Oxidized LDL induces mitochondrially associated reactive oxygen/nitrogen species formation in endothelial cells

Exposure of cells to complex mixtures of oxidized lipids such as those found in oxidized low-density lipoprotein (oxLDL) induce reactive oxygen and nitrogen species (ROS/RNS) formation. The source of the ROS/RNS within cells is unknown; it is thought they may be involved in redox cell signaling. Although this possibility was initially overlooked, it is becoming clear that mitochondria, which are a source of superoxide and hydrogen peroxide, may play a critical role in the response of cells on exposure to oxidized lipids. In this study, we tested the possibility that mitochondria are a potential source of oxLDL-dependent formation of ROS/RNS in endothelial cells. Using confocal microscopy, we demonstrated that a significant proportion of oxLDL-dependent dichlorodihydrofluorescein (DCF) fluorescence is colocalized to mitochondria. In support of this concept, rho0 endothelial cells showed a substantial decrease in ROS/RNS formation stimulated by oxLDL. In contrast, mostly nonmitochondrial DCF fluorescence was detected in cells exposed to an extracellular source of hydrogen peroxide. The exposure of cells to a nitric oxide synthase inhibitor and urate resulted in a decrease in oxLDL-induced DCF fluorescence that was restored by addition of nitric oxide donors to the medium. Taken together, these results suggest that oxLDL-dependent DCF fluorescence is mitochondrially associated and may be due to the formation of peroxynitrite.     

Mass spectrometric characterization of the oxidation of the fluorescent lipid peroxidation reporter molecule C11-BODIPY(581/591)

C11-BODIPY^581/591 is a fluorescent lipid peroxidation reporter molecule that shifts its fluorescence from red to green when challenged with oxidizing agents, i.e., reactive oxygen species (ROS) or reactive nitrogen species (RNS). To understand the molecular mechanism responsible for this shift, we studied the molecular rearrangements leading to the shift in fluorescence in C11-BODIPY^581/591. Furthermore, we aimed to determine if these rearrangements were dependent on the nature of the applied ROS, in homogenous solution, bilayer vesicles, and living cells. C11-BODIPY^581/591 was challenged with various ROS- or RNS-generating systems, including peroxynitrite, NO₂^√, peroxides, and hydroxyl, alkoxyl, tyrosyl, and peroxyl radicals. The reaction products were subsequently analyzed by means of mass spectrometry. Our results show that the initial target for free radical-mediated oxidation is the conjugated diene interconnection between the BODIPY core and the terminal phenyl moiety, which already explains the shift in fluorescence properties of the probe. After oxidative challenge, three different stable products were identified, one of which was specific for oxidation by peroxynitrite. The two other stable end products had lost the entire phenyl moiety, irrespective of the type of radical generating system used. These products were also recovered from Rat-1 fibroblasts stressed either by GSH depletion/serum starvation or by exposure to peroxynitrite, and were the only C11-BODIPY^581/591 oxidation products detectable in these cells.     

Unravelling how plants benefit from ROS and NO reactions,while resisting oxidative stress

Background and Aims Reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as nitric oxide (NO), play crucial roles in the signal transduction pathways that regulate plant growth, development and defence responses, providing a nexus of reduction/oxidation (redox) control that impacts on nearly every aspect of plant biology. Here we summarize current knowledge and concepts that lay the foundations of a new vision for ROS/RNS functions – particularly through signalling hubs – for the next decade.Scope Plants have mastered the art of redox control using ROS and RNS as secondary messengers to regulate a diverse range of protein functions through redox-based, post-translational modifications that act as regulators of molecular master-switches. Much current focus concerns the impact of this regulation on local and systemic signalling pathways, as well as understanding how such reactive molecules can be effectively used in the control of plant growth and stress responses.Conclusions The spectre of oxidative stress still overshadows much of our current philosophy and understanding of ROS and RNS functions. While many questions remain to be addressed – for example regarding inter-organellar regulation and communication, the control of hypoxia and how ROS/RNS signalling is used in plant cells, not only to trigger acclimation responses but also to create molecular memories of stress – it is clear that ROS and RNS function as vital signals of living cells.     

