepsis was induced in two inbred mouse strains, C57BL/6J (B6) and A/J, by cecal ligation and single puncture (CLP). Frequency of mortality was significantly higher in B6 than A/J mice from 36 to 132 h after CLP. Plasma TNF-alpha, IL-1beta, and IL-6 levels were similar in both strains after CLP. IL-10 plasma levels were significantly higher in B6 mice as opposed to A/J mice after 24 h of CLP. Similarly, hepatic myeloperoxidase activity, an index of polymorphonuclear leukocytes, was elevated in B6 mice as compared with A/J mice after 24 h of CLP. On the contrary, metallothionein mRNA levels were higher in A/J mice compared with B6 mice. Finally, leptin levels were also higher in A/J than B6 mice within 19 h of CLP. This study demonstrates a genetic contribution in the response to sepsis.

Topics: Animals; Disease Models, Animal; Fibrinogen; Interleukin-1; Interleukin-10; Interleukin-6; Leptin; Liver; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Peroxidase; Sepsis; Species Specificity; Survival Rate; Tumor Necrosis Factor-alpha

Many diabetic patients suffer from cardiomyopathy, even in the absence of vascular disease. This diabetic cardiomyopathy predisposes patients to heart failure and mortality from myocardial infarction. Evidence from animal models suggests that reactive oxygen species play an important role in the development of diabetic cardiomyopathy. Our laboratory previously developed a transgenic mouse model with targeted overexpression of the antioxidant protein metallothionein (MT) in the heart. In this study we used MT-transgenic mice to test whether an antioxidant protein can reduce cardiomyopathy in the OVE26 transgenic model of diabetes. OVE26 diabetic mice exhibited cardiomyopathy characterized by significantly altered mRNA expression, clear morphological abnormalities, and reduced contractility under ischemic conditions. Diabetic hearts appeared to be under oxidative stress because they had significantly elevated oxidized glutathione (GSSG). Diabetic mice with elevated cardiac MT (called OVE26MT mice) were obtained by crossing OVE26 transgenic mice with MT transgenic mice. Hyperglycemia in OVE26MT mice was indistinguishable from hyperglycemia in OVE26 mice. Despite this, the MT transgene significantly reduced cardiomyopathy in diabetic mice: OVE26MT hearts showed more normal levels of mRNA and GSSG. Typically, OVE26MT hearts were found to be morphologically normal, and elevated MT improved the impaired ischemic contractility seen in diabetic hearts. These results demonstrate that cardiomyocyte-specific expression of an antioxidant protein reduces damage to the diabetic heart.

Topics: Actins; Animals; Antioxidants; Atrial Natriuretic Factor; Base Sequence; Blood Glucose; Blotting, Northern; Cardiomyopathies; Diabetic Angiopathies; Disease Models, Animal; Gene Expression Regulation; Glutathione Disulfide; Insulin; Metallothionein; Mice; Mice, Transgenic; Molecular Sequence Data; Myocardial Contraction; Oligonucleotide Probes; RNA, Messenger; Transcription, Genetic; Triglycerides

Copper is both essential for life and toxic. Aberrant regulation of copper at the level of intracellular transport has been associated with inherited diseases, including Wilson's disease (WND) in humans. WND results in accumulation of copper and the copper and zinc-binding protein metallothionein (MT) in liver and other tissues, liver degeneration, and neurological dysfunction. The toxic milk (TX) mutation in mice results in a phenotype that mimics human WND, and TX has been proposed to be a model of the disease. We characterized TX mice as a model of altered metal ion and MT levels during development, and after treatment with the metal ion chelators tetrathiomolybdate (TTM) and deferiprone (L1). We report that hepatic, renal and brain copper and MT are elevated in TX mice at 3 and 12 months of age. Zinc was significantly higher in TX mouse liver, but not brain and kidney, at both time points. Nodules appeared spontaneously in TX mouse livers at 8-12 months that maintained high copper levels, but with more normal morphology and decreased MT levels. Treatment of TX mice with TTM significantly reduced elevated hepatic copper and MT. Transient increases in blood and kidney copper accompanied TTM treatment and indicated that renal excretion was a significant route of removal. Treatment with L1, on the other hand, had no effect on liver or kidney copper and MT, but resulted in increased brain copper and MT levels. These data indicate that TTM, but not L1, may be useful in treating diseases of copper overload including WND.

Topics: Animals; Copper; Deferiprone; Disease Models, Animal; Hepatolenticular Degeneration; Metallothionein; Mice; Mice, Mutant Strains; Molybdenum; Phenotype; Pyridones

Topics: Animals; Brain; Brain Injuries; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Gene Expression; Humans; Inflammation Mediators; Metallothionein; Mice; Mice, Knockout; Multiple Sclerosis; Neurodegenerative Diseases; Oxidative Stress; Rats; RNA, Messenger

The effect of oral Zn treatment was studied in the liver and kidneys of 26 male Long-Evans Cinnamon (LEC) rats (mutant animals, 5 weeks old) in relation to both the interaction between Zn and Cu and the localisation and concentration of metallothionein (MT). Rats receiving 80 mg zinc acetate daily by gavage and control rats receiving no treatment were killed after 1 or 2 weeks. By immunohistochemical and analytical chemical techniques we revealed that treated rats had higher levels of MT in the hepatic and renal cells compared to untreated ones. Tissue Zn concentrations were significantly higher in treated rats compared to untreated whereas Cu concentrations decreased in the liver and kidneys as indicated by analytical chemical analyses. MT levels also decreased with treatment period. A histochemical procedure, obtained using autofluorescence of Cu-metallothioneins, confirms these findings: after 2 weeks, the signal decreased in both the liver and kidney sections. This gives a greater understanding of the mechanism of Cu metabolism in the two tissues considered. These results suggest that Zn acts both to compete for absorption on the luminal side of the intestinal epithelium and to induce the synthesis of MT.

Topics: Animals; Copper; Disease Models, Animal; Drug Interactions; Hepatolenticular Degeneration; Immunohistochemistry; Kidney; Liver; Male; Metallothionein; Microscopy, Fluorescence; Rats; Rats, Inbred LEC; Zinc

To study the antagonism of Zn-metallothionein (Zn-MT) to oxidative stress of rats with severe scalding after delayed resuscitation.. 27 Wistar rats were randomly divided into three groups: normal control; burn with delayed fluid resuscitation group; and Zn-MT protected group. The rats were scalded with 30% total body surface area (TBSA) third degree burn. Four hours after injury, they were resuscitated, but Zn-MT (1 x 10(-5) mol/L) was given in the third group rats. 24 hours after they were scalded, blood was taken, RBCs were separated and labeled with electron spin resonance (ESR) spin label 3-maleimido-proxy l to detect membrane protein conformation. The serum was collected and added into the reaction system of xanthine, xanthine oxidase and ESR spin trap 5, 5-dimethylpyroline-N-oxide to assay the levels of superoxide dismutase (SOD) by ESR.. The ratios of strong solidity and weak solidity of RBC membrane proteins in the three groups were 0.352 +/- 0.043, 0.409 +/- 0.027, and 0.386 +/- 0.062 respectively, and the revolved times of membrane proteins were 1.300 +/- 0.210, 1.576 +/- 0.190 and 1.381 +/- 0.210 seconds respectively. Compared to the first group, the membrane protein conformation in the second group was changed remarkably, and the speed of membrane protein movement decreased significantly. However, those changes were alleviated obviously by Zn-MT. The SOD levels in serum of the three groups rats were 73.2% +/- 1.4%, 48.8% +/- 3.8% and 66.8% +/- 3.2% respectively. Compared to the normal control group, the SOD levels in serum of the second group decreased obviously. However, an excess of oxygen free radicals produced after injury could be scavenged by Zn-MT.. RBC membrane proteins of rats with severe scalding after delayed resuscitation were injured by oxygen free radicals produced after injury, and Zn-MT could alleviate this injury.

Topics: Animals; Burns; Disease Models, Animal; Erythrocyte Membrane; Female; Male; Metallothionein; Protective Agents; Rats; Rats, Wistar; Reactive Oxygen Species; Superoxide Dismutase; Zinc

The aim of this work was to study oxidative stress and the compensating mechanisms implicated in severe thermal injury using the burned rat model. Results showed that after thermal injury glutathione (GSH) level was decreased, oxidized glutathione (GSSG) and the ratio of GSSG/GSH increased both at 24 and 48 h in the liver. The activities of GSH-reductase (GSH-Rx) in the liver and GSH-peroxidase (GSH-Px) both in the liver and erythrocytes increased at 24 h and then decreased at 48 h. The level of alpha-tocopherol in plasma was reduced at 24 h. Lipid peroxide levels increased both at 24 and 48 h in the liver. The serum zinc level decreased, reaching a minimum at 12h, whereas liver zinc level was elevated and reached the maximum at 12 h. After severe thermal injury enhancement of metallothionein (MT) expression has been discovered for the first time. MT content in the liver increased both at 24 and 48 h. Expression of MT-I mRNA was activated at 3 h and reached the top at 24 h postburn. The conclusion is that severe thermal injury gives rise to oxidative stress and dramatic enhancement of MT expression could be one of the important compensative mechanisms of natural defense system postburn.

