4-hydroxy-2-nonenal and thiazolyl-blue

Reactive aldehydes including methyl glyoxal, acrolein and 4-hydroxy-2-nonenal (4-HNE) have been implicated in the progression of neurodegenerative diseases. The reduction of aldehydes to alcohols by the aldo-keto reductase (AKR) family of enzymes may represent an important detoxication route within neuronal cells. In this study, the ability of AKR enzymes to protect human neuroblastoma SH-SY5Y cells against reactive aldehydes was assessed. Using gene-specific RNA interference (RNAi), we report that AKR7A2 makes a significant contribution to the reduction of methyl glyoxal in SH-SY5Y cells, with its knockdown altering the IC(50) from 410 to 25.8μM, and that AKR1C3 contributes to 4-HNE reduction, with its knockdown lowering the IC(50) from 1.25 to 0.58μM. In addition, we have shown that pretreatment of cells with sub-lethal concentrations of 4-HNE or methyl glyoxal leads to a significant increase in IC(50) when cells are exposed to higher concentrations of the toxic aldehyde. The IC(50) for methyl glyoxal increased from 410μM to 1.9mM, and the IC(50) for 4-HNE increased from 120 to 690nM. To investigate this protection, we show that pretreatment of cells with the AKR inhibitor sorbinil lead to decreased resistance to aldehydes. We show that AKR1C can be induced 8-fold in SH-SY5Y cells by treatment with sub-lethal concentrations of methyl glyoxal, and 5-fold by 4-HNE treatment. AKR1B is not induced by methyl glyoxal but is induced 10-fold by 4-HNE treatment. Furthermore, we have shown that this adaptive response can also be induced using the chemoprotective agent tert-butyl hydroquinone (t-BHQ), and that this also evokes an increase in the expression and activity of AKR1B and AKR1C. These findings highlight the potential for the interventional upregulation of AKR via non-toxic derivatives or natural compounds as a novel therapeutic approach towards the detoxication of aldehydes, with the aim of halting the progression of aldehyde-dependent neurodegenerative diseases.

Topics: Adaptation, Physiological; Alcohol Oxidoreductases; Aldehyde Reductase; Aldehydes; Aldo-Keto Reductases; Blotting, Western; Cell Line, Tumor; Coloring Agents; Enzyme Induction; Humans; Hydroquinones; Inactivation, Metabolic; Nerve Tissue Proteins; Pyruvaldehyde; RNA Interference; Tetrazolium Salts; Thiazoles

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. Oxidative damage has been associated with pathological neuronal loss in HD. The therapeutic modulation of oxidative stress and mitochondrial function using low molecular weight compounds may be an important strategy for delaying the onset and slowing the progression of HD. In the present study, we found a marked increase of 4-hydroxy-2-nonenal (4-HNE) adducts, a lipid peroxidation marker, in the caudate and putamen of HD brains and in the striatum of HD mice. Notably, 4-HNE immunoreactivity was colocalized with mutant huntingtin inclusions in the striatal neurons of R6/2 HD mice. Administration of nordihydroguaiaretic acid (NDGA), an antioxidant that functions by inhibiting lipid peroxidation, markedly reduced 4-HNE adduct formation in the nuclear inclusions of R6/2 striatal neurons. NDGA also protected cultured neurons against oxidative stress-induced cell death by improving ATP generation and mitochondrial morphology and function. In addition, NDGA restored mitochondrial membrane potential, mitochondrial structure, and synapse structure in the striatum of R6/2 mice and increased their lifespan. The present findings suggest that further therapeutic studies using NDGA are warranted in HD and other neurodegenerative diseases characterized by increased oxidative stress and altered mitochondrial function.