氧化应激与幽门螺杆菌感染相关性胃炎

幽门螺杆菌(Helicobacter pylori,H.pylori)是一种选择性定植于胃上皮细胞的革兰氏阴性菌,是一种广泛传染的病原菌,也是诱导产生慢性胃炎的主要因素之一。近年来研究表明幽门螺杆菌感染诱导机体产生氧化应激反应,并通过各种逃逸机制避免被杀灭。幽门螺杆菌能不断刺激中性粒细胞和巨噬细胞表达活性氧和活性氮,导致体内活性氧和活性氮的过度积累,致使细胞的凋亡和胃粘膜损伤的加剧,这是导致胃炎发生及加重的重要因素。本文对幽门螺杆感染引起氧化应激反应的研究进展作简要综述。     

Redox signalling: from nitric oxide to oxidized lipids

Cellular redox signalling is mediated by the post-translational modification of proteins in signal-transduction pathways by ROS/RNS (reactive oxygen species/reactive nitrogen species) or the products derived from their reactions. NO is perhaps the best understood in this regard with two important modifications of proteins known to induce conformational changes leading to modulation of function. The first is the addition of NO to haem groups as shown for soluble guanylate cyclase and the newly discovered NO/cytochrome c oxidase signalling pathway in mitochondria. The second mechanism is through the modification of thiols by NO to form an S-nitrosated species. Other ROS/RNS can also modify signalling proteins although the mechanisms are not as clearly defined. For example, electrophilic lipids, formed as the reaction products of oxidation reactions, orchestrate adaptive responses in the vasculature by reacting with nucleophilic cysteine residues. In modifying signalling proteins ROS/RNS appear to change the overall activity of signalling pathways in a process that we have termed 'redox tone'. In this review, we discuss these different mechanisms of redox cell signalling, and give specific examples of ROS/RNS participation in signal transduction.     

Dynamic determination of Ox-LDL-induced oxidative/nitrosative stress in single macrophage by using fluorescent probes

Increased oxidative/nitrosative stress, resulting from generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears to play an important role in the inflammatory responses to atherosclerosis. By using MitoTracker Orange CM-H(2)TMRos, CM-H(2)DCFDA (DCF-DA), Dihydrorhodamine 123 (DHR123), DAF-FM, Dihydroethidium (DHE) and JC-1 alone or in all combinations of red and green probes, the present study was designed to monitor the ROS and RNS generation in acute exposure of single monocyte U937-derived macrophage to oxidized low density lipoprotein (Ox-LDL). Acute Ox-LDL (100 microg/ml) treatment increased time-dependently production of intracellular nitric oxide (NO), superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)), and decreased mitochondrial membrane potential (Deltapsi) in single cell. Pretreatment of aminoguanidine (an inhibitor of inducible nitric oxide synthase (iNOS), 10 microM) and vitamin C (an antioxidant agent, 100 microM) for 2h, reduced significantly the Ox-LDL-induced increase of NO and O2*-, and vitamin C completely inhibited increase of intracellular NO and O2*-. In contrast to aminoguanidine, Vitamin C pretreatment significantly prevented Ox-LDL-induced overproduction of NO and O2*- (P<0.01), indicating that antioxidant may be more effective in therapeutic application than iNOS inhibitor in dysfunction of ROS/RNS. By demonstrating a complex imbalance of ROS/RNS via fluorescent probes in acute exposure of single cell to Ox-LDL, oxidative/nitrosative stress might be more detected in the early atherosclerotic lesions.     

Epithelial,dendritic, and CD4 T cell regulation of and by reactive oxygen and nitrogen species in allergic sensitization

Background

While many of the contributing cell types and mediators of allergic asthma are known, less well understood are the factors that induce allergy in the first place. Amongst the mediators speculated to affect initial allergen sensitization and the development of pathogenic allergic responses to innocuous inhaled antigens and allergens are exogenously or endogenously generated reactive oxygen species (ROS) and reactive nitrogen species (RNS).

