metallothionein and thiazolyl-blue

Zinc (Zn) is an essential mineral that is required for various cellular functions. Its abnormal metabolism is related to certain disorders such as diabetic complications. Oxidative stress has been considered as the major causative factor for diabetic cardiomyopathy. Zn has a critical antioxidant action in protecting the heart from various oxidative stresses. Zn deficiency was found to be a risk factor for cardiac oxidative damage and supplementation with Zn provides a significant prevention of oxidative damage to the heart. Diabetes causes a significant systemic oxidative stress and also often is accompanied by Zn deficiency that increases the susceptibility of the heart to oxidative damage. Therefore, there is a strong rationale to consider the strategy of Zn supplementation to prevent or delay diabetic cardiomyopathy. This short article collects the preliminary evidence, based on our own studies and those by others, for a preventive effect of Zn supplementation on diabetes-induced injury to the heart in animals and under in vitro conditions. Possible mechanisms by which Zn supplementation prevents diabetic heart disease are discussed. They include an antioxidant action of Zn, insulin function and metallothionein induction. In the final section, the future of Zn supplementation for diabetic patients is also briefly discussed. Although Zn supplementation has not been clinically used to prevent diabetic complications, because several issues need to be addressed, the fact that Zn supplementation is being used clinically for other disorders encourages us to explore its direct clinical application for the prevention of diabetic cardiomyopathy.

Topics: Animals; Antioxidants; Biological Transport; Cardiomyopathies; Cell Line; Coloring Agents; Diabetes Complications; Diabetes Mellitus, Experimental; Dietary Supplements; Glutathione; Glutathione Peroxidase; Humans; Metallothionein; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase; Tetrazolium Salts; Thiazoles; Time Factors; Triglycerides; Zinc

Several studies have shown intracellular Zn(2+) release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn(2+) release and subsequent cell death. Cortical neurons were loaded with the Zn(2+) selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn(2+) levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn(2+). Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn(2+) induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn(2+) release appeared to peak at 1 microM spermine NONOate (123.8 +/- 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn(2+) levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn(2+) in cortical neurons. The possibility that release of intracellular Zn(2+) in response to NO could play a role in intracellular signaling is discussed.

Topics: Animals; Cells, Cultured; Hemoglobins; Metallothionein; Microscopy, Fluorescence; Neurons; Nitric Oxide; Rats; Rats, Sprague-Dawley; Signal Transduction; Spermine; Tetrazolium Salts; Thiazoles; Time Factors; Zinc

Epothilone B (EpoB) is a non-taxane microtubule-stabilizing agent with a mode of action similar to that of paclitaxel, but with the advantage of being active in paclitaxel-resistant cells. Knowledge regarding other mechanisms of EpoB action is limited. The purpose of this study was to identify gene expression profiles associated with the biological response to EpoB in an ovarian cancer cell line (SKOV3).. SKOV3 cells were maintained in McCoy's 5A media. Equal densities cells were treated with or without EpoB, and were evaluated for cell growth arrest and apoptosis. mRNA expression was evaluated by cDNA microarrays and quantitative, real time reverse transcription polymerase chain reaction (QRTPCR).. EpoB (10 nM) led to cell cycle arrest and apoptosis in SKOV3 cells. Microarray analysis, comparing EpoB-treated to untreated cells, revealed altered expression of 41 genes. There was a predominance of sequences related to the TNFalpha stress response pathway. Differential expression of selected genes was confirmed by QRTPCR.. We demonstrated that cDNA microarrays are a useful tool to rapidly screen for patterns of gene expression that characterize drug response. The microarray data suggest that the microtubule-stabilizing agent, EpoB, triggers stress-related signal transduction pathways related to TNFalpha. These pathways may contribute to mechanisms of EpoB action and potential mechanisms of resistance in ovarian cancer.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Epothilones; Female; Flow Cytometry; Fluorescent Antibody Technique; G2 Phase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Mitosis; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha; Up-Regulation