Topics: alpha-Tocopherol; Animals; Burns; Disease Models, Animal; Glutathione; Glutathione Reductase; Liver; Male; Metallothionein; Oxidative Stress; Rats; Rats, Wistar; Time Factors; Zinc

We previously reported that abnormal copper release from mutated Cu, Zn-superoxide dismutase (SOD1) proteins might be a common toxic gain-of-function in the pathogenesis of familial amyotrophic lateral sclerosis (FALS) [Ogawa et al. (1997) Biochem. Biophys. Res. Commun., 241, 251-257.]. In the present study, we first examined metallothioneins (MTs), known to bind copper ions and decrease oxidative toxicity, and found a twofold increase in MTs in the spinal cord of the SOD1 transgenic mice with a FALS-linked mutation (G93A), but not in the spinal cord of wild-type SOD1 transgenic mice. We then investigated whether the clinical course of FALS mice could be modified by the reduced expression of MTs, by crossing the FALS mice with MT-I- and MT-II-deficient mice. FALS mice clearly reached the onset of clinical signs and death significantly earlier in response to the reduction of protein expression. These results indicated that the copper-mediated free radical generation derived from mutant SOD1 might be related to the degeneration of motor neurons in FALS and that MTs might play a protective role against the expression of the disease.

Topics: Amyotrophic Lateral Sclerosis; Animals; Copper; Disease Models, Animal; Gene Dosage; Gene Expression Regulation, Enzymologic; Humans; Hydroxyl Radical; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Transgenic; Oxidative Stress; Spinal Cord; Superoxide Dismutase

Because oxidative stress may be involved in arterial hypertension by affecting the balance between relaxing and contracting factors of vascular smooth muscle, the training-induced adaptation of antioxidant defenses could be implicated in the antihypertensive effect of chronic exercise. It has been suggested that metallothionein (MT), a metal-binding protein, plays an antioxidant role in mammals. The aim of this experiment was to study whether chronic exercise (swimming) influences both the development of arterial hypertension in spontaneously hypertensive rats (SHR) and the modification of MT levels.. Male SHR and Wistar Kyoto (WKY) rats as control were trained to swim 1 h.d-1 5 d.wk-1 for 8 wk and sacrificed 72 h after the last exercise period. MT and total thiol levels were then measured.. Exercise training 1) reduced systolic blood pressure and heart rate in both SHR WKY rats, and 2) was associated with a decrease in hepatic and cardiac MT levels; there was an increase in the aortic MT amounts in exercised SHR only. No modifications were noted in the gastrocnemius muscle or kidneys. In exercised animals, total thiols were lower in the liver but not in kidneys.. Chronic exercise induced a reduction in arterial hypertension development in SHR rats and an adaptation of the MT levels in cardiac, hepatic, and aortic tissues. Further experiments are needed to pinpoint the role of the MT in these two cases in which oxidative stress occurs.

Topics: Animals; Aorta; Disease Models, Animal; Heart Rate; Hypertension; Liver; Male; Metallothionein; Myocardium; Physical Conditioning, Animal; Rats; Rats, Inbred SHR; Rats, Wistar

Zinc enhances cell protection against infection and injury and the healing processes themselves. We evaluated the effect of zinc supplementation at different doses on a model of experimental colitis in the rat.. Colitis, induced by intra-rectal instillation of dinitrobenzen-sulphonic acid, was assessed at 1 week by examining: general outcome and macroscopic damage, myeloperoxidase activity, mucosal zinc, iron and metallothionein concentrations. Rats received zinc sulphate, 2 mg/kg or 30 mg/kg, twice a day by gavage for 9 days, starting 3 days before the induction of colitis, or intrarectal instillation of zinc (20 mg/kg) once daily starting 8 hours after the induction of colitis and for 6 days thereafter. Zinc-treated rats had less diarrhoea, higher body weight and lower colonic weight than untreated rats but no effect was observed on macroscopic inflammation, adhesions, colonic distension and neutrophil infiltration of the colonic mucosa. Zinc supplementation did not affect mucosal iron and zinc concentrations or plasma zinc levels in colitic rats. Metallothionein synthesis was induced in control rats and to a lesser extent in colitic rats.. Zinc administration induces metallothionein synthesis but has little effect on the short-term course of experimental colitis.

Topics: Administration, Oral; Administration, Rectal; Analysis of Variance; Animals; Colitis; Dietary Supplements; Disease Models, Animal; Dose-Response Relationship, Drug; Intestinal Mucosa; Male; Metallothionein; Peroxidase; Probability; Random Allocation; Rats; Rats, Sprague-Dawley; Trace Elements; Zinc

A bolus dose of cadmium metallothionein (CdMT) produces renal proximal tubular dysfunction because it accumulates in the tubular epithelial cells and undergoes rapid degradation, releasing Cd. Morphologically, mitochondria appear to be the target organelle. The present study examined changes in renal cortical mitochondrial function following CdMT administration and investigated whether some of these effects could be ascribed to Cd2+ accumulation in the mitochondria. Sprague-Dawley rats were injected ip with 0.3 mg Cd as CdMT/kg and the animals were sacrificed after 6, 8, or 12 h. Two- to threefold increases in urinary protein excretion and LDH activity were evident at 8 h, with marked elevations (11- and 29-fold) thereafter. Renal cortical mitochondria were swollen and rounded at 12 h. The mitochondrial Cd level was 399 pmol/mg protein at 6 h and did not change significantly during the next 6 h; however, mitochondrial respiratory function declined with time. At 12 h, state 3 oxygen consumption, respiratory control ratio (RCR), and ADP:O (P/O) ratio were 48, 49, and 76% of control values, respectively, indicating inhibition of electron transfer and oxidative phosphorylation. The direct effect of Cd on mitochondrial function was examined by incubating mitochondria from untreated rats with 0.1-2 microM CdCl2. Rapid uptake of Cd resulted in concentration-dependent effects on respiration. After 1 min of incubation with 2 microM Cd, the mitochondria contained 262 microgCd/mg protein and state 3 respiration and RCR values were 75 and 33% of control levels, respectively. Thus, renal proximal tubular cell damage following a bolus dose of CdMT involves perturbations in mitochondrial respiration, brought on by the accumulation of Cd.

Topics: Analysis of Variance; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Kidney Cortex; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Metallothionein; Mitochondria; Mitochondrial Swelling; Oxidative Phosphorylation; Oxygen Consumption; Proteins; Rats; Rats, Sprague-Dawley; Time Factors

Cadmium is a potent hepatotoxicant for which neither effective preventive methods nor the mechanism of toxicity has been established. We investigated the preventive effect of dexamethasone against cadmium toxicity on cadmium-induced liver injury in rabbits. Pretreatment with dexamethasone at 1 mg/kg increased the rate of survival in rabbits administered 2.5 mg/kg iv cadmium. Cadmium induced acute severe liver injury characterized by hepatocellular necrosis, infiltration by inflammatory cells, and increases of plasma GOT, GPT, LDH, and LDH5. Dexamethasone mitigated the acute hepatotoxic effect of cadmium, but exacerbated cadmium-induced kidney dysfunction, with destruction of renal tubular cells and increases in excretion of protein, glucose, and amino acids into urine. The cadmium concentration in liver and kidney of rabbits administered cadmium was not changed by dexamethasone pretreatment. Although metallothionein mRNA expression induced by cadmium was not affected by dexamethasone in liver or kidney, cadmium-induced metallothionein protein production was augmented at the early phase in liver and decreased at the later phase in kidney. Neutrophilia observed after cadmium administration was enhanced initially by dexamethasone pretreatment. These results indicate that dexamethasone pretreatment potently prevented cadmium-induced liver injury, but exacerbated renal tubular dysfunction.

Topics: Amino Acids; Animals; Anti-Inflammatory Agents; Blood Urea Nitrogen; Cadmium; Chemical and Drug Induced Liver Injury; Creatinine; Dexamethasone; Disease Models, Animal; Drug Interactions; Female; Kidney; Kidney Diseases; Liver; Metallothionein; Proteinuria; Rabbits; Survival Rate; Time Factors

Oxidative damage has long been related to carcinogenesis in human cancers and animal cancer models. Recently a rat esophageal adenocarcinoma (EAC) model was established in our laboratory by using esophagoduodenal anastomosis (EDA) plus iron supplementation. Our previous study suggested that iron supplementation enhanced inflammation and the production of reactive nitrogen species in the esophageal epithelium, which could contribute to esophageal adenocarcinogenesis. Here we further characterized oxidative damage in this model. We were particularly interested in how excess iron was deposited in the esophagus, and which cells were targeted by oxidative damage. Male Sprague-Dawley rats received iron supplementation (50 mg Fe/kg/month, i.p.) starting 4 weeks after EDA. The animals were killed at 11, 30 or 35 weeks after surgery. EAC appeared as early as week 11 after surgery, and increased over time, up to 60% at 35 weeks after surgery. All EACs were well-differentiated mucinous adenocarcinoma at the squamocolumnar junction. Iron deposition was found at the squamocolumnar junction and in the area with esophagitis. Esophageal iron overload could result from transient increase of blood iron after i.p. injection, and the overexpression of transferrin receptor in the premalignant columnar-lined esophagus (CLE) cells. Oxidative damage to DNA (8-hydroxy-2'-deoxyguanosine), protein (carbonyl content) and lipid (thiobarbituric acid reactive substance) in the esophagus was significantly higher than that of the non-operated control. CLE cells were believed to be the target cells of oxidative damage because they overexpressed heme oxygenase 1 and metallothionein, both known to be responsive to oxidative damage. We propose that oxidative damage plays an important role in the formation of EAC in the EDA model, and a similar situation may occur in humans with gastroesophageal reflux and iron over-nutrition.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenocarcinoma, Mucinous; Anastomosis, Surgical; Animals; Barrett Esophagus; Cocarcinogenesis; Deoxyguanosine; Disease Models, Animal; DNA Adducts; Duodenum; Epithelial Cells; Esophageal Neoplasms; Esophagitis; Esophagus; Gastroesophageal Reflux; Heme Oxygenase (Decyclizing); Humans; Iron; Isoenzymes; Male; Metallothionein; Oxidative Stress; Postoperative Complications; Precancerous Conditions; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Transferrin; Thiobarbituric Acid Reactive Substances