Scope of review

The interactions between ROS/RNS, dendritic cells (DCs), and CD4⁺ T cells, as well as their modulation by lung epithelium, are of critical importance for the genesis of allergies that later manifest in allergic asthma. Therefore, this review will primarily focus on the initiation of pulmonary allergies and the role that ROS/RNS may play in the steps therein, using examples from our own work on the roles of NO₂ exposure and airway epithelial NF-κB activation.

Major conclusions

Endogenously generated ROS/RNS and those encountered from environmental sources interact with epithelium, DCs, and CD4⁺ T cells to orchestrate allergic sensitization through modulation of the activities of each of these cell types, which quantitiatively and qualitatively dictate the degree and type of the allergic asthma phenotype.

General significance

Knowledge of the effects of ROS/RNS at the molecular and cellular levels has the potential to provide powerful insight into the balance between inhalational tolerance (the typical immunologic response to an innocuous inhaled antigen) and allergy, as well as to potentially provide mechanistic targets for the prevention and treatment of asthma.     

Determination of reactive oxygen and nitrogen species in rat aorta using the dichlorofluorescein assay

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH₂) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH₂-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.     

Influence of vitamin C and vitamin E on redox signaling: Implications for exercise adaptations

The exogenous antioxidants vitamin C (ascorbate) and vitamin E (α-tocopherol) often blunt favorable cell signaling responses to exercise, suggesting that redox signaling contributes to exercise adaptations. Current theories posit that this antioxidant paradigm interferes with redox signaling by attenuating exercise-induced reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. The well-documented in vitro antioxidant actions of ascorbate and α-tocopherol and characterization of the type and source of the ROS/RNS produced during exercise theoretically enable identification of redox-dependent mechanisms responsible for the blunting of favorable cell signaling responses to exercise. This review aimed to apply this reasoning to determine how the aforementioned antioxidants might attenuate exercise-induced ROS/RNS production. The principal outcomes of this analysis are (1) neither antioxidant is likely to attenuate nitric oxide signaling either directly (reaction with nitric oxide) or indirectly (reaction with derivatives, e.g., peroxynitrite); (2) neither antioxidant reacts appreciably with hydrogen peroxide, a key effector of redox signaling; (3) ascorbate but not α-tocopherol has the capacity to attenuate exercise-induced superoxide generation; and (4) alternate mechanisms, namely pro-oxidant side reactions and/or reduction of bioactive oxidized macromolecule adducts, are unlikely to interfere with exercise-induced redox signaling. Out of all the possibilities considered, ascorbate-mediated suppression of superoxide generation with attendant implications for hydrogen peroxide signaling is arguably the most cogent explanation for blunting of favorable cell signaling responses to exercise. However, this mechanism is dependent on ascorbate accumulating at sites rich in NADPH oxidases, principal contributors to contraction-mediated superoxide generation, and outcompeting nitric oxide and superoxide dismutase isoforms. The major conclusions of this review are: (1) direct evidence for interference of ascorbate and α-tocopherol with exercise-induced ROS/RNS production is lacking; (2) theoretical analysis reveals that both antioxidants are unlikely to have a major impact on exercise-induced redox signaling; and (3) it is worth considering alternate redox-independent mechanisms.     

An investigation of the genotoxic effects of N-nitrosomorpholine in mammalian cells

N-nitrosomorpholine (NMOR) is a well-known hepatocarcinogen. Since this compound is representative of the group of indirect-acting N-nitrosamines, its metabolic activation should be essential. However, the mechanism of NMOR-induced carcinogenesis is still not completely clear. In this paper we tried to further our understanding of the genotoxic effects of NMOR. The central aim of this study was to elucidate to what extent NMOR requires metabolic activation. For evaluation of the mutagenicity of NMOR, V79 cells were used either in the presence or absence of the microsomal S9 fraction in the mutation assay and formation of reactive oxygen/nitrogen species (ROS/RNS) in Caco-2 cells treated with NMOR was measured by a fluorescent assay. A very weak rise of 6-thioguanine resistant mutations was observed in both NMOR-treated model cells, V79/-S9 and V79/+S9. A significant difference between the level of mutations in V79/-S9 and V79/+S9 cells was recorded on the 7th day of expression only. Data obtained by the fluorescent assay confirmed that NMOR caused generation of ROS/RNS. In summary, the presented results showed that NMOR might induce DNA damage not only indirectly by its activation by drug-metabolizing enzymes but also via direct formation of ROS/RNS.     

