metallothionein and 4-hydroxy-2-nonenal

The present study was to explore the effect of metallothionein (MT) on intermittent hypoxia (IH) induced aortic pathogenic changes. Markers of oxidative damages, inflammation, and vascular remodeling were observed by immunohistochemical staining after 3 days and 1, 3, and 8 weeks after IH exposures. Endogenous MT was induced after 3 days of IH but was significantly decreased after 8 weeks of IH. Compared with the wild-type mice, MT knock-out mice exhibited earlier and more severe pathogenic changes of oxidative damages, inflammatory responses, and cellular apoptosis, as indicated by the significant accumulation of collagen, increased levels of connective tissue growth factor, transforming growth factor β1, tumor necrosis factor-alpha, vascular cell adhesion molecule 1,3-nitrotyrosine, and 4-hydroxy-2-nonenal in the aorta. These findings suggested that chronic IH may lead to aortic damages characterized by oxidative stress and inflammation, and MT may play a pivotal role in the above pathogenesis process.

Topics: Aldehydes; Animals; Aorta; Apoptosis; Connective Tissue Growth Factor; Hypoxia; Metallothionein; Mice; Mice, Knockout; NADPH Oxidases; Nitric Oxide Synthase Type III; Oxidative Stress; Time Factors; Tumor Necrosis Factor-alpha; Tyrosine

Ferric nitrilotriacetate (Fe-NTA) is a potent nephrotoxicant and a renal carcinogen that induces its effect by causing oxidative stress. The present study was undertaken to explore protective effect of silymarin, a flavonolignan from milk thistle (Silybum marianum), against Fe-NTA mediated renal oxidative stress, inflammation and tumor promotion response along with elucidation of the implicated mechanism(s). Administration of Fe-NTA (10 mg/kg bd wt, i.p.) to Swiss albino mice induced marked oxidative stress in kidney, evident from augmentation in renal metallothionein (MT) expression, depletion of glutathione content and activities of antioxidant and phase II metabolizing enzymes, and enhancement in production of aldehyde products such as 4-hydroxy-2-nonenal. Fe-NTA also significantly activated nuclear factor kappa B (NFkappaB) and upregulated the expression of downstream genes: cyclooxygenase 2 and inducible nitric oxide synthase and enhancing the production of proinflammatory cytokines: tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). However, feeding of 0.5% and 1% silymarin diet conferred a significant protection against Fe-NTA induced oxidative stress and inflammation. It further augmented MT expression, restored the antioxidant armory, ameliorated NFkappaB activation and decreased the expression of proinflammatory mediators. Silymarin also suppressed Fe-NTA induced hyperproliferation in kidney, ameliorating renal ornithine decarboxylase activity and DNA synthesis. From these results, it could be concluded that silymarin markedly protects against chemically induced renal cancer and acts plausibly by virtue of its antioxidant, anti-inflammatory and antiproliferative activities.

Topics: Aldehydes; Animals; Antioxidants; Cyclooxygenase 2; Cytokines; Dietary Supplements; DNA, Neoplasm; Female; Ferric Compounds; Inflammation; Inflammation Mediators; Kidney; Kidney Neoplasms; Lipid Peroxidation; Metabolic Detoxication, Phase II; Metallothionein; Mice; NF-kappa B; Nitric Oxide Synthase Type II; Nitrilotriacetic Acid; Ornithine Decarboxylase; Protective Agents; Silymarin; Treatment Outcome

The tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has been studied for chronic disease preventive effects, and is marketed as part of many dietary supplements. However, case-reports have associated the use of green tea-based supplements with liver toxicity. We studied the hepatotoxic effects of high dose EGCG in male CF-1 mice. A single dose of EGCG (1500 mg/kg, i.g.) increased plasma alanine aminotransferase (ALT) by 138-fold and reduced survival by 85%. Once-daily dosing with EGCG increased hepatotoxic response. Plasma ALT levels were increased 184-fold following two once-daily doses of 750 mg/kg, i.g. EGCG. Moderate to severe hepatic necrosis was observed following treatment with EGCG. EGCG hepatotoxicity was associated with oxidative stress including increased hepatic lipid peroxidation (5-fold increase), plasma 8-isoprostane (9.5-fold increase) and increased hepatic metallothionein and gamma-histone 2AX protein expression. EGCG also increased plasma interleukin-6 and monocyte chemoattractant protein-1. Our results indicate that higher bolus doses of EGCG are hepatotoxic to mice. Further studies on the dose-dependent hepatotoxic effects of EGCG and the underlying mechanisms are important given the increasing use of green tea dietary supplements, which may deliver much higher plasma and tissue concentrations of EGCG than tea beverages.

