cytochrome-c-t and sodium-arsenite

The present study reports beneficial effect of hydroxytyrosol (HT) against arsenic (As)-induced oxidative stress in the rat brain. Rats were orally administered with sodium arsenite dissolved in distilled water (25 ppm, by oral gavage) for 8 weeks or HT (10 mg/kg b. wt.) in combination with As. Results showed increase in protein oxidation and lipid peroxidation, while catalase and superoxide dismutase (SOD) activities as well as GSH content were decreased after As exposure in rat brain. Fourier transform infrared analysis showed significant alteration in peak area values that also validated the oxidative damage to lipids and proteins. In addition, As exposure caused increase in protein expression of caspase-3 and Bax, while Bcl-2 expression was downregulated resulting in translocation of cytochrome c from mitochondria to cytosol. Treatment of HT with As reversed protein oxidation, lipid peroxidation, and increased GSH content as well as catalase and SOD activities. Administration of HT also prevented translocation of cytochrome c from mitochondria and increased mitochondria/cytosol ratio of cytochrome c. Hence, treatment of HT with As improved antioxidant system and efficiently lowered the generation of oxidative stress in rat brain.

Topics: Administration, Oral; Animals; Antioxidants; Arsenites; Blotting, Western; Brain; Caspase 3; Catalase; Cytochromes c; Cytosol; Down-Regulation; Glutathione; Lipid Metabolism; Lipid Peroxidation; Male; Mitochondria; Nerve Tissue Proteins; Oxidative Stress; Phenylethyl Alcohol; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Rats, Wistar; Sodium Compounds; Spectroscopy, Fourier Transform Infrared; Superoxide Dismutase

Chronic arsenic toxicity is a global health problem that affects more than 100 million people worldwide. Long-term health effects of inorganic sodium arsenite in drinking water may result in skin, lung and liver cancers and in severe neurological abnormalities. We investigated in the present study whether sodium arsenite affects signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). We demonstrated that the critical signaling pathway, which was suppressed by sodium arsenite in NSC, was the protective PI3K-AKT pathway. Sodium arsenite (2-4μM) also caused down-regulation of Nanog, one of the key transcription factors that control pluripotency and self-renewal of stem cells. Mitochondrial damage and cytochrome-c release induced by sodium arsenite exposure was followed by initiation of the mitochondrial apoptotic pathway in NSC. Beside caspase-9 and caspase-3 inhibitors, suppression of JNK activity decreased levels of arsenite-induced apoptosis in NSC. Neuronal differentiation of NSC was substantially inhibited by sodium arsenite exposure. Overactivation of JNK1 and ERK1/2 and down-regulation of PI3K-AKT activity induced by sodium arsenite were critical factors that strongly affected neuronal differentiation. In conclusion, sodium arsenite exposure of human NSC induces the mitochondrial apoptotic pathway, which is substantially accelerated due to the simultaneous suppression of PI3K-AKT. Sodium arsenite also negatively affects neuronal differentiation of NSC through overactivation of MEK-ERK and suppression of PI3K-AKT.

Topics: Apoptosis; Arsenites; Caspase 3; Caspase 8; Caspase Inhibitors; Cell Proliferation; Cell Survival; Chromones; Cytochromes c; Homeodomain Proteins; Humans; Immunohistochemistry; MAP Kinase Signaling System; Mitochondria; Morpholines; Nanog Homeobox Protein; Neural Stem Cells; Neurogenesis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Sodium Compounds; Transcription, Genetic

This study was conducted to evaluate the possible involvement of mitochondrial pathway in NaAsO2-induced apoptosis and the role of reactive oxygen species (ROS) and reduced glutathione (GSH) in the apoptotic effect in Chang human hepatocytes. The MTT assay demonstrated that sodium arsenite (NaAsO2) treatment for 24 h caused a dose-dependent decrease of cell viability. NaAsO2 treatment (0-30 microM) was also found to induce phosphatidylserine externalization, a hallmark of apoptosis; to disrupt the mitochondrial membrane potential (Deltapsi ( m )); to cause the release of cytochrome c into the cytosol, and to trigger cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner. All these changes were accompanied with the enhanced generation of intracellular ROS and malondialdehyde (MDA). Increase of intracellular GSH also coincided unexpectedly. Moreover, the extracellular addition of N-acetyl-L-cysteine (NAC, 5 mM) effectively reduced the generation of ROS and MDA, and rescued the cells from NaAsO2 induced apoptosis and related alteration of mitochondria. These data suggest that the arsenic-induced cell apoptosis occurs though the mitochondrial pathway, and is mostly dependent on generation of ROS rather than GSH depletion in Chang human hepatocytes.

Topics: Acetylcysteine; Apoptosis; Arsenic; Arsenites; Cell Survival; Cells, Cultured; Cytochromes c; Glutathione; Hepatocytes; Humans; Malondialdehyde; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Sodium Compounds

Arsenic is an environmental toxicant that recently has been shown to have anticancer activity against a number of types of cancer cells by inducing apoptosis. Glycogen synthase kinase-3 (GSK3), a serine/threonine kinase, is an important pro-apoptotic signaling enzyme. Although GSK3 has been shown to promote apoptosis caused by a wide variety of insults, a role for GSK3 in arsenic-induced apoptosis has not yet been identified. Investigation of the involvement of GSK3 in arsenite-induced apoptosis demonstrated that arsenite induced apoptosis in SH-SY5Y human neuroblastoma cells, activating the executioner caspase-3 which caused cleavage of poly-ADP ribose-polymerase (PARP). Two selective GSK3 inhibitors, lithium and SB216763, attenuated caspase-3 activation and PARP cleavage induced by arsenite treatment indicating that GSK3 contributed to arsenite-induced apoptosis. Apoptotic signaling following exposure to arsenite involved cytochrome C release from mitochondria, and this was reduced by inhibition of GSK3 indicating that GSK3 promotes arsenite-induced apoptotic signaling upstream of mitochondrial disruption. Moreover, arsenite induced the translocation of Bax and p53 to the mitochondria and the activation-associated oligomerization of Bax, and these crucial events were reduced by inhibition of GSK3, indicating that GSK3 promotes arsenite-induced apoptosis by facilitating signals leading to mitochondrial apoptotic events. Taken together, the findings from this study reveal that GSK3 promotes arsenite-induced apoptosis by facilitating signaling leading to disruption of mitochondria.