Lipopolysaccharide (LPS) is a component of the gram-negative bacterial cell wall that is known to activate inflammatory cells and enhance the production of inflammatory mediators in the lung. As it is a ubiquitous compound, inhalation exposure is highly likely in the human environment. Adaptation is a phenomenon by which a previous exposure results in improved survival or reduced injury as compared to a single exposure alone. We hypothesized that the basic proinflammatory effects of LPS in the lung could result in the development of adaptation in animals. Based on evidence of age- and species-related differences in lung injury, we used an acute lung injury model with inhaled LPS to compare the development of adaptation in young and old Fisher 344 rats and C57Bl/6J mice. Animals were exposed to low-dose (predicted lung deposition approximately 20 ng in rats and approximately 5 ng in mice) LPS aerosols for 10 min on 3 consecutive days; on day 4, a high dose (rats approximately 200 ng; mice approximately 25 ng) was delivered. Another group of animals received only the high LPS dose on day 4, whereas controls were unexposed. Twenty-four hours after the last exposure, cellular and inflammatory parameters in bronchoalveolar lavage (BAL) were determined. An adaptive response was found in both rats and mice. Adapted animals showed significantly fewer BAL neutrophils compared to nonadapted ones; there was also a significantly lower release of oxidants from phorbol methyl ester-stimulated BAL cells from adapted compared to nonadapted animals, which, in turn, showed a greater response than controls. Furthermore, studies in old animals (21 mo of age) showed that adaptation also occurs in this age group. The adaptive response is clear in old mice; in rats, there is greater variability in the response, but an adaptive trend is apparent. Therefore, we have demonstrated that inhaled low-dose LPS can induce adaptation to subsequent higher doses, much as has been shown for other toxicants that induce oxidative lung injury.

Topics: Administration, Inhalation; Aging; Animals; Bronchoalveolar Lavage Fluid; Cell Count; Cells, Cultured; Cytokines; Disease Models, Animal; Immune Tolerance; Lipopolysaccharides; Lung; Male; Metallothionein; Mice; Mice, Inbred C57BL; Neutrophils; Pseudomonas aeruginosa; Rats; Rats, Inbred F344; Respiratory Burst; RNA, Messenger; Specific Pathogen-Free Organisms; Tetradecanoylphorbol Acetate

Missense mutations in the gene encoding copper zinc superoxide dismutase (SOD1) have been found to cause one form of familial amyotrophic lateral sclerosis (FALS). Although the exact mechanism of disease is unknown, abnormalities in the ability of mutant SOD1 to bind zinc or copper ions may be crucial in the pathogenesis of disease. Because members of the metallothionein (MT) family of zinc and copper binding proteins function as important cellular regulators of metal ion bioavailability in the central nervous system, we used in situ hybridization and immunohistochemistry to study the expression pattern of these molecules in a transgenic mouse model of familial ALS. In adult wild-type mouse spinal cord, expression of MT-I and MT-II is restricted to ependymal cells and a subset of astrocytes located in white matter tracts, while MT-III synthesis is limited to neurons within gray matter. Compared to wild-type littermates, transgenic mice carrying the G93A SOD1 mutation demonstrate markedly increased expression of MT-I and MT-II within astrocytes in both white and gray matter as weakness develops. MT-III synthesis in neurons is also greatly upregulated as G93A SOD1 animals age, with glial cell expression of MT-III evident by later stages of the disease. Changes in MT expression occur before the onset of motor deficits or significant motor neuron pathology in G93A SOD1 mice and remarkably extend beyond ventral horn populations of neurons and glia. These results are consistent with the hypothesis that metallothioneins may serve an early and important protective function in FALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Disease Models, Animal; Gene Expression; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; In Situ Hybridization; Inclusion Bodies; Metallothionein; Mice; Mice, Transgenic; Motor Neurons; Neurofilament Proteins; RNA, Messenger; Spinal Cord; Superoxide Dismutase; Superoxide Dismutase-1; Ubiquitins; Zinc

Cadmium (Cd) is an environmental pollutant and is toxic to a number of organs. Chronic exposure to Cd causes loss of bone mass and increased incidence of bone fractures, as seen in Itai-itai patients and laboratory animals. Metallothionein (MT), a low-molecular weight, cysteine-rich, metal-binding protein, has been shown to play an important role in the detoxication of Cd. Thus, this study was designed to test the hypothesis that MT protects against Cd-induced bone injury. Wild-type and MT-I/II knockout (MT-null) mice were given repeated sc injections of CdCl(2) over a wide range of doses for 10 weeks, and Cd-induced bone injury was examined. Cd produced dose- and time-dependent increases in bone Cd content. However, the concentration of Cd in bone was much lower than that found in the liver and kidney (11 vs 400 and 120 microg/g, respectively) of the same mice. There was no difference in bone Cd content between wild-type and MT-null mice. Repeated Cd injections produced a dose-dependent loss of bone mass (up to 25%), as shown by analysis of the femur, tibia, and lumbar vertebrae. The loss of bone mass was more marked in MT-null mice than in wild-type mice, as shown by dry bone weight, defatted bone weight, bone ash weight, and total calcium content. X-ray photography showed decreasing bone density along the entire bone length with increasing dose and time of Cd exposure. The decrease in bone density was more marked in MT-null mice than in wild-type mice at the same dose and time points. Histopathology showed dilatation of haversian canals with increased osteoid seams, rounded osteocytes with expanded pericellular space, and expansion of hyperplastic bone marrow into metaphyseal cortical bone. Again, these lesions were more marked in MT-null mice. In conclusion, this study demonstrates that deficiency in MT renders animals more susceptible to Cd-induced bone mass loss and bone injury, and thus indicates that MT plays a protective role in Cd-induced toxicity in bone, as it does in other tissues.

Topics: Animals; Bone and Bones; Bone Density; Cadmium; Cadmium Chloride; Disease Models, Animal; Female; Genetic Predisposition to Disease; Homozygote; Injections, Subcutaneous; Male; Metallothionein; Mice; Mice, Knockout; Osteomalacia

Global hypoxia preconditioning provides neuroprotection against a subsequent, normally damaging challenge. While the mechanistic pathways are unknown, changes in the expression of stress-related proteins are implicated. Hypoxia preconditioning attenuates the brain edema and neuropathology associated with kainic acid-induced status epilepticus in a protein synthesis-dependent manner when a kainic acid challenge is given up to one week post-preconditioning. Kainic acid initiates a glutamate-driven status epilepticus causing a Ca2+ and oxidative stress, resulting in injury to the piriform cortex and hippocampus. Stress-related gene expression [e.g. metallothioneins (MTs), heme oxygenase-1 (HO-1)] is enhanced during seizures in vulnerable brain areas, (e.g. piriform cortex). This study explores the effects of hypoxia preconditioning on expression of MT-1, MT-2 and HO-1 before and after kainic acid-induced seizures. Analysis of MT-1, MT-2 and HO-1 expression, through Western and Northern blotting, indicates that there is a variable pattern of induction and suppression of these two genes following hypoxia preconditioning alone as well as after kainic acid-induced seizures compared to non-preconditioned animals. These findings suggest that hypoxia preconditioning induces an adaptive response that prevents kainic acid seizure-associated neuropathology even when robust seizures occur. This may involve a variety of stress-related proteins, working in concert, each with their own individual expression profiles. Induction of this type of neuroprotection pharmacologically, or through preconditioning, will provide a better understanding of the stress response in brain.