In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012

Production of reactive oxygen and nitrogen species (ROS/RNS) is an important part of the inflammatory response, but prolonged elevated levels of ROS/RNS as under chronic inflammation can contribute to the development of disease. Monitoring ROS/RNS in living animals is challenging due to the rapid turnover of ROS/RNS and the limited sensitivity and specificity of ROS/RNS probes. We have explored the use of the chemiluminescent probe L-012 for noninvasive imaging of ROS/RNS production during inflammation in living mice. Various inflammatory conditions were induced, and L-012-dependent luminescence was recorded with an ultrasensitive CCD camera. Strong luminescent signals were observed from different regions of the body corresponding to inflammation. The signal was reduced by administration of the SOD mimetic tempol, the NADPH oxidase inhibitor apocynin, and the inhibitor of nitric oxide synthesis L-NAME, signifying the requirement for the presence of ROS/RNS. Additionally, the L-012 signal was abolished in mice with a mutation in the Ncf1 gene, encoding a protein in the NADPH oxidase complex 2, which generates ROS/RNS during inflammation. In conclusion, L-012 is well distributed in the mouse body and mediates a strong ROS/RNS-dependent luminescent signal in vivo and is useful for monitoring the development and regulation of inflammation in living organisms.     

Quantitative Assessment of Protein-bound Tyrosine Nitration in Airway Secretions from Patients with Inflammatory Airway Disease

Because reactive nitrogen species (RNS) have potent inflammatory activity, they may be involved in the inflammatory process in pulmonary diseases. We recently reported increased numbers of 3-nitrotyrosine immunopositive cells, which are evidences of RNS production, in the sputum of patients with chronic obstructive pulmonary disease (COPD) and patients with asthma compared with healthy subjects. In the present study, we attempted to quantify this protein nitration in the airways by means of high-performance liquid chromatography (HPLC) used together with an electrochemical detection system that we developed. Sputum samples were obtained from 15 stable COPD patients, 9 asthmatic patients and 7 healthy subjects by using hypertonic saline inhalation. The values for the molar ratio of protein-bound 3-nitrotyrosine/tyrosine in patients with asthma (4.31±1.13?×?10^-6, p<0.05) and patients with COPD (3.04±0.36?×?10^-6, p<0.01) were significantly higher than those in healthy subjects (1.37±0.19?×?10^-6). The levels of protein-bound 3-nitrotyrosine in the airways were not significantly different in asthmatic patients and COPD patients. A significant negative correlation was found between values for protein-bound 3-nitrotyrosine/tyrosine and % FEV¹ values in patients with COPD (r=-0.53, p<0.05) but not in patients with asthma. These results suggest that our HPLC-electrochemical method is useful for quantifying RNS production in human airways. More importantly, they show that increased RNS production in the airways seems to contribute in a critical way to the pathogenesis of COPD, and that the effects of RNS in airways may differ in asthma and COPD.     

D-Pinitol treatment induced the apoptosis in human leukemia MOLT-4 cells by improved apoptotic signaling pathway

Cancer is still remain as a global burden with the 18.1 million and 9.6 million new cases and mortlities, respectively estimated globally. Leukemia may arise at all ages varied from the infants to elders. In this exploration, we planned to evaluate the antiproliferative effect of D-pinitol on human leukemia MOLT-4 cells. Anticancer potential of D-pinitol was examined using MTT assay. Reactive oxygen species (ROS) generation was studied by fluorescence microscopic method using DCFH-DA staining. Apoptotic morphological alterations were determined by dual staining (acridine orange and ethidium bromide). Western blot and ELISA methods were employed to study apoptotic protein expression. D-pinitol treatment significantly induced cytotoxicity in human leukemia MOLT-4 cells. We observed that D-pinitol induces the generation of ROS in MOLT-4 cells. Further, we noticed that D-pinitol significantly induced apoptosis in a dosage dependent manner. Moreover, western blot and ELISA based analysis revealed that D-pinitol elevated the Bax, Caspase-3, Caspase-9 and attenuated the Bcl-2 expression in leukemic cancer cell. Our findings suggest that D-pinitol treatment induces the apoptosis in human leukemic cells by generating intracellular ROS and modulating apoptotic protein expression.     