The robust induction of metallothionein-I and II (MT-I and MT-II) genes by several heavy metals such as zinc and cadmium requires the specific transcription factor metal-responsive transcription factor 1 (MTF1). Chromium (VI), a major environmental carcinogen, not only failed to activate these genes but also inhibited their induction by Zn2+ or Cd2+. The heavy metal-induced expression of another MTF1 target gene, zinc transporter 1 (ZnT-1), was also down-regulated by Cr6+. By contrast, the expression of two MTF1-independent Cd2+-inducible genes, heme oxygenase 1 (HO-1) and HSP-70, was not sensitive to Cr6+. Cr6+ did not also affect the expression of housekeeping genes such as GAPDH or beta-actin. Stable cell lines overexpressing variable levels of MTF1, the key transactivator of the MT genes, demonstrated differential resistance toward the inhibitory effect of Cr6+, indicating MTF1 as a target of chromium toxicity. The basal and inducible binding of MTF1 to metal response elements was not affected by treatment of cells with Cr6+. Transient transfection studies showed that the ability of MTF1 to transactivate the MT-I promoter was significantly compromised by Cr6+. The fusion protein consisting of a Gal-4 DNA binding domain and one or more of the three transactivation domains of MTF1, namely the acidic domain, proline-rich domain, and serine-threonine rich domain, activated the GAL-4-driven luciferase gene to different degrees, but all were sensitive to Cr6+. MTF1 null cells were prone to apoptosis after exposure to Zn2+ or Cd2+ that was augmented in presence Cr6+, whereas the onset of apoptosis was significantly delayed in cells overexpressing MTF1.

Topics: Annexin A5; Apoptosis; Blotting, Northern; Blotting, Western; Cell Line; Chromium; Coloring Agents; DNA Footprinting; DNA-Binding Proteins; Down-Regulation; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; HSP70 Heat-Shock Proteins; Humans; In Situ Nick-End Labeling; Membrane Proteins; Metallothionein; Metals, Heavy; Promoter Regions, Genetic; Protein Structure, Tertiary; Recombinant Fusion Proteins; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles; Time Factors; Transcription Factor MTF-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Zinc

The mechanism of cadmium-mediated hepatotoxicity has been the subject of numerous investigations, principally in hepatocytes. Although, some uncertainties persist, sufficient evidence has emerged to provide a reasonable account of the toxic process in parenchymal cells. However, there is no information about the effect of cadmium in other hepatic cell types, such as stellate cells (fat storing cells, Ito cells, perisinusoidal cells, parasinusoidal cells, lipocytes). Hepatic stellate cells (HSC) express a quiescent phenotype in a healthy liver and acquire an activated phenotype in liver injury. These cells play an important role in the fibrogenic process. The objective of this study was to investigate the effect of a 24 h treatment of low Cd concentrations in glutathione content, lipid peroxidation damage, cytosolic free Ca, antioxidant enzyme activities: glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase along with the capacity of this heavy metal to induce metallothionein II and alpha(1)collagen (I) in an hepatic stellate cell line (CFSC-2G). Cd-treated cells increased lipid peroxidation and the content of cytosolic free calcium, decreased glutathione content and superoxide dismutase, glutathione peroxidase and catalase activity. Cd was able to induce the expression of the metallothionein II and alpha(1)collagen (I) gene, that was not described in this cell type. Cadmium may act as a pro-fibrogenic agent in the liver probably by inducing oxidative damage by enhancing lipid peroxidation and altering the antioxidant system of the cells. Although, the exact role metallothionein induction plays in this process is unknown, it probably, provides a cytosolic pool of potential binding sites to sequester ionic Cd, thereby decreasing its toxicity.