Topics: Animals; Cerebral Cortex; Disease Models, Animal; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hippocampus; Hypoxia; Kainic Acid; Male; Metallothionein; Rats; Rats, Wistar; Seizures; Time Factors

Early effects of experimental cholestasis on the homeostasis of zinc (Zn) and metallothionein (MT) were studied in rats which had undergone bile duct ligation for 0, 3, 6, 9, 12, 16, 20, and 24 h. Transient increases in hepatic Zn levels were observed at 9 h but returned to control values at 12 h. Serum Zn levels increased at 24 h. Cholestasis was confirmed by increased serum alkaline phosphatase (AP) activity. MT increased at 3 h and reached a maximum level at 12 h and remained elevated even at 24 h after the onset of experimental cholestasis. No hepatocellular damage was detected according to the results of alanine aminotransferase (ALT) activities in serum. These results shown that the increases in Zn observed in liver are related to bile stagnation rather than to a hepatocellular damage and that increased MT occurs concurrently with increased hepatic Zn. These observations suggest that the cellular levels of Zn in cholestasis is regulated by homeostatic mechanisms, of which one could be mediated by MT.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Cholestasis; Disease Models, Animal; Female; Liver; Metallothionein; Rats; Rats, Wistar; Zinc

AA amyloidosis can be induced in mice experimentally through injection of certain chemical or biological compounds. However, the usefulness of this approach is limited by its dependence on exogenous inflammatory agents that stimulate cytokines to increase the synthesis of precursor serum amyloid A (SAA) protein and the transitory nature of the pathological fibrillar deposits. We now report that transgenic mice carrying the human interleukin 6 gene under the control of the metallothionein-I promoter had markedly increased concentrations of SAA and developed amyloid in the spleen, liver, and kidneys by 3 months of age. At the time of death about 6 months later, organs obtained from these animals had extensive amyloid deposits. This disease process was apparent radiographically using small-animal computer axial tomography and magnetic resonance imaging equipment. The AA nature of the amyloid was evidenced immunohistochemically and was unequivocally established by sequence analysis of protein extracted from the fibrils. The availability of this unique in vivo experimental model of AA amyloidosis provides the means to assess the therapeutic efficacy of agents designed to reduce or prevent the fibrillar deposits found in AA and other types of amyloid-associated disease.

Topics: Amino Acid Sequence; Amyloidosis; Animals; Bone and Bones; Disease Models, Animal; Female; Humans; Interleukin-6; Kidney; Liver; Magnetic Resonance Imaging; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Molecular Sequence Data; Serum Amyloid A Protein; Spleen; Tomography, X-Ray Computed

The present work was performed to examine the effect of tetrathiomolybdate on Cu and Fe metabolism, especially redistribution of Cu and Fe in the brains of Long-Evans Cinnamon rats, with inherently abnormal Cu deposition in the liver. The drug was injected subcutaneously at 5 mg/kg of body weight twice a week for 65 days (total dose of 20 mg) into 40-day-old Long-Evans Cinnamon rats. In Long-Evans Cinnamon rats treated with tetrathiomolybdate, the hepatic Cu concentration was 60 microg/g wet weight, compared to 170 microg/g in untreated rats. In seven brain regions (cerebellum, medulla oblongata, hypothalamus, striatum, midbrain, hippocampus and cortex) of the Long-Evans Cinnamon rats treated with tetrathiomolybdate. the Cu concentration (1.5 to 2.3 microg/g) was slightly lower (1.6 to 2.7 microg/g) than in untreated rats. A significant difference between the two groups was found only in the midbrain. Brain Fe concentrations in regions other than the striatum were not changed significantly by the tetrathiomolybdate injections. The hepatic Fe concentration was about 120 microg/g in Long-Evans Cinnamon rats without tetrathiomolybdate. Tetrathiomolybdate injection further increased the concentration to about 250 microg/g. Our results indicated that subcutaneous tetrathiomolybdate injection did not have an effect that stimulated redistribution of Cu and Fe in the seven brain regions examined, although hepatic Cu was markedly decreased and the removed Cu was deposited in kidneys, spleen and testes. The increased hepatic Fe level should be taken into account when considering side effects of the compound.

Topics: Animals; Brain; Ceruloplasmin; Copper; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Ferritins; Hepatolenticular Degeneration; Injections, Subcutaneous; Iron; Kidney; Liver; Male; Metallothionein; Molybdenum; Rats; Rats, Inbred LEC

Rats of Long-Evans Cinnamon (LEC) strain were used as a hepatorenal syndrome model of fulminant Wilson's disease. Copper levels in the kidneys increased markedly from 16 to 126 microg Cu/g from 12 to 16 weeks, and remained at the same level at 16 and 19 weeks when the rats suffered from severe renal dysfunction and also at 20 weeks in some other normal rats. The above findings imply that the renal dysfunction may have been induced independently of the copper level in the kidneys. The present study suggested the following mechanism: immediately after copper-induced hepatic dysfunction, plasma copper-metallothionein (CuMT), which was released from the liver, became elevated. The elevation was closely related to the increases in alkaline phosphatase, glucose and amino acids, all in the urine. The above findings suggest that plasma CuMT, which was released from the liver into the blood upon copper-induced hepatic dysfunction, was subsequently filtered at the glomeruli due to its smaller molecular weight, and then caused dysfunction of the brush border membrane of the renal proximal tubules probably after splitting into radical copper and amino acids in acidic vesicles close to the membrane. The critical concentration of plasma CuMT required to induce renal dysfunction was estimated as 1 microg Cu/l.

Topics: Animals; Copper; Disease Models, Animal; Hepatolenticular Degeneration; Hepatorenal Syndrome; Jaundice; Kidney; Liver; Male; Metallothionein; Rats; Rats, Long-Evans

Zinc may have an antioxidant effect mediated by induction of metallothionein. Based on the assumption that metallothionein can scavenge oxygen free radicals, we examined whether zinc administration prior to renal ischemia would improve renal dysfunction caused by ischemia-reperfusion injury in rats. Wistar rats weighing 265 g were treated with an intraperitoneal injection of 20 mg/kg zinc 24 h prior to the renal ischemia-reperfusion procedure, which was achieved by a 30-min clamping of the bilateral renal vessels and subsequent 90-min reperfusion. Thirty-minute renal clearance tests were performed before and after renal ischemia in zinc- (n = 11) and saline-treated (n = 8) rats. Thiobarbituric acid reactive substance, conjugated diene, and metallothionein levels in the renal tissues were also determined. Sham-operated rats (n = 5 in each treatment) served as control for the ischemia-reperfusion rats. Ischemia-reperfusion resulted in significantly lower glomerular filtration rate values and marked increases in tissue concentrations of thiobarbituric acid reactive substance and conjugated diene compared with sham-operation. Zinc administration improved the reduced glomerular filtration rate values seen after the ischemia-reperfusion procedure, but not to the extent of pre-ischemic levels. Zinc pretreatment significantly reduced the increased levels of thiobarbituric acid reactive substance and conjugated diene during ischemia-reperfusion and increased metallothionein levels compared with saline injection. These findings suggest that zinc has an antioxidant effect mediated through the induction of metallothionein, but appears only to have a minor protective effect on renal function induced by renal ischemia-reperfusion injury.

Topics: Acute Kidney Injury; Animals; Antioxidants; Disease Models, Animal; Glomerular Filtration Rate; Kidney; Lipid Peroxidation; Male; Metallothionein; Rats; Rats, Wistar; Reactive Oxygen Species; Renal Circulation; Reperfusion Injury; Thiobarbiturates; Treatment Outcome; Zinc

In the study described here we have revealed an abnormal accumulation of porphyrin derivatives in the kidneys of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. In addition, we have confirmed that the derivatives emitted red-orange light in renal sections under UV excitation. This renal red-orange emission has previously been identified as luminescence from cuprous metallothioneins [Cu(I)-MTs], which also accumulate in both the kidneys and liver of LEC rats. In this study, we measured the emission spectra of the luminescence in the kidneys using microspectrophotometry. The spectra of the renal red-orange emission resembled those of porphyrin derivatives rather than those of Cu(I)-MTs. We then extracted these derivatives from the kidneys. An abundance of porphyrin derivatives was established. A significant increase in the levels of the derivatives in the liver and urine of the LEC rats was also confirmed. These results provide evidence of a heme-metabolism abnormality in LEC rats.

Topics: Animals; Copper; Disease Models, Animal; Hepatolenticular Degeneration; Kidney; Male; Metallothionein; Microscopy, Fluorescence; Microspectrophotometry; Porphyrins; Rats; Rats, Mutant Strains

Topics: Animals; Copper; Disease Models, Animal; Hepatolenticular Degeneration; Humans; Metallothionein; Rats; Rats, Mutant Strains

The uptake of tetrathiomolybdate (TTM) by the liver and the removal of copper (Cu) accumulating in the liver in a form bound to metallothionein (MT) by TTM were studied in Long-Evans cinnamon (LEC) rats, an animal model of Wilson disease, in order to develop better treatments for the disease and Cu toxicity. Although molybdenum (Mo) was incorporated in a dose-dependent manner into the livers of both LEC and Long-Evans agouti (LEA) rats, the original strain of LEC rats used as a reference animal, the uptake into the liver of LEC rats was 13 times higher than that in LEA rats. The concentration of Mo in the soluble fraction plateaued and it was distributed more in the insoluble fraction with a higher dose in LEC rats. The concentration of Cu in the whole livers of LEC rats was decreased by TTM in a dose-dependent manner only at lower doses. However, the concentration of Cu in the soluble fraction continued to decrease with the dose of TTM. The results can be explained in terms of complex formation. Namely, TTM forms a complex with Cu, tentatively referred to a Cu/TTM complex, that can be effluxed into the bloodstream, and then binds selectively to albumin when the dose of TTM is low. On the other hand, TTM forms an insoluble complex, named as a Cu/TTM polymer that is precipitated in the liver when the dose is high. The results further indicate that TTM taken up by a cell is immobilized in the cell through the dose-dependent formation of a complex containing Cu, Mo and sulfur (S), which causes further uptake of TTM. TTM injected into rats or incubated in vitro with serum does not remove Cu from ceruloplasmin. TTM is, thus, suggested to target a cell accumulating excess Cu as Cu-MT, and to remove Cu selectively without interacting with Cu in Cu-enzymes. The results indicate that TTM is taken up by the liver depending on the amount of Cu accumulating in the form of MT, and then Cu is effluxed together with Mo in the form of Cu/TTM complex into the bloodstream.

