Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species

In mammalian cells, reactive oxygen species (ROS) are produced via a variety of cellular oxidative processes, including the activity of NADPH oxidases (NOX), the activity of xanthine oxidases, the metabolism of arachidonic acid (AA) by lipoxygenases (LOX) and cyclooxygenases (COX), and the mitochondrial respiratory chain. Although NOX-generated ROS are the best characterized examples of ROS in mammalian cells, ROS are also generated by the oxidative metabolism (e.g., via LOX and COX) of AA that is released from the membrane phospholipids via the activity of cytosolic phospholipase A₂ (cPLA₂). Recently, growing evidence suggests that LOX- and COX-generated AA metabolites can induce ROS generation by stimulating NOX and that a potential signaling connection exits between the LOX/COX metabolites and NOX. In this review, we discuss the results of recent studies that report the generation of ROS by LOX metabolites, especially 5-LOX metabolites, via NOX stimulation. In particular, we have focused on the contribution of leukotriene B₄ (LTB₄), a potent bioactive eicosanoid that is derived from 5-LOX, and its receptors, BLT1 and BLT2, to NOX stimulation through a signaling mechanism that leads to ROS generation.     

Protein oxidation and cellular homeostasis: Emphasis on metabolism

Reactive oxygen species (ROS) are generated as the result of a number of physiological and pathological processes. Once formed ROS can promote multiple forms of oxidative damage, including protein oxidation, and thereby influence the function of a diverse array of cellular processes. This review summarizes the mechanisms by which ROS are generated in a variety of cell types, outlines the mechanisms which control the levels of ROS, and describes specific proteins which are common targets of ROS. Additionally, this review outlines cellular processes which can degrade or repair oxidized proteins, and ultimately describes the potential outcomes of protein oxidation on cellular homeostasis. In particular, this review focuses on the relationship between elevations in protein oxidation and multiple aspects of cellular metabolism. Together, this review describes a potential role for elevated levels of protein oxidation contributing to cellular dysfunction and oxidative stress via impacts on cellular metabolism.     

The roles of NADPH oxidase and phospholipases A2 in oxidative and inflammatory responses in neurodegenerative diseases

Reactive oxygen species (ROS) are produced in mammalian cells through enzymic and non-enzymic mechanisms. Although some ROS production pathways are needed for specific physiological functions, excessive production is detrimental and is regarded as the basis of numerous neurodegenerative diseases. Among enzymes producing superoxide anions, NADPH oxidase is widespread in mammalian cells and is an important source of ROS in mediating physiological and pathological processes in the cardiovascular and the CNS. ROS production is linked to the alteration of intracellular calcium homeostasis, activation of Ca(2+)-dependent enzymes, alteration of cytoskeletal proteins, and degradation of membrane glycerophospholipids. There is evolving evidence that ROS produced by NADPH oxidase regulate neuronal functions and degrade membrane phospholipids through activation of phospholipases A(2) (PLA(2)). This review is intended to cover recent studies describing ROS generation from NADPH oxidase in the CNS and its downstream activation of PLA(2), namely, the group IV cytosolic cPLA(2) and the group II secretory sPLA(2). A major focus is to elaborate the dual role of NADPH oxidase and PLA(2) in mediating the oxidative and inflammatory responses in neurodegenerative diseases, including cerebral ischemia and Alzheimer's disease. Elucidation of the signaling pathways linking NADPH oxidase with the multiple forms of PLA(2) will be important in understanding the oxidative and degradative mechanisms that underline neuronal damage and glial activation and will facilitate development of therapeutic intervention for prevention and treatment of these and other neurodegenerative diseases.     