Topics: Adenosine Triphosphate; Age Factors; Aldehydes; Analysis of Variance; Animals; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Embryo, Mammalian; Glutamic Acid; Humans; Huntingtin Protein; Huntington Disease; Imaging, Three-Dimensional; In Situ Nick-End Labeling; Indoles; Lipid Peroxidation; Lipoxygenase Inhibitors; Male; Masoprocol; Membrane Potential, Mitochondrial; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Mitochondria; Neostriatum; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Oxidative Stress; Synapses; Tetrazolium Salts; Thiazoles; Trinucleotide Repeat Expansion

Previously, we reported that genipin, a herbal iridoid, had neuritogenic and neuroprotective actions on PC12 cells. Although nitric oxide (NO)-activated signalings were proposed to be neuritogenic, the neuroprotective action of genipin remains to be elucidated. From the standpoint of NO activation, we tested a possible protective mechanism through the nitrosative Kelch-like ECH-associated protein (Keap1)/NF-E2-related factor 2 (Nrf2)-antioxidant response element pathway in rat retinal ganglion cells (RGC-5 cells) in culture, and in vivo, against hydrogen peroxide and optic nerve injury (ONI), respectively, using a long-acting (1R)-isoPropyloxygenipin (IPRG001). IPRG001 induced NO generation and the expressions of antioxidative enzymes, such as heme oxygenase-1 (HO-1), in RGC-5 cells. The protective action of IPRG001 depended on HO-1 and NO induction. We found that S-nitrosylation of Keap1 by IPRG001 may contribute to translocation of Nrf2 to the nucleus and triggered transcriptional activation of antioxidative enzymes. Furthermore, apoptotic cells were increased and 4-hydroxy-2-nonenal was accumulated in rat retina following ONI. Pre-treatment with IPRG001 almost completely suppressed apoptosis and accumulation of 4-hydroxy-2-nonenal in RGCs following ONI accompanied by HO-1 induction. These data demonstrate for the first time that IPRG001 exerts neuroprotective action in RGCs in vitro and in vivo, through the Nrf2/antioxidant response element pathway by S-nitrosylation against oxidative stress.

Topics: Aldehydes; Animals; Antioxidants; Blotting, Western; Cell Death; Cell Line; Chromatin; Coloring Agents; Cysteine Proteinase Inhibitors; Heme Oxygenase-1; Humans; Immunohistochemistry; Immunoprecipitation; In Situ Nick-End Labeling; Intracellular Signaling Peptides and Proteins; Iridoid Glycosides; Iridoids; Kelch-Like ECH-Associated Protein 1; NF-E2-Related Factor 2; Nitric Oxide; Oxidative Stress; Rats; Rats, Sprague-Dawley; Response Elements; Retinal Ganglion Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Small Interfering; Signal Transduction; Tetrazolium Salts; Thiazoles

We have developed transgenic Chinese hamster V79 cell lines in order to examine the potential for a mouse aldo-keto reductase, AKR7A5, to protect against the toxicity of 4-hydroxynonenal (4-HNE) and related toxic aldehydes. Stable expression of mouse AKR7A5 in V79 cells conferred four-fold increased resistance to 4-HNE cytotoxicity using the MTT assay compared to empty vector-transfected V79 cells. Cells expressing AKR7A5 showed a decrease in mutation rate compared to control cells in the presence of 4-HNE as measured by HGPRT mutagenicity assay. Furthermore, the cells expressing AKR7A5 showed decreased 4-HNE-induced caspase-3 activity in both a time and dose-dependent manner compared to control cells. These results show that in V79 cells 4-HNE mediates apoptosis via caspase-3 activation and that the AKR7A5 enzyme is able to metabolize 4-HNE in cells, thereby attenuating 4-HNE-induced apoptosis. AKR7A isozymes may therefore be important in protecting against toxic aldehydes derived from lipid peroxidation in vivo.

Topics: Alcohol Oxidoreductases; Aldehyde Reductase; Aldehydes; Aldo-Keto Reductases; Animals; Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Line; Cricetinae; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Fibroblasts; Lipid Peroxidation; Mice; Mutagenicity Tests; Tetrazolium Salts; Thiazoles; Transfection

Hypoglycemia sometimes occurs in patients with diabetes mellitus who receive excessive doses of insulin. Severe hypoglycemia has been known to induce mitochondrial swelling followed by neuronal death in the brain. Since L-carnitine effectively preserves mitochondrial function in various cells both in vitro and in vivo, we investigated its effects on the neuronal damage induced by hypoglycemic insult in male Wistar rats. Animals were given L-carnitine-containing water (0.1%) for 1 week and then received insulin (20 U/kg, i.p.) to induce hypoglycemia. Although L-carnitine did not affect the mortality of animals that developed hypoglycemic shock, it improved the cognitive function of the survived animals as assessed by the Morris water-maze test. L-carnitine effectively inhibited the increase in oxidized glutathione and mitochondrial dysfunction in the hippocampus and prevented neuronal injury. L-carnitine also inhibited the decrease in mitochondrial membrane potential and the generation of reactive oxygen species in hippocampal neuronal cells cultured in glucose-deprived medium. These results suggest that L-carnitine prevents hypoglycemia-induced neuronal damage in the hippocampus, presumably by preserving mitochondrial functions. Thus, L-carnitine may have therapeutic potential in patients with hypoglycemia induced by insulin overdose.

Topics: Aldehydes; Analysis of Variance; Animals; Apoptosis; Benzimidazoles; Brain Injuries; Carbocyanines; Carnitine; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Embryo, Mammalian; Glucose; Glutathione; Hippocampus; Hypoglycemia; Immunohistochemistry; In Situ Nick-End Labeling; Insulin; Male; Maze Learning; Membrane Potentials; Mitochondria; Neurons; Rats; Rats, Wistar; Reaction Time; Reactive Oxygen Species; Respiration; Tetrazolium Salts; Thiazoles; Time Factors

Oxidative stress has been demonstrated in Alzheimer's disease (AD) brain and may affect glutamate transport (GT), thereby leading to excitotoxic neuronal death. Since oxidative stress markers have been shown also in peripheral tissues, we investigated possible GT alterations in fibroblast cultures obtained from 18 patients with AD and 15 control patients and analyzed the effects of the lipoperoxidation product 4-hydroxynonenal (4-HNE) and antioxidants. Basal GT was decreased by 60% in fibroblasts from patients with AD versus control patients. Exposure to HNE did not affect GT in control patients, but it reduced GT by 50% in patients with AD, without any concomitant change in cell viability; conversely, HNE exposure induced a larger increase in ROS intracellular levels in AD than in control fibroblasts. Glutathione and N-acetylcysteine completely blocked 4-HNE effects and also increased basal uptake in AD cells. Moreover, inhibition of glutathione synthesis in control fibroblasts by pretreatment with buthionine sulfoximine resulted in GT reduction (40%) and an increase in ROS levels after exposure to 4-HNE. Nevertheless, since there are no differences between GSH basal level in controls and patients with AD, the alteration of other antioxidant systems cannot be excluded. Our study supports the hypothesis of a systemic impairment of GT in AD, possibly linked to oxidative stress and to reduced antioxidant defenses, which may be partially reversed by antioxidant treatment. Therefore, we suggest fibroblast cultures as a tool for exploring pathogenetic mechanisms and possible therapeutic strategies in patients with AD.

Topics: Acetylcysteine; Adenosine Triphosphate; Aged; Aged, 80 and over; Aldehydes; Alzheimer Disease; Animals; Antioxidants; Case-Control Studies; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Fibroblasts; Free Radicals; Glutamic Acid; Glutathione; Humans; L-Lactate Dehydrogenase; Lipid Peroxidation; Middle Aged; Oxidative Stress; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Time Factors

Oxidative stress has been implicated in the pathogenesis of cancer and prominent neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Apoptosis and cell cycle deregulation appear to be the mode of cell death in these disorders. Green tea polyphenol, epigallocatechin-3-gallate (EGCG) has been shown to be a potent antiinflammatory, apoptotic and cancer chemopreventive agent. 4-Hydroxynonenal (HNE), a by-product of lipid peroxidation (LPO), has been reported to induce apoptosis and inhibit growth in many cell systems including neuroglial cultures. We have studied both the dose and time dependent effects of HNE and EGCG on the viability of primary astrocyte cell cultures prepared from neonatal rats. HNE was found to be cytotoxic at a higher dose (0.1 mM) and markedly reduced (up to 80%) the astrocyte viability while EGCG did not appear to be cytotoxic under similar conditions. In addition, we have also studied the alterations in glutathione (GSH) and LPO levels and the activities of GSH metabolizing enzymes after treatment with HNE and EGCG. A 40% decrease in GSH level and a moderate increase in LPO were observed in HNE treated cells suggesting an increase in oxidative stress. HNE treatment caused a 50% decrease in GSH reductase and a 35% increase in GSH peroxidase activities. Although HNE treatment did not lead to any significant alterations in GSH-S-transferase (GST) activity, an increased expression of GST isoenzymes was seen following the exposure to HNE. EGCG treatment caused a significant increase in LPO even in the presence of elevated GSH content. In contrast to HNE, EGCG treatment resulted in a significant decrease (50%) in the activity and expression of GSTs. Treatment of astrocyte cultures with HNE, resulted in a severe impairment in mitochondrial respiration as measured by MTT exclusion assay, while treatment with EGCG had no effect on mitochondrial respiratory activity. Both HNE and EGCG were found to initiate apoptosis in astrocytes as measured by DNA fragmentation assay. However, HNE seems to be a stronger apoptotic and cytotoxic agent than EGCG. These results suggest that HNE and EGCG differentially modulate oxidative stress and regulate the growth and survival of astrocytes.

Topics: Aldehydes; Animals; Apoptosis; Astrocytes; Blotting, Western; Catechin; Flavonoids; Glutathione; Glutathione Transferase; Immunohistochemistry; Isoenzymes; Lipid Peroxidation; Mitochondria; Oxidative Stress; Phenols; Polymers; Rats; Rats, Wistar; Tea; Tetrazolium Salts; Thiazoles

A major pathway for detoxification of the highly reactive lipid peroxidation product, 4-hydroxy-2,3-trans-nonenal (HNE) is through the conjugation with glutathione (GSH). We have studied the metabolism of GS-HNE conjugate by the enzyme gamma-glutamyltranspeptidase (GGT) using its purified form, as well as a GGT-overexpressing fibroblast cell line (V79 GGT). Using mass spectrometry analysis we identified for the first time cysteinylglycine-HNE (CysGly-HNE) as the GGT metabolite of GS-HNE. Furthermore, the GGT-dependent metabolism of GS-HNE in the V79 GGT cell line was associated with a considerable increase of cytotoxicity as compared to a control cell line which does not express GGT (V79 Cl). The cytotoxic effect was dose- and time-dependent (100% cellular death at 200 microM GS-HNE after 24 h incubation) in V79 GGT cells, whereas no decrease of viability was observed in V79 Cl cells. A similar cytotoxic effect was obtained when cells were incubated directly with CysGly-HNE, demonstrating that this GGT-dependent metabolite unlike GS-HNE, exhibits cytotoxic properties.

Topics: Aldehydes; Animals; Cell Line; Cell Survival; Chromatography, High Pressure Liquid; Coloring Agents; Cricetinae; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Fibroblasts; gamma-Glutamyltransferase; Glutathione; Humans; Kinetics; Mass Spectrometry; Models, Chemical; Protein Binding; Spectrophotometry; Tetrazolium Salts; Thiazoles; Time Factors

Glutathione-S-transferases (GSTs) are a superfamily of enzymes that function to catalyze the nucleophilic attack of glutathione on electrophilic groups of a second substrate. GSTs are present in many organs and have been implicated in the detoxification of endogenous alpha, beta unsaturated aldehydes, including 4-hydroxynonenal (HNE). Exogenous GST protects hippocampal neurons against HNE in culture. To test the hypothesis that overexpression of GST in cells would increase resistance to exogenous or endogenous HNE induced by oxidative stress, stable transfectants of SY5Y neuroblastoma cells with GST were established. Stable GST transfectants demonstrated enzyme activities 13.7 times (Clone 1) and 30 times (Clone 2) higher than cells transfected with vector alone. GST transfectants (both Clones 1 and 2) demonstrated significantly (p <.05) increased resistance to ferrous sulfate/hydrogen peroxide (20.9% for Clone 1; 46.5% for Clone 2), amyloid beta-peptide (12.2% for Clone 1; 27.5.% for Clone 2), and peroxynitrite (24.3% for Clone 1; 43.9% for Clone 2), but not to exogenous application of HNE in culture medium. GST transfectants treated with 1,1,4-tris (acetyloxy)nonane, a nontoxic derivative of HNE that is degraded to HNE intracellularly, demonstrated a statistically significant (p <.05) increase in viability in a dose-dependent manner compared with SY5Y cells transfected with vector alone. These results suggest that overexpression of GST increases resistance to endogenous HNE induced by oxidative stress or released in the degradation of 1,1,4-tris (acetyloxy)nonane, but not to exogenous application of HNE.

Topics: Aldehydes; Amyloid beta-Peptides; Blotting, Western; Cell Survival; Drug Resistance, Neoplasm; Ferrous Compounds; Gene Expression; Glutathione; Glutathione Transferase; Humans; Hydrogen Peroxide; Isoenzymes; L-Lactate Dehydrogenase; Neuroblastoma; Oxidative Stress; Tetrazolium Salts; Thiazoles; Transfection; Tumor Cells, Cultured

Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. Thiamine deficiency (TD) is an animal model in which chronic oxidative stress and inflammation lead to selective neuronal death, whereas other cell types show an inflammatory response. Therefore, the current studies determined the response of different brain cell types to TD and/or inflammation in vitro and tested whether their responses reflect inherent properties of the cells. The cells that have been implicated in TD-induced neurotoxicity, including neurons, microglia, astrocytes, and brain endothelial cells, as well as neuroblastoma and BV-2 microglial cell lines, were cultured in either thiamine-depleted media or in normal culture media with amprolium, a thiamine transport inhibitor. The activity levels of a key mitochondrial enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), were uniquely distributed among different cell types: The highest activity was in the endothelial cells, and the lowest was in primary microglia and neurons. The unique distribution of the activity did not account for the selective response to TD. TD slightly inhibited general cellular dehydrogenases in all cell types, whereas it significantly reduced the activity of KGDHC exclusively in primary neurons and neuroblastoma cells. Among the cell types tested, only in neurons did TD induce apoptosis and cause the accumulation of 4-hydroxy-2-nonenal, a lipid peroxidation product. On the other hand, chronic lipopolysaccharide-induced inflammation significantly inhibited cellular dehydrogenase and KGDHC activities in microglia and astrocytes but not in neurons or endothelial cells. The results demonstrate that the selective cell changes during TD in vivo reflect inherent properties of the different brain cell types.

Topics: Aldehydes; Amprolium; Animals; Apoptosis; Brain; Cell Death; Cells, Cultured; Coloring Agents; Ketoglutarate Dehydrogenase Complex; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Neurons; Oxidative Stress; Oxidoreductases; Tetrazolium Salts; Thiamine Deficiency; Thiazoles

Amyloid beta-peptide (Abeta) is a key factor in the neurotoxicity of Alzheimer's disease (AD). Recent research has shown that Abeta-mediated neurotoxicity involves free radicals and that Abeta peptides can initiate multiple membrane alterations, including protein oxidation and lipid peroxidation, eventually leading to neuronal cell death. Research also has emphasized the role of 4-hydroxynonenal (HNE), a downstream product of lipid peroxidation, in being able to mimic some of the effects of Abeta peptides. In the current investigation, electron paramagnetic resonance (EPR) studies of spin labeled cortical synaptosomal membrane proteins has been employed to study conformational changes in proteins, spectrophotometric methods have been used to measure protein carbonyl content, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for mitochondrial function has been used to study the effect of vitamin E on samples that were treated with Abeta or HNE. The free radical dependence of beta-amyloid-associated toxicity was confirmed by the ability of the free radical scavenger vitamin E to prevent the toxic effects of Abeta. In contrast, HNE was still toxic in the presence of vitamin E. These results support our Abeta-associated free radical model for neurotoxicity in AD brain and are discussed with reference to potential therapeutic strategies for AD.

Topics: Aldehydes; Alzheimer Disease; Amyloid beta-Peptides; Animals; Antioxidants; Biomarkers; Cells, Cultured; Cerebral Cortex; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Neuroprotective Agents; Oxidation-Reduction; Peptide Fragments; Rats; Rats, Sprague-Dawley; Synaptosomes; Tetrazolium Salts; Thiazoles; Vitamin E