Topics: Animals; Biological Transport, Active; Ceruloplasmin; Copper; Disease Models, Animal; Female; Hepatolenticular Degeneration; Liver; Metallothionein; Molybdenum; Protein Binding; Rats; Rats, Mutant Strains

We report here on a novel metallothionein-I (MT)/ret transgenic mouse line in which skin melanosis, benign melanocytic tumor and malignant melanoma metastasizing to distant organs develop stepwise. The process of tumor development and its malignant transformation in this line may resemble that of the human giant congenital melanocytic nevus that is present at birth and that frequently gives rise to malignant melanoma during aging. We observed an increase in the expression level and activity of the ret transgene during the disease progression. That increase in transgene expression accompanied an activation of mitogen-activated protein kinases (MAPKs) and c-Jun as well as matrix metalloproteinases. These results suggest that progressive dysregulation of the expression level of the ret transgene might play a crucial role in the malignant transformation of melanocytic tumors developed in the MT/ret transgenic mouse line.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Disease Models, Animal; Female; Humans; Male; Melanoma, Experimental; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase 1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-jun; Skin Neoplasms

It was found that intoxication of rats with cadmium results in a significant increase of metallothioneins content in various organs, especially in kidneys and liver and in lower degree in spleen, muscle, brain and heart.

Topics: Animals; Brain; Cadmium; Culture Techniques; Disease Models, Animal; Heart; Kidney; Liver; Lung; Metallothionein; Myocardium; Rats; Rats, Wistar

Exposure of humans to toxic metals and metalloids is a major environmental problem. Many metals, such as cadmium, can be hepatotoxic. However, the mechanisms by which metals cause acute hepatic injury are in many cases unknown. Previous reports suggest a major role for inflammation in acute cadmium induced hepatotoxicity. In initial experiments we found that a non-hepatotoxic dose of cadmium chloride (CdCl2; 2.0 mg/kg, i.v.) markedly increased the clearance rate of colloidal carbon from the blood, which is indicative of enhanced phagocytic activity by Kupffer cells (resident hepatic macrophages). Thus. the objective these studies was to determine the involvement of Kupffer cells in cadmium induced liver injury by inhibiting their function with gadolinium chloride (GdCl3). Male Sprague-Dawley rats were administered GdCl3 (10 mg/kg, i.v.) followed 24 h later by a single dose of CdCl2 (3.0 and 4.0 mg/kg, i.v.). Twenty four hours after CdCl2 administration animals were killed and the degree of liver toxicity was assessed using plasma alanine aminotransferase (ALT), as well as light microscopy. Cadmium chloride administration produced multifocal hepatocellular necrosis and increased plasma ALT activity. Pretreatment with GdCl3 significantly reduced both the morphological changes and hepatic ALT release caused by CdCl2. However, the protection was specific to the liver, and did not alter CdCl2 induced testicular injury, as determined by histopathological damage. In many cases, the inducible cadmium-binding protein, metallothionein (MT) is often an essential aspect of the acquisition of cadmium tolerance in the liver. Although cadmium caused a dramatic induction of hepatic MT (32-fold), GdCl3 caused only a minor increase (2-fold). Combined CdCl2 and GdCl3 treatment did not induce levels to an extent greater than CdCl2 alone. As expected, GdCl3 also caused a slight increase in the amount of cadmium associated with the liver. In cultured hepatocytes isolated from GdCl3 pretreated rats, CdCl2 induced cytotoxicity was not significantly altered compared to control hepatocytes, indicating that the mechanism of tolerance required the presence of other cell types. Thus, GdCl3 attenuation of CdCl2 induced hepatotoxicity does not appear to be caused by increased tissue MT content or a decreased susceptibility of hepatocytes to cadmium. From these data, we concluded that tolerance to cadmium induced hepatotoxicity involves the inhibition of Kupffer cell function which resu

Topics: Alanine Transaminase; Animals; Cadmium Chloride; Carcinogens; Cells, Cultured; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Dose-Response Relationship, Drug; Gadolinium; Gout Suppressants; Injections, Intravenous; Kupffer Cells; Lethal Dose 50; Liver; Liver Diseases; Male; Metallothionein; Necrosis; Phagocytosis; Rats; Rats, Sprague-Dawley; Testis; Tissue Distribution

Menkes disease is an X-linked disorder of copper metabolism. Excess amounts of copper in the kidney of Macular mice, a model for this disease, were found as copper-metallothionein (Cu-MT) from kidney of the mice. Histochemical studies of Cu-MT based on its autofluorescent emission properties showed that the protein was predominant in the proximal convoluted tubule (PCT) cells of the cortex. PCT cells are known to be the primary site of the nephrotoxicity caused by heavy metals. MT mRNA was also observed in the cortex, indicating that the protein was biosynthesized in this region. On the basis of these results, we suggest that biosynthesis and degradation of Cu-MT occur repeatedly in the PCT cells of the cortex. We also compared the histochemical localization of Cu-MT in Macular mice and Long-Evans cinnamon rats, a model for Wilson's disease. The significance of this comparison is discussed.

Topics: Acid Phosphatase; Animals; Carrier Proteins; Copper; Disease Models, Animal; Histocytochemistry; Injections, Subcutaneous; Kidney; Kidney Cortex; Kidney Tubules, Proximal; Lysosomes; Male; Menkes Kinky Hair Syndrome; Metallothionein; Mice; Mice, Inbred C3H; Mice, Inbred Strains; Rats; RNA, Messenger

Repeated exposures to high doses of chemotherapy are often required to eradicate solid tumors. The success of such high-dose therapy is often limited by the myelosuppressive and toxic effects of these drugs on bone marrow cells and by the intrinsic resistance of the cancer cells to chemotherapy. To test ways of using genetic modification of somatic cells to circumvent both of these problems, we first genetically modified normal bone marrow cells with multidrug resistance-1 (MDR-1) cDNA retroviral vectors to render these cells more resistant to p-glycoprotein-transported agents. Experiments conducted previously in a mouse model in our laboratory (E. G. Hanania et al., Cancer Gene Ther., 2: 251-261, 1995; E. G. Hanania and A. B. Deisseroth, Cancer Gene Ther., 1: 21-25, 1994), which involve transplantation of mouse marrow cells modified with the human MDR-1 cDNA, showed that the majority of the marrow cells of these animals were resistant to repetitive administration of myelotoxic doses of Taxol, a MDR-1-transported drug. Next, to test the effects of genetically modifying marrow cells to make them resistant to chemotherapy, and genetically modifying tumor cells to make them more sensitive to chemotherapy, a mouse breast cancer cell line was transfected with a plasmid expression vector that contained a wild-type p53 chemosensitization transcription unit. Others have shown that restoration of the p53 gene can lead to decreased proliferation, reduced tumorigenicity, and increased sensitivity to chemotherapy-induced apoptosis. In this animal model, the simultaneous use of both chemoprotection and chemosensitization vectors, which provided protection of the normal cells to the chemotherapy and at the same time sensitized the tumor cells to the toxic effects of the chemotherapy, resulted in levels of in vivo tumor reduction that were not possible when either genetic chemoprotection of marrow cells or chemosensitization of tumor cells was used alone. These data should be of interest to those who are studying ways of using genetic modification to improve the outcome of established chemotherapy treatment programs for solid tumors.

Topics: Animals; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bone Marrow Cells; Chemoprevention; Disease Models, Animal; DNA, Complementary; Drug Resistance, Neoplasm; Female; Genetic Therapy; Humans; Mammary Neoplasms, Animal; Metallothionein; Mice; Mice, Inbred BALB C; Paclitaxel; Promoter Regions, Genetic; Retroviridae; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Human Menkes disease and the murine Mottled phenotype are X-linked diseases that result from copper deficiency due to mutations in a copper-effluxing ATPase, designated ATP7A. Male mice with the Mottled-Brindled allele (Mo-brJ) accumulate copper in the intestine, fail to export copper to peripheral organs and die a few weeks after birth. Much of the intestinal copper is bound by metallothionein (MT). To determine the function of MT in the presence of Atp7a deficiency, we crossed Mo-brJ females with males that bear a targeted disruption of the Mt1 and Mt2 genes (Mt-/-). On an Mt -/- background, most Mo-brJ males as well as heterozygous Mo-brJ females die before embryonic day 11. The lethality in Mo-brJ females can be explained by preferential inactivation of the paternal X chromosome in extraembryonic tissues and resultant copper toxicity in the absence of MT. In support of this hypothesis, cell lines derived from Mt -/-, Mo-brJ embryos are very sensitive to copper toxicity.