Intraperoxisomal redox balance in mammalian cells: oxidative stress and interorganellar cross-talk

Reactive oxygen species (ROS) are at once unsought by-products of metabolism and critical regulators of multiple intracellular signaling cascades. In nonphotosynthetic eukaryotic cells, mitochondria are well-investigated major sites of ROS generation and related signal initiation. Peroxisomes are also capable of ROS generation, but their contribution to cellular oxidation-reduction (redox) balance and signaling events are far less well understood. In this study, we use a redox-sensitive variant of enhanced green fluorescent protein (roGFP2-PTS1) to monitor the state of the peroxisomal matrix in mammalian cells. We show that intraperoxisomal redox status is strongly influenced by environmental growth conditions. Furthermore, disturbances in peroxisomal redox balance, although not necessarily correlated with the age of the organelle, may trigger its degradation. We also demonstrate that the mitochondrial redox balance is perturbed in catalase-deficient cells and upon generation of excess ROS inside peroxisomes. Peroxisomes are found to resist oxidative stress generated elsewhere in the cell but are affected when the burden originates within the organelle. These results suggest a potential broader role for the peroxisome in cellular aging and the initiation of age-related degenerative disease.     

Involvement of cAMP-response element binding protein-1 in arachidonic acid-induced vascular smooth muscle cell motility

In addition to their role in many vital cellular functions, arachidonic acid (AA) and its eicosanoid metabolites are involved in the pathogenesis of several diseases, including atherosclerosis and cancer. To understand the potential mechanisms by which these lipid molecules could influence the disease processes, particularly cardiovascular diseases, we studied AA's effects on vascular smooth muscle cell (VSMC) motility and the role of cAMP-response element binding protein-1 (CREB-1) in this process. AA exerted differential effects on VSMC motility; at lower doses, it stimulated motility, whereas at higher doses, it was inhibitory. AA-induced VSMC motility requires its conversion via the lipoxygenase (LOX) and cyclooxygenase (COX) pathways. AA stimulated the phosphorylation of extracellular signal-regulated kinases (ERKs), Jun N-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38MAPK) in a time-dependent manner, and blockade of these serine/threonine kinases significantly attenuated AA-induced VSMC motility. In addition, AA stimulated CREB-1 phosphorylation and activity in a manner that was also dependent on its metabolic conversion via the LOX and COX pathways and the activation of ERKs and p38MAPK but not JNKs. Furthermore, suppression of CREB-1 activation inhibited AA-induced VSMC motility. 15(S)-Hydroxyeicosatetraenoic acid and prostaglandin F2alpha, the 15-LOX and COX metabolites of AA, respectively, that are produced by VSMC at lower doses, were also found to stimulate motility in these cells. Together, these results suggest that AA induces VSMC motility by complex mechanisms involving its metabolism via the LOX and COX pathways as well as the ERK- and p38MAPK-dependent and JNK-independent activation of CREB-1.     

Reactive oxygen species in cell signaling

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When cellular production of ROS overwhelms its antioxidant capacity, damage to cellular macromolecules such as lipids, protein, and DNA may ensue. Such a state of "oxidative stress" is thought to contribute to the pathogenesis of a number of human diseases including those of the lung. Recent studies have also implicated ROS that are generated by specialized plasma membrane oxidases in normal physiological signaling by growth factors and cytokines. In this review, we examine the evidence for ligand-induced generation of ROS, its cellular sources, and the signaling pathways that are activated. Emerging concepts on the mechanisms of signal transduction by ROS that involve alterations in cellular redox state and oxidative modifications of proteins are also discussed.     

Dual oxidase in the intestinal epithelium of zebrafish larvae has anti-bacterial properties

Reactive oxygen species (ROS) function in a range of physiological processes such as growth, metabolism and signaling, and also have a pathological role. Recent research highlighted the requirement for ROS generated by dual oxidase (DUOX) in host-defence responses in innate immunity and inflammatory disorders such as inflammatory bowel disease (IBD), but in vivo evidence to support this has, to date, been lacking. In order to investigate the involvement of Duox in gut immunity, we characterized the zebrafish ortholog of the human DUOX genes. Zebrafish duox is highly expressed in intestinal epithelial cells. Knockdown of Duox impaired larval capacity to control enteric Salmonella infection.     

Control of endothelial cell proliferation by calcium influx and arachidonic acid metabolism: a pharmacological approach

In physiological conditions, endothelial cell proliferation is strictly controlled by several growth factors, among which bFGF and VEGF are the most effective. Both bind to specific tyrosine kinase receptors and trigger intracellular signal cascades. In particular, bFGF stimulates the release of arachidonic acid (AA) and its metabolites in many types of endothelial cells in culture. In bovine aortic endothelial cells, it has been suggested that AA is released by the recruitment of cytosolic phospholipase A2 (cPLA2). AA metabolites are involved in the control of both endothelial cell motility (mostly via the cyclooxygenase pathway) and proliferation (via the lipoxygenase (LOX) cascade). On the other hand, evidence has been provided for a proliferative role of AA-induced calcium influx. By using a pharmacological approach, we have tried to elucidate the contribution to bovine aortic endothelial proliferation of the different pathways leading to production of AA and its metabolites. Two main informations were obtained by our experiments: first, AA release is not entirely due to cPLA2 involvement, but also to DAG lipase recruitment; second, cyclooxygenase derivatives play a role in the control of cell proliferation, and not only of motility. Moreover, by combining proliferation assays and single cell calcium measurements, we show that the blocking effect of carboxyamido-triazole (CAI), an inhibitor of tumor growth and angiogenesis acting on calcium influx-dependent pathways, including AA metabolism, is at least in part due to a direct effect on AA-induced calcium influx.     

Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha, although the events through which TNF-alpha induces ROS generation are not yet well characterized. We therefore investigated selected candidates likely to mediate TNF-alpha-induced ROS generation. Consistent with the role of Rac in that process, stable expression of Rac(Asn-17), a dominant negative Rac1 mutant, completely blocked TNF-alpha-induced ROS generation. To understand better the mediators downstream of Rac, we investigated the involvement of cytosolic phospholipase A(2) (cPLA(2)) activation and metabolism of the resultant arachidonic acid (AA) by 5-lipoxygenase (5-LO). TNF-alpha-induced ROS generation was blocked by inhibition of cPLA(2) or 5-LO, but not cyclooxygenase, suggesting that TNF-alpha-induced ROS generation is dependent on synthesis of AA and its subsequent metabolism to leukotrienes. Consistent with that hypothesis, TNF-alpha Rac-dependently stimulated endogenous production of leukotriene B(4) (LTB(4)), while exogenous application of LTB(4) increased levels of ROS. In contrast, application of leukotrienes C(4), D(4), and E(4) or prostaglandin E(2) had little effect. Our findings suggest that LTB(4) production by 5-LO is situated downstream of the Rac-cPLA(2) cascade, and we conclude that Rac, cPLA(2), and LTB(4) play pivotal roles in the ROS-generating cascade triggered by TNF-alpha.     

The roles of cellular reactive oxygen species,oxidative stress and antioxidants in pregnancy outcomes

Reactive oxygen species (ROS) are generated as by-products of aerobic respiration and metabolism. Mammalian cells have evolved a variety of enzymatic mechanisms to control ROS production, one of the central elements in signal transduction pathways involved in cell proliferation, differentiation and apoptosis. Antioxidants also ensure defenses against ROS-induced damage to lipids, proteins and DNA. ROS and antioxidants have been implicated in the regulation of reproductive processes in both animal and human, such as cyclic luteal and endometrial changes, follicular development, ovulation, fertilization, embryogenesis, embryonic implantation, and placental differentiation and growth. In contrast, imbalances between ROS production and antioxidant systems induce oxidative stress that negatively impacts reproductive processes. High levels of ROS during embryonic, fetal and placental development are a feature of pregnancy. Consequently, oxidative stress has emerged as a likely promoter of several pregnancy-related disorders, such as spontaneous abortions, embryopathies, preeclampsia, fetal growth restriction, preterm labor and low birth weight. Nutritional and environmental factors may contribute to such adverse pregnancy outcomes and increase the susceptibility of offspring to disease. This occurs, at least in part, via impairment of the antioxidant defense systems and enhancement of ROS generation which alters cellular signalling and/or damage cellular macromolecules. The links between oxidative stress, the female reproductive system and development of adverse pregnancy outcomes, constitute important issues in human and animal reproductive medicine. This review summarizes the role of ROS in female reproductive processes and the state of knowledge on the association between ROS, oxidative stress, antioxidants and pregnancy outcomes in different mammalian species.     

