Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide

A fragment of the amyloid beta protein, βA(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that βA(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, βA(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by βA(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to βA(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 • - ) - and hydroxyl ( •OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of •OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of •OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to βA(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that βA(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 • - can lead to HOCl and further formation of •OH, which both have a cytotoxic potential.     

Genetic Dissection of the Amyloid Precursor Protein in Developmental Function and Amyloid Pathogenesis

Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, β-amyloid (Aβ) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aβ sequence was replaced by the human Aβ. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aβ pathogenesis.     

Effect of 900 MHz Radio Frequency Radiation on Beta Amyloid Protein,Protein Carbonyl,and Malondialdehyde in the Brain

Recently, many studies have been carried out in relation to 900 MHz radiofrequency radiation (RF) emitted from a mobile phone on the brain. However, there is little data concerning possible mechanisms between long-term exposure of RF radiation and biomolecules in brain. Therefore, we aimed to investigate long-term effects of 900 MHz radiofrequency radiation on beta amyloid protein, protein carbonyl, and malondialdehyde in the rat brain. The study was carried out on 17 Wistar Albino adult male rats. The rat heads in a carousel were exposed to 900 MHz radiofrequency radiation emitted from a generator, simulating mobile phones. For the study group (n: 10), rats were exposed to the radiation 2 h per day (7 days a week) for 10 months. For the sham group (n: 7), rats were placed into the carousel and the same procedure was applied except that the generator was turned off. In this study, rats were euthanized after 10 months of exposure and their brains were removed. Beta amyloid protein, protein carbonyl, and malondialdehyde levels were found to be higher in the brain of rats exposed to 900 MHz radiofrequency radiation. However, only the increase of protein carbonyl in the brain of rats exposed to 900 MHz radiofrequency radiation was found to be statistically significant (p < 0.001).

In conclusion, 900 MHz radiation emitted from mobile/cellular phones can be an agent to alter some biomolecules such as protein. However, further studies are necessary.     

The Impact of Different Antioxidant Agents alone or in Combination on Reactive Oxygen Species,Antioxidant Enzymes and Cytokines in a Series of Advanced Cancer Patients at Different Sites: Correlation with Disease Progression

In the present study we tested the ability of different antioxidant agents, used alone or in combination, to reduce the reactive oxygen species (ROS) levels and to increase the glutathione peroxidase (GPx) activity. Moreover, we tested the ability of such antioxidant agents to reduce the serum levels of proinflammatory cytokines IL-6 and TNF &#102 . Fifty-six advanced stage cancer patients with tumors at different sites were included in the study: they were mainly stage III (12.5%) and stage IV (82.1%). The study was divided into two phases. In the 1 st phase 28 patients were divided into five groups and a single different antioxidant agent was administered to each group. The selected antioxidant agents were: alpha lipoic acid or carboxycysteine-lysine salt, amifostine, reduced glutathione, vitamin A plus vitamin E plus Vitamin C. In the 2 nd phase of the study 28 patients were divided into five groups and a combination of two different antioxidant agents was administered to each group. The antioxidant treatment was administered for 10 consecutive days. The patients were studied at baseline and after antioxidant treatment. Our results show that all single antioxidants tested were effective in reducing the ROS levels and three of them in increasing GPx activity, too. Among the combinations of antioxidant agents, three were effective in reducing ROS, while three were effective in increasing GPx activity (arm 4 was effective in both instances). Comprehensively, the "antioxidant treatment" was found to be effective both on ROS levels and GPx activity. Moreover, the antioxidant treatment was able to reduce serum levels of IL-6 and TNF &#102 . Furthermore, a correlation was shown between the Eastern Cooperative Oncology Group Performance Status of patients and blood levels of ROS, GPx activity, serum levels of proinflammatory cytokines.     

A Minimal Cysteine Motif Required to Activate the SKOR K+ Channel of Arabidopsis by the Reactive Oxygen Species H2O2

Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca²⁺ and K⁺ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K⁺ channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H₂O₂. We show that H₂O₂ rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H₂O₂ and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H₂O₂-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys¹⁶⁸ located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H₂O₂. The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys¹⁶⁸ as a critical target for H₂O₂, and implicate ROS-mediated control of the K⁺ channel in regulating mineral nutrient partitioning within the plant.     