Topics: Animals; Antioxidants; Blotting, Northern; Cadmium; Calcium; Catalase; Cell Line; Chemical and Drug Induced Liver Injury; Collagen Type I; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Hepatocytes; Lipid Peroxidation; Liver; Metallothionein; Neutral Red; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxide Dismutase; Tetrazolium Salts; Thiazoles

The importance of axonal damage in multiple sclerosis (MS) has been recently stressed in proton magnetic resonance spectroscopy and pathological studies, but the exact mechanism producing this damage is unknown. The aim of our study was to ascertain whether soluble mediators present in the cerebrospinal fluid (CSF) of patients with relapsing-remitting MS could induce neuron injury in culture. Different biochemical and cytochemical parameters were determined in primary embryonal rat neuron cultures following 8 days of exposure to CSF. Cytotoxic activity was evaluated with a blue formazan production colorimetric assay. Morphological and immunocytochemical studies performed with antibodies against beta-tubulin revealed neuritic fragmentation, axonal damage and cellular shrinkage indicating apoptosis. Detection of apoptosis was carried out using the fluorescent DNA-binding dye Hoechst 33342, as well as by a Terminal deoxynucleotidyl transferase-mediated dUTP Nick End-Labeling assay. We observed that soluble factors in CSF from patients with "aggressive" MS i.e, those with poor recovery after relapses, induced neurite breakdown and neuronal apoptosis in cultures. Neuron injury is not related with blood-brain barrier dysfunction nor with IgG index. Interestingly, CSF from patients with "non-aggressive" MS i.e., relapsing-remitting patients with a good recovery after relapses, did not induce any damage. In conclusion, we report that CSF from patients with aggressive MS bears soluble mediators that induce axonal damage and apoptosis of neurons in culture. These mediators can be present during the first attack of the disease, and the neuronal damage caused could be related to the functional deficit of these MS patients.

Topics: Adolescent; Adult; Animals; Apoptosis; Axons; Cells, Cultured; Cerebrospinal Fluid; Coloring Agents; Humans; Metallothionein; Microscopy, Confocal; Middle Aged; Multiple Sclerosis; Multiple Sclerosis, Relapsing-Remitting; Neurons; Rats; Tetrazolium Salts; Thiazoles

Cytotoxicity studies using a 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT)-based in vitro toxicity assay revealed that McCoy cells exposed to low concentrations of mercuric (0.7 microM), cadmium (1 microM) and cupric chloride (3 microM) exhibited significant increases in cellular activity. This increased activity, previously termed hormesis, coincided with the production of high levels of the stress proteins, heat shock protein 70 (Hsp 70) and metallothionein, while the high constitutive expression of these proteins in cadmium-resistant mutant (CRM) cells corresponded to constitutive hormetic activity. Hormesis was found to obey uniform kinetics allowing for a mathematical description of this increased activity. These results suggest that hormetic activity is a specific cellular response, and most likely, a stress response to low but harmful levels of toxic agents and may therefore provide a rapid test for the presence of toxicants at concentrations associated with chronic toxicity.

Topics: Algorithms; Animals; Cadmium; Cells, Cultured; Colorimetry; Coloring Agents; HSP70 Heat-Shock Proteins; Metallothionein; Metals, Heavy; Mice; Mutation; Proteins; Stimulation, Chemical; Stress, Physiological; Tetrazolium Salts; Thiazoles

Human cervical carcinoma HeLa cells were made resistant to cisplatin by one of two schedules; "acute" (cells repeatedly treated with cisplatin for 1 h in serum-free medium--CA cells) or "continuous" (cells treated repeatedly for 24 h in complete medium--CK cells). The sensitivity of CA and CK sublines to cisplatin and various other antineoplastic drugs was determined by the modified MTT staining procedure. The possible involvement of glutathione (GSH), glutathione S-transferases (GST) and metallothioneins (MT) in cisplatin resistance was examined. The results show that acutely treated CA cells became more resistant to cisplatin than CK cells. The resistance to cisplatin does not involve either glutathione or glutathione transferase. The increased levels of metallothioneins might be involved in the development of resistance. The sensitivity of CA and CK sublines to the selected drugs was different from that of the parent cells. Both sublines became cross-resistant to vincristine and methotrexate, but only CA cells exhibited cross resistance to etoposide doxorubicin and 5-fluorouracil. Thus, the development of resistance to cisplatin is a consequence of numerous intracellular alterations that are reflected in cell response to a variety of anticancer drugs. The response depends on the schedule of resistance development and on the nature of the secondary agent.

Topics: Antineoplastic Agents; Cell Separation; Cisplatin; Dose-Response Relationship, Drug; Drug Resistance; HeLa Cells; Humans; Metallothionein; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured