Novel probes for visualizing reactive oxygen species in lipid membranes

This work describes the rationale behind the preparation of fluorescent probes for imaging lipid peroxyl radicals within membranes of living cells (fluorescent lipophilic antioxidants). The new probes are based on BODIPY dyes tethered to phenol moieties. We discuss the spectroscopic properties of these novel probes, specifically the BODIPY-α-tocopherol analogue B-TOH, and present a molecular level explanation, based on photoinduced electron transfer, that accounts for the significant emission enhancement that the probe BTOH experiences upon reaction with peroxyl free radicals. In addition to the spectroscopy results in homogeneous media, we also describe studies performed in model lipid membranes which show that the sensitivity of BTOH towards lipid peroxyl radicals is somewhat reduced when the probe is membrane embedded. Solutions to increase the sensitivity of the free radical probes are discussed based on the redox potential of BODIPY dyes.

Reactive nitrogen species in cellular signaling

The transduction of cellular signals occurs through the modification of target molecules. Most of these modifications are transitory, thus the signal transduction pathways can be tightly regulated. Reactive nitrogen species are a group of compounds with different properties and reactivity. Some reactive nitrogen species are highly reactive and their interaction with macromolecules can lead to permanent modifications, which suggested they were lacking the specificity needed to participate in cell signaling events. However, the perception of reactive nitrogen species as oxidizers of macromolecules leading to general oxidative damage has recently evolved. The concept of redox signaling is now well established for a number of reactive oxygen and nitrogen species. In this context, the post-translational modifications introduced by reactive nitrogen species can be very specific and are active participants in signal transduction pathways. This review addresses the role of these oxidative modifications in the regulation of cell signaling events.

Deterministic Chaos and Mitochondrial Synthesis of Reactive Oxygen Species

We tested the Lotan hypothesis that two potent anticancer retinoids, all-trans retinoic acid (atRA) and N-(4-hydroxyphenyl) retinamide (4-HPR) interfere with mitochondrial electron transport enhancing synthesis of reactive oxygen species (ROS). Mitochondria isolated from normal rat liver and three human uroepithelial cell lines T24, TCC-Sup and RT4 were used in this study. Membrane potential (Δψ) was measured simultaneously with oxygen consumption when mitochondria were incubated with 10 µM atRA or 5µM 4-HPR. Subsequently, synthesis of ROS was monitored by spin trap electroparamagnetic resonance (EPR) and spectrofluorescence (SF). Uncoupling of mitochondrial respiration by atRA or 4-HPR with succinate as oxidizable substrate was observed (i.e. decrease in both ADP/O and respiratory control (RC) ratio). Thus, the hypothesis must be rejected. Mathematical model indicates that chaotic oscillations of the concentrations of superoxide anion observed in the experiment are caused by some autocatalytic interactions between free radicals and oxygen molecules. Those interactions lead to the emergence of teleological, complex, fractal dynamics in both mitochondrial electron transport chain and ATP synthesis. From the perspective of deterministic chaotic dynamics, electron transport is not a simplistic phenomenon, nor is ROS synthesis its by-process. The observed uncoupling leads to the ATP deficit and may protect tissues against damage, e.g., caused by radiotherapy.

Chemoprevention of cancer and cardiovascular disease by resveratrol.

Resveratrol (trans-3,4',5-trihydroxystibene) is a phytopolyphenol isolated from the seeds and skins of grapes. Recent studies indicate that resveratrol can block the process of multistep carcinogenesis, namely, tumor initiation, promotion and progression. Resveratrol can also reduce the risk of cardiovascular disease in man. The molecular mechanisms of resveratrol in chemoprevention of cancer and cardiovascular disease are interesting and under intensive investigation. Resveratrol was found to strongly inhibit nitric oxide (NO) generation in activated macrophages, as measured by the amount of nitrite released into the culture medium, and resveratrol strongly reduced the amount of cytosolic inducible nitric oxide synthase (iNOS) protein. The activation of nuclear factor kappa B (NF kappa B) induced by lipopolysaccharide (LPS) was inhibited by resveratrol. The phosphorylation and degradation of nuclear factor inhibitor kappa B alpha (I kappa B alpha) were inhibited by resveratrol simultaneously. Reactive oxygen species (ROS) are regarded as having carcinogenic potential and have been associated with tumor promotion. Resveratrol may act as a reactive oxygen species scavenger to suppress tumor development. In addition, resveratrol may block multistep carcinogenesis through mitotic signal transduction blockade. Reactive oxygen species are pivotal factors in the genesis of heart disease. Meanwhile, efficient endogenous antioxidants, including superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase, are present in tissues. A fine balance between reactive oxygen species and endogenous antioxidants is believed to exist. Any disturbance of this balance in favor of reactive oxygen species causes an increase in oxidative stress and initiates subcellular changes, leading to cardiomyopathy and heart failure. The experimental results indicate that exogenous antioxidant resveratrol is of value in chemopreventing the development of heart disease. It is urgent that more efforts be made to investigate newer therapies employing antioxidants for the chemoprevention of cardiovascular disease and cancer.

Imaging of cellular oxygen and analysis of metabolic responses of mammalian cells.

Many parameters reflecting mitochondrial function and metabolic status of the cell, including the mitochondrial membrane potential, reactive oxygen species, ATP, NADH, ion gradients, and ion fluxes (Ca(2+), H(+)), are amenable for analysis by live cell imaging and are widely used in many labs. However, one key metabolite - cellular oxygen - is currently not analyzed routinely. Here we present several imaging techniques that use the phosphorescent oxygen-sensitive probes loaded intracellularly and which allow real-time monitoring of O(2) in live respiring cells and metabolic responses to cell stimulation. The techniques include conventional wide-field fluorescence microscopy to monitor relative changes in cell respiration, microsecond FLIM format which provides quantitative readout of O(2) concentration within/near the cells, and live cell array devices for the monitoring of metabolic responses of individual suspension cells. Step by step procedures of typical experiments for each of these applications and troubleshooting guide are given.

Classification of reactive sites on the surface of polycrystalline tin dioxide

A new method is introduced for study of the nature of reactive sites on oxide surfaces, which is derived from the study of gas sensors and is based on the correlation between combustion catalysis and the effects of gases on electrical conductivity at elevated temperature. Chemical modification of the surface, using a gaseous siloxane to react progressively with the surface sites, is employed to discriminate and classify different types of site. Both the electrical response and the combustion rate are altered by the titration of the surface sites against the siloxane. The method comprises the sequential application of test gas (hexamethyldisiloxane, HMDS, at low concentration in air) and probe gas (methane, water vapour), monitoring methane combustion rate, resistivity in pure, dry air and electrical response to methane and water vapour, as a function of HMDS dose. The measurements require the material to be fabricated into a gas sensor device having an array of electrodes designed to probe conductivity variations throughout the porous ceramic body, which allows the combustion rate and the electrical response to be obtained simultaneously. Four different types of reactive surface site have thereby been distinguished on polycrystalline SnO2: an electrically neutral oxygen site which catalyses methane and HMDS combustion, two electrically charged oxygen sites which respectively mediate methane response and dissociative chemisorption of water, and a site for molecular chemisorption of water. By comparison with other literature, the neutral site which catalyses combustion could be attributed to a coordinatively unsaturated lattice oxygen. The two different electrically charged sites could be associated with oxygen adsorbed on or at two different types of surface oxygen vacancy associated with reduced (Sn2+) surface cations.

Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut

Relationship between redox activity and chemical speciation of size-fractionated particulate matter

BackgroundAlthough the mechanisms of airborne particulate matter (PM) related health effects remain incompletely understood, one emerging hypothesis is that these adverse effects derive from oxidative stress, initiated by the formation of reactive oxygen species (ROS) within affected cells. Typically, ROS are formed in cells through the reduction of oxygen by biological reducing agents, with the catalytic assistance of electron transfer enzymes and redox active chemical species such as redox active organic chemicals and metals. The purpose of this study was to relate the electron transfer ability, or redox activity, of the PM samples to their content in polycyclic aromatic hydrocarbons and various inorganic species. The redox activity of the samples has been shown to correlate with the induction of the stress protein, hemeoxygenase-1.ResultsSize-fractionated (i.e. < 0.15; < 2.5 and 2.5 – 10 μm in diameter) ambient PM samples were collected from four different locations in the period from June 2003 to July 2005, and were chemically analyzed for elemental and organic carbon, ions, elements and trace metals and polycyclic aromatic hydrocarbons. The redox activity of the samples was evaluated by means of the dithiothreitol activity assay and was related to their chemical speciation by means of correlation analysis. Our analysis indicated a higher redox activity on a per PM mass basis for ultrafine (< 0.15 μm) particles compared to those of larger sizes. The PM redox activity was highly correlated with the organic carbon (OC) content of PM as well as the mass fractions of species such as polycyclic aromatic hydrocarbons (PAH), and selected metals.ConclusionThe results of this work demonstrate the utility of the dithiothreitol assay for quantitatively assessing the redox potential of airborne particulate matter from a wide range of sources. Studies to characterize the redox activity of PM from various sources throughout the Los Angeles basin are currently underway.

c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: a mechanism for oncogene-induced genetic instability.

Oncogene overexpression activates p53 by a mechanism posited to involve uncharacterized hyperproliferative signals. We determined whether such signals produce metabolic perturbations that generate DNA damage, a known p53 inducer. Biochemical, cytological, cell cycle, and global gene expression analyses revealed that brief c-Myc activation can induce DNA damage prior to S phase in normal human fibroblasts. Damage correlated with induction of reactive oxygen species (ROS) without induction of apoptosis. Deregulated c-Myc partially disabled the p53-mediated DNA damage response, enabling cells with damaged genomes to enter the cycle, resulting in poor clonogenic survival. An antioxidant reduced ROS, decreased DNA damage and p53 activation, and improved survival. We propose that oncogene activation can induce DNA damage and override damage controls, thereby accelerating tumor progression via genetic instability.

Scavenging of reactive oxygen and nitrogen species with nanomaterials

Reactive oxygen and nitrogen species (RONS) are essential for normal physiological processes and play important roles in cell signaling, immunity, and tissue homeostasis. However, excess radical species are implicated in the development and augmented pathogenesis of various diseases. Several antioxidants may restore the chemical balance, but their use is limited by disappointing results of clinical trials. Nanoparticles are an attractive therapeutic alternative because they can change the biodistribution profile of antioxidants, and possess intrinsic ability to scavenge RONS. Herein, we review the types of RONS, how they are implicated in several diseases, and the types of nanoparticles with inherent antioxidant capability, their mechanisms of action, and their biological applications.

The complex role of physical exercise and reactive oxygen species on brain

Abstract Reactive oxygen species (ROS) are continuously generated during aerobic metabolism and at moderate level. They play a role in redox signaling, but in significant concentration they cause oxidative damage and neurodegeneration. Because of the enhanced sensitivity of brain to ROS, it is especially important to maintain the normal redox state in different types of neuron cells. In last decade it became clear that regular exercise beneficially affects brain function, and can play an important preventive and therapeutic role in stroke, Alzheimer, and Parkinson diseases. The effects of exercise appear to be very complex and could include neurogenesis via neurotrophic factors, increased capillariszation, decreased oxidative damage, and increased proteolytic degradation by proteasome and neprilysin. Data from our and other laboratories indicate that exercise-induced modulation of ROS levels plays a role in the protein content and expression of brain-derived neurotrophic factor, tyrosine-related kinase B (TrkB), and cAMP response element binding protein, resulting in better function and increased neurogenesis. Therefore, it appears that exercise-induced modulation of the redox state is an important means, by which exercise benefits brain function, increases the resistance against oxidative stress, facilitates recovery from oxidative stress, and attenuates age-associated decline in cognition.

S-nitrosylation of NADPH oxidase regulates cell death in plant immunity

Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.

Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. A review

Abstract In recent years there has been enormous interest in researching oxidative stress. Reactive oxygen species (ROS) are derived from the metabolism of oxygen as by-products of cell respiration, and are continuously produced in all aerobic organisms. Oxidative stress occurs as a consequence of an imbalance between ROS production and the available antioxidant defence against them. Nowadays, a variety of diseases and degenerative processes such as cancer, Alzheimer’s and autoimmune diseases are mediated by oxidative stress. Heat stress was suggested to be an environmental factor responsible for stimulating ROS production because of similarities in responses observed following heat stress compared with that occurring following exposure to oxidative stress. This manuscript describes the main mitochondrial sources of ROS and the antioxidant defences involved to prevent oxidative damage in all the mitochondrial compartments. It also deals with discussions concerning the cytotoxic effect of heat stress, mitochondrial heat-induced alterations, as well as heat shock protein (HSP) expression as a defence mechanism.

Crosstalk of reactive oxygen species and NF-κB signaling

Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress

Reactive oxygen species are required for cell proliferation but can also induce apoptosis. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans, and we conclude that this model also extends to mammalian systems.

Fluorescent Gold Nanoprobe Sensitive to Intracellular Reactive Oxygen Species

Gold nanoprobes immobilized with fluorescein‐hyaluronic acid (HA) conjugates are fabricated and utilized for monitoring intracellular reactive oxygen species (ROS) generation in live cells via nanoparticle surface energy transfer. A bio‐inspired adhesive molecule, dopamine, is used to robustly end‐immobilize HA onto the surface of gold nanoparticles (AuNPs) for securing intracellular stability against glutathione. ROS induces cleavage and fragmentation of the HA chains immobilized on the surface of the AuNPs allows rapid and specific detection of intracellular ROS by emitting strong fluorescence‐recovery signals. In particular, fluorescence‐quenched gold nanoprobes exhibit selective and dose‐dependent fluorescence‐recovery signals upon exposure to certain oxygen species such as superoxide anion (${\rm O}_{2}^{\cdot{{-\!\!\!\!\!-}}}$) and hydroxyl radical (·OH). The fluorescent gold nanoprobe is usefully exploited for real‐time intracellular ROS detection and antioxidant screening assay, and has exciting potential for various biomedical applications as a new class of ROS imaging probes.