Topics: Adenosine Triphosphatases; Animals; Base Sequence; Carrier Proteins; Cation Transport Proteins; Cell Survival; Cells, Cultured; Copper; Copper-Transporting ATPases; Crosses, Genetic; Culture Media; Disease Models, Animal; Embryo, Mammalian; Female; Fetal Death; Intestinal Mucosa; Intestines; Liver; Male; Menkes Kinky Hair Syndrome; Metallothionein; Mice; Mice, Inbred Strains; Molecular Sequence Data; Recombinant Fusion Proteins; Tissue Distribution; X Chromosome

1. Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumours, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. 2. Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human transforming growth factor (TGF-alpha) cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumourigenesis. 3. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumours were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. 4. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. 5. We have now extended these studied and examined the interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumour promotion by phenobarbitone was completely inhibited in the c-myc/HGF double transgenic mice whereas phenobarbitone was an effective tumour promoter in the c-myc single transgenic mice. 6. The results indicate that HGF may function as a tumour suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c-myc with HGF or TGF-alpha results in profoundly different outcomes of the neoplastic process in the liver.

Topics: Albumins; Animals; Disease Models, Animal; Female; Genes, myc; Hepatocyte Growth Factor; Liver Neoplasms, Experimental; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Phenobarbital; Transforming Growth Factor alpha

The manipulation of stress gene expression by heavy metals provides protection against the lethal effects of endotoxemia in murine models of septic shock. Recent in vitro studies with alveolar macrophages or monocytes show that induction of the stress response in these cells is followed by a decreased liberation of major cytokines [tumor necrosis factor-alpha (TNF alpha) and interleukin-1 (IL-1)] after endotoxin challenge. These findings suggest that the increased resistance to endotoxin in vivo after stress protein induction could be explained by an altered pattern of inflammatory mediator release. Therefore, we measured the time course of thromboxane-B2 (TxB2), 6-keto-PGF1 alpha, platelet activating factor (PAF), TNF alpha, interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) formation with and without induction of the stress response in an established porcine model of recurrent endotoxemia (Klosterhalfen et al., Biochem Pharmacol 43: 2103-2109, 1992). Induction of the stress response was done by a pretreatment with Zn2+ (25 mg/kg zinc-bis-(DL-hydrogenasparate = 5 mg/kg Zn2+). Pretreatment with Zn2+ prior to lipopolysaccharide (LPS) infusion induced an increased heat shock protein 70 and metallothionein expression in the lungs, liver, and kidneys and increased plasma levels of TNF alpha, IL-1 beta, IL-6, and TxB2 as opposed to untreated controls. After LPS infusion, however, pretreated animals showed significantly decreased peak plasma levels of all mediators as opposed to the untreated group. The time course of mediator release was identical with the decreasing and increasing three peak profiles described previously. Hemodynamic data presented significantly decreased peak pulmonary artery pressures and significantly altered hypodynamic/hyperdynamic cardiac output levels in the pretreated group. In conclusion, the data show that the induction of stress proteins by Zn2+ could be a practicable strategy to prevent sepsis.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Aspartic Acid; Blood Pressure; Cardiac Output; Disease Models, Animal; Endotoxemia; Gene Expression; HSP70 Heat-Shock Proteins; Inflammation Mediators; Interleukin-1; Interleukin-6; Kidney; Lipopolysaccharides; Metallothionein; Platelet Activating Factor; Pulmonary Artery; Recurrence; Swine; Thromboxane B2; Tumor Necrosis Factor-alpha; Zinc

Long-Evans Cinnamon (LEC) rats, characterized by a gross accumulation of hepatic Cu and the spontaneous onset of hepatitis, have been established to be an animal model for Wilson disease. They were used to estimate the relationships among copper (Cu), metallothionein (MT), and reduced glutathione (GSH) in biliary excretion in this study. Even though a huge amount of MT existed in the LEC rat liver (5016 micrograms/g liver) compared to that (63 micrograms/g liver) of controls (Fischer rats), the biliary excretion of MT (65 ng/ml bile) did not reflect the accumulated MT level in LEC rats. It seems likely that MT does not excrete intrinsically into the bile. Biliary excretion of Cu (0.17 microgram/ml) in LEC rats was significantly lower than that (0.57 microgram/ml) in Fischer rats. The difference in biliary excretion of GSH between the two groups was significant but slight. The reduced excretion of GSH into bile in LEC rats may be due to increased hepatic gamma-glutamyltransferase but not to hepatic GSH levels. There were no differences in biliary potassium and inorganic phosphorous between the two groups. On the other hand, excretion of lysosomal enzymes such as beta-N-acetylglucosaminidase into bile was much lower in LEC rats (15.6 units/liter) than in controls (42.5 units/liter). The defective biliary excretion of Cu may be due to impaired lysosomal exocytosis, rather than canalicular membrane impairment. The LEC rat is very useful for research into the dynamics of metal excretion via the hepatobiliary system.

Topics: Acetylglucosaminidase; Aminopeptidases; Animals; Bile; Copper; Disease Models, Animal; Female; gamma-Glutamyltransferase; Glutathione; Hepatolenticular Degeneration; Metallothionein; Rats; Rats, Inbred F344; Rats, Mutant Strains

The bcr/abl chimeric oncoprotein is considered to be implicated in the pathogenesis of Philadelphia chromosome-positive human leukemias. To investigate its biological function and the role in leukemogenesis in vivo, we generated transgenic mice expressing p210bcr/abl driven by the metallothionein promoter. Two of six founder mice and the transgenic progeny of one leukemic founder mouse developed leukemias several months after birth. Phenotypically, each leukemic mouse showed a thymic enlargement, a marked splenomegaly, and/or lymphnode swellings. Pathological examination revealed that leukemic cells were infiltrated in all tissues examined, especially in thymus, spleen, liver, and lymphnode. Expression of the p210bcr/abl transgene product and increased phosphorylation of cellular proteins in leukemic tissues were detected by the Western blot analysis. In addition, the expressed p210bcr/abl protein was demonstrated to possess an enhanced kinase activity by the in vitro immunecomplex kinase assay. These results indicate that hematopoietic precursor cells expressing the p210bcr/abl transgene product acquired a proliferative advantage and eventually developed leukemias in transgenic mice. The p210bcr/abl transgenic mice are considered to be an excellent animal model to investigate p210bcr/abl function and its role in leukemogenesis in vivo.

Topics: Animals; Disease Models, Animal; Female; Fusion Proteins, bcr-abl; Humans; Leukemia, Experimental; Male; Metallothionein; Mice; Mice, Transgenic; Pedigree; Philadelphia Chromosome; Phosphotransferases

Hepatic lipid peroxidation, metallothioneins, collagen and proline hydroxylase activity were investigated in 16 ethanol-fed rats and in 16 control animals. The rats were further divided into three groups to receive either a standard diet, a zinc-deficient diet or a zinc-supplemented diet. The animals were sacrificed at week 12 of the experiment for histological and biochemical assessments. Hepatic tissue examination indicated that oral zinc supplementation was associated with a decrease in lipid peroxidation, collagen deposition and proline hydroxylase activity together with an increase in metallothionein concentration in alcoholic rats. There were no significant differences in lipid peroxidation in the control group in relation to the diet. Zinc supplementation was associated with increased concentrations of hepatic metallothioneins together with decreased concentrations of proline-hydroxylase and collagen but to a lesser degree than in alcoholic animals. These results indicate that zinc is an efficient hepato-protective agent against lipid peroxidation in alcoholic rats and its effect may be, in part, mediated by the activation of metallothionein synthesis. Also, lipid peroxidation may be related to changes in hepatic collagen synthesis.

Topics: Alcoholism; Animals; Collagen; Disease Models, Animal; Ethanol; Food, Fortified; Lipid Peroxidation; Liver; Male; Metallothionein; Procollagen-Proline Dioxygenase; Rats; Rats, Wistar; Zinc

Hyperzincuria and low Zn absorption in diabetic animals and humans have prompted speculation that diabetics are more susceptible to Zn deficiency. There is little information, however, describing the effects of diabetes on the biochemical mechanisms of intestinal Zn transport. We evaluated Zn absorption in streptozotocin-induced diabetic rats based on a model of Zn transport in which cysteine-rich intestinal protein serves as an intracellular carrier that is inhibited by metallothionein (MT). Apparent absorption and retention of Zn and Cu in rats fed a purified diet were measured in a balance study 15-17 d after induction of diabetes. The rate of 65Zn absorption from isolated intestinal segments, molecular distribution of 65Zn in mucosal cytosol, and tissue MT levels were measured on d 20-22. Food consumption, and thus Zn and Cu intake, by diabetic rats was twice that of controls. Although fractional absorption (percent) of Zn and Cu was lower in the diabetic rats, net absorption (micrograms/100 g body weight/d) was higher. The higher net absorption in the diabetic group was offset, however, by higher urinary excretion, so that Zn and Cu retention was similar in both groups of animals. Low fractional absorption is attributable to the down-regulation of intestinal Zn transport, as indicated by the lower rate of 65Zn absorption from isolated intestinal segments in the diabetic rats. Down-regulation of intestinal transport is in turn attributable to higher concentrations of intestinal MT, which resulted in more 65Zn in the mucosal cytosol bound to MT, an inhibitor of Zn transport, and less to cysteine-rich intestinal protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blood Glucose; Carrier Proteins; Copper; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Intestinal Absorption; LIM Domain Proteins; Male; Metallothionein; Rats; Rats, Sprague-Dawley; Streptozocin; Zinc

DNA constructs encoding BCR/ABL P210 have been introduced into the mouse germ line using microinjection of one-cell fertilized eggs. Kinetics of BCR/ABL P210 expression in transgenic mice were very similar to those of BCR/ABL P190 constructs in transgenic mice. mRNA transcripts were detectable early in embryonic development and also in hematopoietic tissue of adult animals. Expression of BCR/ABL in peripheral blood preceded development of overt disease. P210 founder and progeny transgenic animals, when becoming ill, developed leukemia of B, T-lymphoid, or myeloid origin after a relatively long latency period. In contrast, P190-transgenic mice exclusively developed leukemia of B-cell origin, with a relatively short period of latency. The observed dissimilarities are most likely due to intrinsically different properties of the P190 and P210 oncoproteins and may also involve sequences that control transgene expression. The delayed progression of BCR/ABL P210-associated disease in the transgenic mice is consistent with the apparent indolence of human chronic myeloid leukemia during the chronic phase. We conclude that, in transgenic models, comparable expression of BCR/ABL P210 and BCR/ABL P190 results in clinically distinct conditions.