Topics: Aldehydes; Animals; Antioxidants; Biomarkers; Catechin; Chemical and Drug Induced Liver Injury; Chromatography, High Pressure Liquid; Cysteine; Cytokines; Dose-Response Relationship, Drug; Immunohistochemistry; Lipid Peroxidation; Liver Function Tests; Male; Metallothionein; Mice; Oxidants; Oxidative Stress; Spectrometry, Mass, Electrospray Ionization; Survival Analysis

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

Copper is an essential metal that is able to produce reactive oxygen species and to induce intracellular oxidative stress. Several studies have examined the effects of excessive copper and oxidative stress on various organisms and tissues, but few have addressed the molecular mechanisms by which copper affects transcription. Our results demonstrated that, in COS-7 cells, copper treatment caused an increase in the binding of nuclear proteins to activating protein-1 and antioxidant response elements. The level of copper-inducible nuclear protein binding was modulated by increasing or decreasing the level of intracellular oxidative stress. Copper exposure also led to an increase in the steady-state levels of c-fos, c-jun, and c-myc mRNAs. Exposure to copper resulted in an increase in the levels of phosphorylation and activation of the c-Jun N-terminal kinase/stress-activated protein kinase and p38 pathways. The activation of these pathways resulted in a concomitant increase in c-Jun phosphorylation. We investigated the hypothesis that copper-induced oxidative stress leads to the formation of stable lipid peroxidation by-products that activate mitogen-activated protein kinase (MAPK) pathways, ultimately affecting transcription. While exposure did result in the production of 4-hydroxynonenal, the timing of the increased levels of proto-oncogene mRNA, phosphorylation of c-jun, and phosphorylation and activation of MAPKs, as well as the inability of the lipophilic antioxidant vitamin E to abrogate MAPK phosphorylation, suggest that the formation of stable lipid peroxidation by-products may not be the primary mechanism by which copper activates MAPKs. These results further elucidate the effects of copper on signal transduction pathways to alter gene expression.

Topics: Aldehydes; Animals; Chlorocebus aethiops; Copper; COS Cells; Extracellular Signal-Regulated MAP Kinases; JNK Mitogen-Activated Protein Kinases; Lipid Peroxidation; MAP Kinase Signaling System; Metallothionein; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Response Elements; RNA, Messenger; Transcription Factor AP-1; Transcription, Genetic; Vitamin E

Age-related macular degeneration (ARMD) is the leading cause of blindness in the developed world and yet its pathogenesis remains poorly understood. Retina has high levels of polyunsaturated fatty acids (PUFAs) and functions under conditions of oxidative stress. To investigate whether peroxidative products of PUFAs induce apoptosis in retinal pigmented epithelial (RPE) cells and possibly contribute to ARMD, human retinal pigmented epithelial cells (ARPE-19) were exposed to micromolar concentrations of H2O2, 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). A concentration- and time-dependent increase in H2O2-, HNE-, and HHE-induced apoptosis was observed when monitored by quantifying DNA fragmentation as determined by ELISA, flow cytometry, and Hoechst staining. The broad-spectrum inhibitor of apoptosis Z-VAD inhibited apoptosis. Treatment of RPE cells with a thionein peptide prior to exposure to H2O2 or HNE reduced the formation of protein-HNE adducts as well as alteration in mitochondrial membrane potential and apoptosis. Using 3H-HNE, various metabolic pathways to detoxify HNE by ARPE-19 cells were studied. The metabolites were separated by HPLC and characterized by ElectroSpray Ionization-Mass Spectrometry (ESI-MS) and gas chromatography-MS. Three main metabolic routes of HNE detoxification were detected: (1) conjugation with glutathione (GSH) to form GS-HNE, catalyzed by glutathione-S-transferase (GST), (2) reduction of GS-HNE catalyzed by aldose reductase, and (3) oxidation of HNE catalyzed by aldehyde dehydrogenase (ALDH). Preventing HNE formation by a combined strategy of antioxidants, scavenging HNE by thionein peptide, and inhibiting apoptosis by caspase inhibitors may offer a potential therapy to limit retinal degeneration in ARMD.