Transient Influx of Nickel in Root Mitochondria Modulates Organic Acid and Reactive Oxygen Species Production in Nickel Hyperaccumulator Alyssum murale

Mitochondria are important targets of metal toxicity and are also vital for maintaining metal homeostasis. Here, we examined the potential role of mitochondria in homeostasis of nickel in the roots of nickel hyperaccumulator plant Alyssum murale. We evaluated the biochemical basis of nickel tolerance by comparing the role of mitochondria in closely related nickel hyperaccumulator A. murale and non-accumulator Alyssum montanum. Evidence is presented for the rapid and transient influx of nickel in root mitochondria of nickel hyperaccumulator A. murale. In an early response to nickel treatment, substantial nickel influx was observed in mitochondria prior to sequestration in vacuoles in the roots of hyperaccumulator A. murale compared with non-accumulator A. montanum. In addition, the mitochondrial Krebs cycle was modulated to increase synthesis of malic acid and citric acid involvement in nickel hyperaccumulation. Furthermore, malic acid, which is reported to form a complex with nickel in hyperaccumulators, was also found to reduce the reactive oxygen species generation induced by nickel. We propose that the interaction of nickel with mitochondria is imperative in the early steps of nickel uptake in nickel hyperaccumulator plants. Initial uptake of nickel in roots results in biochemical responses in the root mitochondria indicating its vital role in homeostasis of nickel ions in hyperaccumulation.     

Study on the ability of 1,2,3,4-tetrahydropapaveroline to cause oxidative stress: Mechanisms and potential implications in relation to parkinson's disease

Tetrahydropapaveroline (THP) is a compound derived from dopamine monoamine oxidase-mediated metabolism, particularly present in the brain of parkinsonian patients receiving L-dopa therapy, and is capable of causing dopaminergic neurodegeneration. The aim of this work was to evaluate the potential of THP to cause oxidative stress on mitochondrial preparations and to gain insight into the molecular mechanisms responsible for its neurotoxicity. Our data show that THP autoxidation occurs with a continuous generation of hydroxyl radicals (*OH) and without the involvement of the Fenton reaction. The presence of ascorbate enhances this process by establishing a redox cycle, which regenerates THP from its quinolic forms. It has been shown that the production of *OH is not affected by the presence of either ferrous or ferric iron. Although THP does not affect lipid peroxidation, it is capable of reducing the high levels of thiobarbituric acid-reactive substances obtained in the presence of ascorbate and/or iron. However, THP autoxidation in the presence of ascorbate causes both an increase in protein carbonyl content and a reduction in protein-free thiol content. THP also increases protein carbonyl content when the autoxidation occurs in the presence of iron. The remarkable role played by ascorbate in the production of oxidative stress by THP autoxidation is of particular interest.     

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Characterization of the C584R variant in the mtDNA depletion syndrome gene FBXL4, reveals a novel role for FBXL4 as a regulator of mitochondrial fusion

Mutations in FBXL4 (F-Box and Leucine rich repeat protein 4), a nuclear-encoded mitochondrial protein with an unknown function, cause mitochondrial DNA depletion syndrome. We report two siblings, from consanguineous parents, harbouring a previously uncharacterized homozygous variant in FBXL4 (c.1750 T > C; p.Cys584Arg). Both patients presented with encephalomyopathy, lactic acidosis and cardiac hypertrophy, which are reported features of FBXL4 impairment. Remarkably, dichloroacetate (DCA) administration to the younger sibling improved metabolic acidosis and reversed cardiac hypertrophy. Characterization of FBXL4 patient fibroblasts revealed severe bioenergetic defects, mtDNA depletion, fragmentation of mitochondrial networks, and abnormalities in mtDNA nucleoids. These phenotypes, observed with other pathogenic FBXL4 variants, confirm the pathogenicity of the p.Cys584Arg variant. Although treating FBXL4 fibroblasts with DCA improved extracellular acidification, in line with reduced lactate levels in patients, DCA treatment did not improve any of the other mitochondrial functions. Nonetheless, we highlight DCA as a potentially effective drug for the management of elevated lactate and cardiomyopathy in patients with pathogenic FBXL4 variants. Finally, as the exact mechanism through which FBXL4 mutations lead to mtDNA depletion was unknown, we tested the hypothesis that FBXL4 promotes mitochondrial fusion. Using a photo-activatable GFP fusion assay, we found reduced mitochondrial fusion rates in cells harbouring a pathogenic FBXL4 variant. Meanwhile, overexpression of wildtype FBXL4, but not the p.Cys584Arg variant, promoted mitochondrial hyperfusion. Thus, we have uncovered a novel function for FBXL4 in promoting mitochondrial fusion, providing important mechanistic insights into the pathogenic mechanism underlying FBXL4 dysfunction.