Decrease in Mitochondrial Complex I Activity in Ischemic/Reperfused Rat Heart: Involvement of Reactive Oxygen Species and Cardiolipin

Abstract— Reactive oxygen species (ROS) are considered an important factor in ischemia/reperfusion injury to cardiac myocytes. Mitochondrial respiration is an important source of ROS production and hence a potential contributor to cardiac reperfusion injury. In this study, we have examined the effect of ischemia and ischemia followed by reperfusion of rat hearts on various parameters related to mitochondrial function, such as complex I activity, oxygen consumption, ROS production, and cardiolipin content. The activity of complex I was reduced by 25% and 48% in mitochondria isolated from ischemic and reperfused rat heart, respectively, compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. The capacity of mitochondria to produce H2O2 increased on reperfusion. The mitochondrial content of cardiolipin, which is required for optimal activity of complex I, decreased by 28% and 50% as function of ischemia and reperfusion, respectively. The lower complex I activity in mitochondria from reperfused rat heart could be completely restored to the level of normal heart by exogenous added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is proposed that the defect in complex I activity in ischemic/reperfused rat heart could be ascribed to a ROS-induced cardiolipin damage. These findings may provide an explanation for some of the factors responsible for myocardial reperfusion injury.

Regulation of Signal Transduction by Reactive Oxygen Species in the Cardiovascular System

Oxidative stress has long been implicated in cardiovascular disease, but more recently, the role of reactive oxygen species (ROS) in normal physiological signaling has been elucidated. Signaling pathways modulated by ROS are complex and compartmentalized, and we are only beginning to identify the molecular modifications of specific targets. Here, we review the current literature on ROS signaling in the cardiovascular system, focusing on the role of ROS in normal physiology and how dysregulation of signaling circuits contributes to cardiovascular diseases, including atherosclerosis, ischemia–reperfusion injury, cardiomyopathy, and heart failure. In particular, we consider how ROS modulate signaling pathways related to phenotypic modulation, migration and adhesion, contractility, proliferation and hypertrophy, angiogenesis, endoplasmic reticulum stress, apoptosis, and senescence. Understanding the specific targets of ROS may guide the development of the next generation of ROS-modifying therapies to reduce morbidity and mortality associated with oxidative stress.

Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress.

The use of dichlorofluorescin (DCFH) as a measure of reactive oxygen species was studied in aqueous media. Hydrogen peroxide oxidized DCFH to fluorescent dichlorofluorescein (DCF), and the oxidation was amplified by the addition of ferrous iron. Hydrogen peroxide-induced DCF formation in the presence of ferrous iron was completely inhibited by deferoxamine and partially inhibited by ethylenediaminetetraacetic acid, but was augmented by diethylenetriaminepentaacetic acid. Iron-peroxide-induced oxidation of DCFH was partially inhibited by catalase but not by horseradish peroxidase. Nonchelated iron-peroxide oxidation of DCFH was partially inhibited by several hydroxyl radical scavengers, but was independent of the scavenger concentration, and this suggests that free hydroxyl radical is not involved in the oxidation of DCFH in this system. Superoxide anion did not directly oxidize DCFH. Data suggest that H2O2-Fe(2+)-derived oxidant is mainly responsible for the nonenzymatic oxidation of DCFH. In addition, peroxidase alone and oxidants formed during the reduction of H2O2 by peroxidase oxidize DCFH. Since DCFH oxidation may be derived from several reactive intermediates, interpretation of specific reactive oxygen species involved in biological systems should be approached with caution. However, DCFH remains an attractive probe as an overall index of oxidative stress in toxicological phenomena.

Reactive Oxygen Species, Oxidative Damage, and Antioxidative Defense Mechanism in Plants under Stressful Conditions

Reactive oxygen species (ROS) are produced as a normal product of plant cellular metabolism. Various environmental stresses lead to excessive production of ROS causing progressive oxidative damage and ultimately cell death. Despite their destructive activity, they are well-described second messengers in a variety of cellular processes, including conferment of tolerance to various environmental stresses. Whether ROS would serve as signaling molecules or could cause oxidative damage to the tissues depends on the delicate equilibrium between ROS production, and their scavenging. Efficient scavenging of ROS produced during various environmental stresses requires the action of several nonenzymatic as well as enzymatic antioxidants present in the tissues. In this paper, we describe the generation, sites of production and role of ROS as messenger molecules as well as inducers of oxidative damage. Further, the antioxidative defense mechanisms operating in the cells for scavenging of ROS overproduced under various stressful conditions of the environment have been discussed in detail.