Analysis of hydroxylation and nitration products of d-phenylalanine for in vitro and in vivo radical determination using high-performance liquid chromatography and photodiode array detection

d-Phenylalanine is capable of trapping reactive oxygen species (ROS) and reactive nitrogen species (RNS) by forming three major hydroxylation (o-, m-, p-tyrosine) and two major nitration products (nitrophenylalanine, nitrotyrosine). Here, we show how a method for the analysis of these phenylalanine derivatives was established using isocratic HPLC (Nucleosil120, C18 column) coupled with photodiode array detection and validated for cell-free in vitro and in vivo determination of radical formation. An ideal separation was achieved using a mobile phase consisting of 5% acetonitrile, 50 mM KH₂PO₄, pH 3.0, a column temperature of 35 °C and a flow rate of 1.0 mL/min. Limits of detection were in the range of 5–100 nM. Linearity was given within 5 nM–100 μM (correlation coefficient >0.999). Retention times as well as peak heights exhibited a high precision (RSD: ≤0.1% and <1.5%, respectively). The feasibility of d-phenylalanine for ROS/RNS measurement was demonstrated in a cell-free in vitro assay using peroxynitrite and by analysis of brain samples of mice treated with the dopaminergic neurotoxin 6-hydroxydopamine.     

Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.     

Reactive oxygen and nitrogen species are involved in sorbitol-induced apoptosis of human erithroleukaemia cells K562

In this study, we found that production of both reactive oxygen (ROS) and nitrogen (RNS) species is a very early event related to treatment with hyperosmotic concentration of sorbitol. The production of nitric oxide (NO) was paralleled by the increase of the mRNA and protein level of the inducible form of the nitric oxide synthase (iNOS). ROS and RNS enhancement, process concomitant to the failure of mitochondrial trans-membrane potential (ΔΨ), was necessary for the induction of apoptosis as demonstrated by the protection against sorbitol-mediated toxicity observed after treatment with ROS scavengers or NOS inhibitors. The synergistic action of ROS and RNS was finally demonstrated by pre-treatment with rosmarinic acid that, by powerfully buffering both these species, prevents impairment of ΔΨ and cell death. Overall results suggest that the occurrence of apoptosis upon sorbitol treatment is an event mediated by oxidative/nitrosative stress rather than a canonical hyperosmotic shock.     

Oxidative and Nitrosative Modifications of Tropoelastin Prevent Elastic Fiber Assembly in Vitro

Elastic fibers are extracellular structures that provide stretch and recoil properties of tissues, such as lungs, arteries, and skin. Elastin is the predominant component of elastic fibers. Tropoelastin (TE), the precursor of elastin, is synthesized mainly during late fetal and early postnatal stages. The turnover of elastin in normal adult tissues is minimal. However, in several pathological conditions often associated with inflammation and oxidative stress, elastogenesis is re-initiated, but newly synthesized elastic fibers appear abnormal. We sought to determine the effects of reactive oxygen and nitrogen species (ROS/RNS) on the assembly of TE into elastic fibers. Immunoblot analyses showed that TE is oxidatively and nitrosatively modified by peroxynitrite (ONOO⁻) and hypochlorous acid (HOCl) and by activated monocytes and macrophages via release of ONOO⁻ and HOCl. In an in vitro elastic fiber assembly model, oxidatively modified TE was unable to form elastic fibers. Oxidation of TE enhanced coacervation, an early step in elastic fiber assembly, but reduced cross-linking and interactions with other proteins required for elastic fiber assembly, including fibulin-4, fibulin-5, and fibrillin-2. These findings establish that ROS/RNS can modify TE and that these modifications affect the assembly of elastic fibers. Thus, we speculate that oxidative stress may contribute to the abnormal structure and function of elastic fibers in pathological conditions.