Topics: Apoptosis; Arsenites; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Glycogen Synthase Kinase 3; Humans; Indoles; Lithium Compounds; Maleimides; Mitochondria; Neuroblastoma; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins c-akt; Signal Transduction; Sodium Compounds; Tumor Suppressor Protein p53

The mechanism underlying sodium arsenite (arsenite)-induced neurotoxicity was investigated in rat brain. Arsenite was locally infused in the substantia nigra (SN) of anesthetized rat. Seven days after infusion, lipid peroxidation in the infused SN was elevated and dopamine level in the ipsilateral striatum was reduced in a concentration-dependent manner (0.3-5 nmol). Furthermore, local infusion of arsenite (5 nmol) decreased GSH content and increased expression of heat shock protein 70 and heme oxygenase-1 in the infused SN. Aggregation of alpha-synuclein, a putative pathological protein involved in several CNS neurodegenerative diseases, was elevated in the arsenite-infused SN. From the breakdown pattern of alpha-spectrin, both necrosis and apoptosis were involved in the arsenite-induced neurotoxicity. Pyknotic nuclei, cellular shrinkage and cytoplasmic disintegration, indicating necrosis, and TUNEL-positive cells and DNA ladder, indicating apoptosis was observed in the arsenite-infused SN. Arsenite-induced apoptosis was mediated via two different organelle pathways, mitochondria and endoplasmic reticulum (ER). For mitochondrial activation, cytosolic cytochrome c and caspase-3 levels were elevated in the arsenite-infused SN. In ER pathway, arsenite increased activating transcription factor-4, X-box binding protein 1, C/EBP homologues protein (CHOP) and cytosolic immunoglobulin binding protein levels. Moreover, arsenite reduced procaspase 12 levels, an ER-specific enzyme in the infused SN. Taken together, our study suggests that arsenite is capable of inducing oxidative injury in CNS. In addition to mitochondria, ER stress was involved in the arsenite-induced apoptosis. Arsenite-induced neurotoxicity clinically implies a pathophysiological role of arsenite in CNS neurodegeneration.

Topics: alpha-Synuclein; Animals; Apoptosis; Arsenites; Caspase 3; Corpus Striatum; Cytochromes c; Dopamine; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Glutathione; Heme Oxygenase-1; HSP70 Heat-Shock Proteins; Lipid Peroxidation; Male; Mitochondria; Necrosis; Neurotoxicity Syndromes; Oxidative Stress; Rats; Rats, Sprague-Dawley; Sodium Compounds; Substantia Nigra

BVR reduces biliverdin, the HO-1 and HO-2 product, to bilirubin. Human biliverdin (BVR) is a serine/threonine kinase activated by free radicals. It is a leucine zipper (bZip) DNA-binding protein and a regulatory factor for 8/7-bp AP-1-regulated genes, including HO-1 and ATF-2/CREB. Presently, small interference (si) RNA constructs were used to investigate the role of human BVR in sodium arsenite (As)-mediated induction of HO-1 and in cytoprotection against apoptosis. Activation of BVR involved increased serine/threonine phosphorylation but not its protein or transcript levels. The peak activity at 1 h (4-5-fold) after treatment of 293A cells with 5 mum As preceded induction of HO-1 expression by 3 h. The following suggests BVR involvement in regulating oxidative stress response of HO-1: siBVR attenuated As-mediated increase in HO-1 expression; siBVR, but not siHO-1, inhibited As-dependent increased c-jun promoter activity; treatment of cells with As increased AP-1 binding of nuclear proteins; BVR was identified in the DNA-protein complex; and AP-1 binding of the in vitro translated BVR was phosphorylation-dependent and was attenuated by biliverdin. Most unexpectedly, cells transfected with siBVR, but not siHO-1, displayed a 4-fold increase in apoptotic cells when treated with 10 mum As as detected by flow cytometry. The presence of BVR small interference RNA augmented the effect of As on levels of cytochrome c, TRAIL, and DR-5 mRNA and cleavage of poly(ADP-ribose) polymerase. The findings describe the function of BVR in HO-1 oxidative response and, demonstrate, for the first time, not only that BVR advances the role of HO-1 in cytoprotection but also affords cytoprotection independent of heme degradation.

Topics: Acetylcysteine; Apoptosis; Apoptosis Regulatory Proteins; Arsenites; Biliverdine; Blotting, Northern; Blotting, Western; Cell Line; Cell Nucleus; Cell Survival; Cytochromes c; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gene Silencing; Heme; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Luciferases; Membrane Glycoproteins; Membrane Proteins; Oligonucleotides; Oxidative Stress; Oxidoreductases Acting on CH-CH Group Donors; Oxygen; Phosphorylation; Poly(ADP-ribose) Polymerases; Promoter Regions, Genetic; Protein Binding; Protein Biosynthesis; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Retroviridae; RNA, Messenger; RNA, Small Interfering; Serine; Sodium Compounds; Threonine; Time Factors; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor AP-1; Transfection; Tumor Necrosis Factor-alpha