Topics: Animals; B-Lymphocytes; Base Sequence; Blast Crisis; Disease Models, Animal; Embryonal Carcinoma Stem Cells; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Humans; Leukemia, Experimental; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Metallothionein; Mice; Mice, Transgenic; Molecular Sequence Data; Molecular Weight; Neoplastic Stem Cells; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; T-Lymphocytes; Transgenes

Metallothionein is a low molecular mass protein inducible mainly by heavy metals, having high affinity for binding cadmium, zinc and copper. In the present study we investigated the expression of metallothionein in regenerating liver, at different time intervals, in cadmium pretreated partially hepatectomized rats. Liver metallothionein is highly expressed during regeneration induced by partial hepatectomy in rats, providing zinc within the rapidly growing tissue. Cadmium pretreatment caused inhibition of the first peak of liver regeneration, while metallothionein expression was markedly more prominent in the liver residues of cadmium-pretreated rats. These results demonstrate that although metallothionein able to bind temporarily metal ions as zinc and cadmium has been highly expressed, the liver regenerative process was inhibited possibly due to the effects of cadmium on other pivotal events necessary to the DNA replication.

Topics: Animals; Cadmium; Cadmium Chloride; Cadmium Poisoning; Chlorides; Disease Models, Animal; DNA; Hepatectomy; Immunohistochemistry; Linear Models; Liver; Liver Regeneration; Male; Metallothionein; Protein Binding; Rats; Rats, Wistar; Spectrophotometry, Atomic; Thymidine Kinase; Zinc

Cadmium metallothionein (CdMT) was injected subcutaneously into obese hyperglycaemic Umeå ob/ob mice or their lean litter mates (normal mice) at doses of 0, 0.1 and 0.4 mg Cd/kg. Proteinuria and calciuria were induced in both types of mice, but in the ob/ob mice this condition developed at a lower dose of CdMT (0.1 mg Cd/kg) than in the normal mice (0.4 mg Cd/kg). These results show, therefore, that Umeå ob/ob mice are particularly susceptible to CdMT-induced nephrotoxicity. The mechanism underlying this phenomenon needs to be further investigated. After the administration of CdMT, a dose-related increase in glycosuria was observed in both types of mice, in spite of decreased levels of serum insulin and glucose. It is suggested that such glycosuria induced by CdMT could be one of the signs of cadmium nephrotoxicity. The results of the present study thus indicate that metabolic changes like those in diabetes may increase susceptibility to cadmium-induced renal tubular damage.

Topics: Animals; Blood Glucose; Calcium; Diabetes Mellitus, Type 2; Disease Models, Animal; Dose-Response Relationship, Drug; Injections, Subcutaneous; Insulin; Kidney Diseases; Kidney Tubules; Liver; Male; Metallothionein; Mice; Mice, Obese; Obesity; Pancreas; Proteinuria; Tissue Distribution

There is sufficient evidence for pulmonary carcinogenicity of inhaled cadmium (Cd) compounds in rats whereas no such evidence was found in mice and hamsters, indicating significant species differences in the pulmonary response to inhaled Cd. We hypothesized that expression of metallothionein (MT) protein in the lung after inhalation of Cd differs between species thereby providing different degrees of sequestration of Cd and protection from its effects. Rats and mice were exposed to 100 micrograms CdCl2 aerosols/m3 for 4 weeks, and the presence of MT was determined in lung and free lung cell homogenates as well as by immunocytochemistry in lung sections up to 28 days post-exposure. Cd exposure significantly increased MT in homogenates of total lung in both species; however, no significant increase of MT in rat lung tissue after removal of free lung cells by lavage was found whereas MT was still significantly increased in lavaged mouse lung tissue throughout the post-exposure time. Histochemical analysis of lung sections revealed that mainly the epithelial cells of the bronchi, bronchioli and alveoli of Cd-exposed mice expressed MT. Baseline MT levels were also greater in the lungs of mice compared to rats. The retained Cd dose per g lung was about 2-fold greater in mice. The greater MT induction upon exposure to the same inhaled Cd concentration and the greater baseline MT levels may offer an explanation for the resistance of mice towards the pulmonary carcinogenic effect of inhaled Cd.

Topics: Administration, Inhalation; Animals; Bromodeoxyuridine; Cadmium; Disease Models, Animal; Humans; Lung; Lung Neoplasms; Male; Metallothionein; Mice; Mice, Inbred BALB C; Rats; Rats, Inbred F344; Species Specificity; Time Factors

Topics: Age Factors; Amyloid Neuropathies; Animals; Disease Models, Animal; Gene Expression Regulation; Genes, Synthetic; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Transgenic; Organ Specificity; Prealbumin; Promoter Regions, Genetic; Recombinant Fusion Proteins

The effect of endotoxin on serum and lung and liver lipid peroxidation, as measured by thiobarbituric acid reactants (TBARs), as well as on lung and liver metallothionein (MT) has been studied in the rat. Endotoxin consistently increased serum and liver TBARs in a time-response manner. The increase in the serum preceded that in the liver, with peaks 3-6 h and 24 h after endotoxin administration, respectively. In contrast, lung TBARs levels did not increase regardless of the experimental approaches studied, suggesting that the rat is not a good model for the adult respiratory distress syndrome. Endotoxin increased both lung and liver MT levels in a time-response manner, although to a lesser degree in the former than in the latter tissue, indicating that this protein may have a significant role in the response of the organism to a septic insult.

Topics: Animals; Disease Models, Animal; Drug Administration Routes; Endotoxins; Lipid Peroxidation; Liver; Lung; Male; Metallothionein; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Thiobarbituric Acid Reactive Substances

The effect of free fatty acids on zinc absorption was studied in a rat model of chronic osmotic diarrhea induced with magnesium citrate and phenolphthalein. In vivo rates of zinc removal from the lumen and analysis of tissue for zinc uptake and metallothionein alterations were monitored. One mmol/L stearate enhanced zinc absorption in rats with or without diarrhea, from 207 +/- 22 and 353 +/- 13 pmol/min x cm to 676 +/- 34 and 610 +/- 26 pmol/min x cm, respectively. Palmitate was only effective in normal rats. Zinc absorption inversely correlated with mucosal zinc content in the perfused intestinal segments, in both type of rats. Hepatic metallothionein was enhanced by zinc and even more by oleate plus zinc in both groups; kidney metallothionein in animals with diarrhea was normalized by either oleate or zinc. The data support previous reports on the effect of long-chain fatty acids on the enhancement of zinc absorption: saturation and a longer chain appear to be positive factors. A membrane modification role of long-chain fatty acids could have nutritional implications in the formulation of special diets.

Topics: Animals; Biological Transport; Diarrhea; Disease Models, Animal; Fatty Acids; Intestinal Absorption; Kidney; Liver; Male; Metallothionein; Osmosis; Perfusion; Rats; Rats, Wistar; Sodium; Zinc

These studies were designed to determine if macular mutant mouse, which is a proposed animal model of Menkes' kinky-hair disease, is sensitive to the acute toxic effect of Cu as compared to normal and heterozygote mice. Single sc injection of Cu were administered to 6- to 8-day-old mice, and mortalities were recorded for 30 days. The copper treatment at high doses (12 to 25 mg Cu/kg) was very toxic to mutant mice as compared to normal mice, and almost all mutant mice died within 10 days after injection. The effect of Cu toxicity on heterozygote mice was intermediate. The LD50 values 3 days after injection of Cu were 29.5 mg Cu/kg for normal mice, 23.5 mg Cu/kg for heterozygote mice, and 15.5 mg Cu/kg for mutant mice. In Cu-injected mutant mice (11 and 18 mg Cu/kg), significant elevations in serum aspartate aminotransferase and lactate dehydrogenase activity occurred as compared to Cu-injected normal and heterozygote mice. However, no significant elevations in serum creatinine and urea nitrogen contents in Cu-injected mutant were observed as compared to normal and heterozygote mouse. No significant differences in hepatic metallothionein(MT) and MT-1 mRNA, and serum ceruloplasmin oxidase activity levels were observed between Cu-injected normal and mutant mouse. These results indicated that macular mutant mice was sensitive to the acute toxic or hepatotoxic effects of Cu as compared to normal and heterozygote mice.