Topics: Aldehydes; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Ergothioneine; Humans; Hydrogen Peroxide; Lipid Metabolism; Lipids; Metallothionein; Oxidative Stress; Pigment Epithelium of Eye; Protein Binding; Tetrazolium Salts; Thiazoles; Time Factors; Trypan Blue

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, reacts with histidine, lysine or cysteine residues of proteins to form hemiacetal Michael adducts and thus interferes with the functions of the proteins. Here we undertook to identify HNE-modified proteins in the target organ of a ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis model with histidine-specific HNEJ-2 antibody. Immunoaffinity column separation and sequencing identified one of the major modified proteins as actin. To further explore the characteristics of actin as an HNE acceptor, we produced four novel monoclonal antibodies against HNE-modified keyhole limpet hemocyanin. All these antibodies (HNEJ-1, 3-5) recognized histidine adducts, but were different from HNEJ-2 in recognizing lysine and cysteine adducts to some extent. Actin, albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), metallothionein and superoxide dismutase were treated in vitro with HNE and evaluated with these antibodies. The results revealed that actin was most sensitive to HNE modification and metallothionein most resistant. Furthermore, the residue-specificity of GAPDH was in accord with that shown by our recent mass spectrometry data. Immunohistochemistry with the antibodies revealed cytoplasmic staining with or without nuclear staining in the renal proximal tubules after Fe-NTA administration. The results suggest that actin is a major target protein for HNE modification in vivo, and that our monoclonal antibodies are useful for evaluating the HNE adducts produced.

Topics: Actins; Albumins; Aldehydes; Animals; Antibodies, Monoclonal; Blotting, Western; Cysteine; Ferric Compounds; Glyceraldehyde-3-Phosphate Dehydrogenases; Hemocyanins; Histidine; Kidney; Kidney Neoplasms; Lysine; Male; Metallothionein; Nitrilotriacetic Acid; Rats; Rats, Wistar; Specific Pathogen-Free Organisms; Superoxide Dismutase

Oxidative stress is involved in the pathogenesis of diabetes and its cardiovascular complications. Metallothionein (MT), a stress-response protein, is significantly increased in the liver and kidney of diabetic animals. We examined whether diabetes also induces cardiac MT synthesis through oxidative damage and whether MT overexpression protects the heart from injury. Diabetes was induced in mice by single injection of streptozotocin (STZ), and cardiac MT mRNA and protein levels were measured 2 weeks and 2 months after STZ treatment. Diabetes significantly increased cardiac MT synthesis 2 weeks and 2 months after STZ treatment, with no change in cardiac metals including zinc, copper, and iron. Serum and cardiac vasopeptide endothelin and inflammatory cytokine tumor necrosis factor-alpha were also significantly increased in diabetic hearts, as were the ratio of oxidized to reduced glutathione and the immunohistochemical staining of 3-nitrotyrosine and 4-hydroxynonenal. To explore the biological importance of increased MT synthesis in the heart, MT-overexpressing transgenic mice were treated with STZ and then examined 2 months later. A loss of inotropic reserve, uncovered during beta-adrenergic stimulation, and the presence of cardiac fibrosis, shown by increased Sirius red staining of collagen, were evident in the wild-type diabetic mice but not in the MT-overexpressing transgenic diabetic mice. These results suggest that diabetes-induced cardiac MT expression likely associates with systemic increases in endothelin-1 and tumor necrosis factor-alpha and the resulting cardiac oxidative stress. Overexpressing cardiac MT significantly protects the heart from diabetes-induced injury.

Topics: Aldehydes; Animals; Blotting, Northern; Blotting, Western; Cardiovascular Diseases; Copper; Diabetes Mellitus, Experimental; Endothelin-1; Glutathione; Immunohistochemistry; Interleukin-6; Iron; Metallothionein; Mice; Mice, Transgenic; Myocardium; Oxidative Stress; RNA, Messenger; Tumor Necrosis Factor-alpha; Tyrosine; Zinc