Topics: Animals; Aspartate Aminotransferases; Blood Urea Nitrogen; Ceruloplasmin; Copper; Disease Models, Animal; Heterozygote; L-Lactate Dehydrogenase; Lethal Dose 50; Male; Menkes Kinky Hair Syndrome; Metallothionein; Mice; Mice, Mutant Strains; Tissue Distribution

Levels of metallothionein-1 (MT-1) messenger RNA (mRNA) in various tissues from normal and macular mutant mice at different stages of development (17 days of gestation, 1-, 3-, 7- and 14-day-old) were determined by Northern blot analysis. Renal MT-1 mRNA levels in mutant mice were slightly elevated at 3 stages compared to normal mice, with the exception of mutant fetus and 3-day-old mutant mice. Intestinal MT-1 mRNA levels in mutant mice were elevated at 3 stages compared to normal mice with the exception of mutant fetus and 3-day-old mutant mice. Hepatic MT-1 mRNA levels in mutant fetus and 1-day-old mutant mice were approximately the same compared to normal mice. In 3- and 7-day-old mutant mice, hepatic MT-1 mRNA levels were depressed and in 14-day-old mutant mice, they were increased compared to normal mice. Brain MT-1 mRNA could not be detected in normal and mutant mice at the 4 stages with the exception of 14-day-old mice. MT-1 mRNA in 14-day-old mice was detected and the level of that in mutant mice was slightly elevated compared to normal mice. After injection of Cu, MT-1 mRNA levels in kidney, liver and intestine were determined. The injection of Cu increased the level of MT-1 mRNA in the tissues of normal and mutant mice compared to control (saline-injected) mice. Significant differences in MT-1 mRNA levels in the tissues of both Cu-injected mice were not observed.

Topics: Animals; Brain Chemistry; Copper; Disease Models, Animal; Intestines; Kidney; Liver; Menkes Kinky Hair Syndrome; Metallothionein; Mice; Mice, Mutant Strains; RNA, Messenger; Zinc

Fibroblasts from the brindled mouse model of Menkes disease are known to accumulate excess copper. Most of the copper in the cytosol of these fibroblasts is bound to metallothionein (MT), which is elevated in Menkes or brindled mouse fibroblasts. Copper accumulation by normal fibroblasts containing excess MT was examined to determine if the excess copper accumulation phenotype was secondary to excess MT or associated with the primary defect in fibroblasts from the brindled mice. MT was induced in normal fibroblasts by copper, zinc or dexamethasone to levels comparable with those in brindled mice fibroblasts, as determined by radioimmunoassays. Normal fibroblasts containing excess MT accumulate copper normally, i.e. they do not exhibit the excess copper accumulation phenotype. Consistent with this result, copper efflux from normal fibroblasts containing excess MT was also normal. The data suggest that one function of the protein associated with the primary defect is to help determine how much copper is taken up and retained by fibroblasts and other cell types exhibiting the excess copper phenotype in Menkes disease. The capacity of this protein is apparently exceeded in normal fibroblasts if serum or albumin is not present extracellularly to limit total copper uptake. Consistent with a defect in an intracellular protein, the kinetics of copper transport by brindled mice fibroblasts were found to be normal.

Topics: Animals; Biological Transport; Brain Diseases, Metabolic; Cells, Cultured; Copper; Disease Models, Animal; Female; Fibroblasts; Kinetics; Male; Menkes Kinky Hair Syndrome; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Reference Values; Skin

In certain pathological and experimental conditions associated with hepatic copper overload, an important portion of the excess metal is bound to a copper-thionein and sequestered in hepatocellular lysosomes. The localization can be demonstrated visually by using histochemical and immunocytochemical stains and by extraction of the Cu-protein from particulate fractions of liver homogenates. The livers of Bedlington terriers affected by an inherited defect resulting in the accumulation of large quantities of copper display hepatocellular lysosomes which appear as dense, insoluble, copper-rich granules. Purification and solubilization of the fraction containing these granules yields a copper-thionein with little zinc, which displays homology with other known metallothioneins. Although the primary genetic defect which underlies the pathogenesis of this form of hepatic copper overload is still unknown, circumstantial evidence suggests that sequestration of the protein by lysosomes may be temporarily cytoprotective.

Topics: Aging; Animals; Copper; Disease Models, Animal; Dogs; Hepatolenticular Degeneration; Liver; Lysosomes; Metal Metabolism, Inborn Errors; Metallothionein; Microscopy, Electron

The tissue copper and metallothionein-Cu (MT-Cu) content of kidney and liver were measured in mutant (hemizygous macular male and homozygous macular female), heterozygous macular female and normal mouse. The tissue copper and MT-Cu contents in kidneys from 7-8 day mutants and heterozygotes were significantly greater than those of the normal kidney. Marked elevations in kidney copper and MT-Cu contents were also observed in the 8-9 week mutant (which achieved long-term survival with a single dose of subcutaneous copper administered at day 7) and in the heterozygote. The L-[35S]cystine incorporation experiments also revealed an abnormal synthesis of renal MT in the 8-9 week mutant and in the heterozygote. In contrast to kidney Cu levels, the tissue copper and MT-Cu contents of 7-8 day normal livers were extremely high, whereas the tissue copper and MT-Cu contents of mutant and heterozygote livers were extremely low. The tissue copper contents of livers of 8-9 week mutants and heterozygotes were slightly low compared to normal, and the MT-Cu contents of livers of the 8-9 week mouse were extremely low in all groups. In contrast to the changes in copper content, the changes in tissue zinc and MT-Zn contents in kidney and liver were slight in the 7-8 day and 8-9 week mouse.

Topics: Animals; Brain Diseases, Metabolic; Copper; Cytosol; Disease Models, Animal; Female; Genetic Carrier Screening; Kidney; Liver; Male; Menkes Kinky Hair Syndrome; Metallothionein; Mice; Mice, Mutant Strains; Spectrophotometry, Atomic; Zinc

Type I familial amyloidotic polyneuropathy (FAP) results from the systemic deposition of a plasma transthyretin (TTR) variant with a Val----Met change at position 30. In an attempt to establish a model of this disease, we generated transgenic mice producing the variant TTR. A DNA fragment containing the mouse metallothionein-I promoter fused to the structural gene coding for the human TTR variant was microinjected into fertilized mouse eggs. Among 72 mice that developed from these eggs, ten carried the fusion gene and three of these showed significant concentrations of the variant TTR in their serum. These mice may be useful in elucidating the pathogenesis of FAP and in establishing a therapy for this intractable disorder.

Topics: Animals; Disease Models, Animal; DNA; DNA Restriction Enzymes; Female; Genes; Humans; Lipidoses; Male; Metallothionein; Mice; Prealbumin; Promoter Regions, Genetic

Topics: Animals; Brain; Cadmium; Disease Models, Animal; Gene Expression Regulation; Growth Hormone; Liver; Metallothionein; Mice; Transcription, Genetic; Transformation, Genetic

Comparisons were made between morphologic and biochemical changes occurring in the lungs of rats receiving a single exposure or repeated 2-h exposures of aerosolized 0.1% cadmium chloride, simulating varied exposure conditions potentially experienced by humans. Lung lysyl oxidase activity increases in both models, although less so with repeated exposures. Pulsing lung tissue in vitro with 3H-proline indicated that rates of collagen biosynthesis were elevated preferentially over rates of noncollagen protein synthesis in both exposure models. Lung metallothionein increased nearly linearly over 21 days of repeated exposure. Histologic examination revealed scarred lesions distorting alveolar structure and bronchioles in both models. However, scarring and cell exudation into the airways and interstitium was less in the repeatedly exposed model. The results indicate the activation of a connective tissue repair reaction in both models. Increased levels of metallothionein after repeated exposure may both sequester cadmium and reduce pools of copper available for lysyl oxidase synthesis, thus limiting the fibrotic response.

Topics: Aerosols; Animals; Cadmium; Cadmium Chloride; Collagen; Disease Models, Animal; Lung; Male; Metallothionein; Protein Biosynthesis; Protein-Lysine 6-Oxidase; Rats; Rats, Inbred Strains

Topics: Amino Acids; Animals; Brain Diseases, Metabolic; Cysteine; Disease Models, Animal; Half-Life; Humans; Kidney; Liver; Menkes Kinky Hair Syndrome; Metalloproteins; Metallothionein; Mice; Mice, Inbred Strains

Topics: Anti-Inflammatory Agents; Antineoplastic Agents; Arthritis, Rheumatoid; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Humans; Metallothionein; Receptors, Drug; Structure-Activity Relationship; Tissue Distribution

The copper utilization in mutated Brindled mice is impaired. Copper accumulates in various tissues, e.g., the kidney, of the mutated mice. The renal copper binding protein is characterized as copper-thionein--metallothionein to which copper is bound. The L-[35S]cystine incorporation experiments without prior induction with copper revealed an abnormal synthesis of metallothionein in the mutated mice. Two models are proposed which link the abnormal metallothionein synthesis with an impaired copper utilization. Model 1 is an unrestrained translation of renal mRNA which codes for metallothionein. Model 2 is an impaired renal copper reabsorption resulting in a toxic intracellular copper concentration which induces metallothionein synthesis to sequester copper. The impaired copper utilization results in a fatal copper deficiency in "Menkes" Brindled mice.

Topics: Animals; Brain Diseases, Metabolic; Copper; Disease Models, Animal; Female; Genotype; Heterozygote; Humans; Male; Menkes Kinky Hair Syndrome; Metalloproteins; Metallothionein; Mice; Molecular Weight; Mutation; Phenotype; Sex Factors