Oxidative stress in plant cell culture: a role in production of beta-thujaplicin by Cupresssus lusitanica suspension culture

Oxidative stress is a common physiological stress that often challenges plants. Reactive oxygen species (ROS) are major factors in oxidative stress that significantly affect plant cell growth and secondary metabolism. Here we used beta-thujaplicin production by Cupressus lusitanica cell culture as an example to demonstrate the common occurrence of oxidative stress in cultivated plant cells and its effect on multiple aspects of cell culture process. C. lusitanica cells cultivated under Fe(2+) stress generate a significant level of ROS, and oxidative stress also occurs at late stages of C. lusitanica cell cultures under normal conditions. ROS production inhibited cell growth, induced lipid peroxidation and cell death, and enhanced ethylene and beta-thujaplicin production. It is demonstrated that Fe(2+) stress enhances ROS production via the Fenton reaction and promotes beta-thujaplicin production via ROS-induced lipid peroxidation that may activate cyclic oxylipin and ethylene pathways. Results further indicate that H(2)O(2) is a positive signal for beta-thujaplicin production, whereas superoxide anion radical (O(2) (- )) negatively affects beta-thujaplicin induction and strongly induces cell death. The study suggests that evaluating the oxidative stress and plant responses in a cell culture process is very necessary and important for understanding biochemical processes and for gaining the maximal productivity of target secondary metabolites.     

Determination of mitochondrial reactive oxygen species: methodological aspects

The generation of Reactive Oxygen Species (ROS) as by-products in mitochondria Electron Transport Chain (ETC) has long been admitted as the cost of aerobic energy metabolism with oxidative damages as consequence. The purpose of this methodological review is to present some of the most widespread methods of ROS generation and to underline the limitations as well as some problems, identified with some experiments as examples, in the interpretation of such results. There is now no doubt that besides their pejorative role, ROS are involved in a variety of cellular processes for the continuous adaptation of the cell to its environment. Because ROS metabolism is a complex area (low production, instability of species, efficient antioxidant defense system, several places of production…) bias, variances and limitations in ROS measurements must be recognized in order to avoid artefactual conclusions, and especially to improve our understanding of physiological and pathophysiological mechanisms of such phenomenon.     

NOX isoforms and reactive oxygen species in vascular health

Reactive oxygen species (ROS) are important mediators of cell growth, adhesion, differentiation, migration, senescence, and apoptosis. ROS play an important physiological role in regulating vascular tone and can also contribute to pathological mechanisms related to endothelial dysfunction, vascular reactivity, arterial remodeling, and vascular inflammation. The major source of ROS generated in the cardiovascular system is the NADPH oxidase (NOX) family of enzymes, of which seven members have been characterized. Although each NOX family member is typified by six transmembrane domains along with a cytoplasmic domain that binds NADPH and FAD, each isoform is distinguished by the specific catalytic subunit, interacting proteins, and subcellular localization. We review the current understanding of NOX signaling and regulatory mechanisms related to vascular health and disease.     

Regulation of intracellular signalling through cysteine oxidation by reactive oxygen species

Reactive oxygen species (ROS) have been regarded as harmful molecules that damage various molecules inside cells by oxidation and are responsible for ageing and various human diseases. However, recent studies have revealed an opposite aspect of ROS that these are actively generated in cells and mediate physiological intracellular signalling as second messengers. Several proteins have been shown to function as effectors for ROS, which are sensitively and reversibly oxidized by ROS. Such ROS-effector proteins commonly possess a highly reactive cysteine (Cys) residue, of which oxidation changes the protein function, thus enabling signal transmission to downstream targets. Among the ROS effectors, protein tyrosine phosphatase (PTP), thioredoxin (TRX) and peroxiredoxin (PRX) family proteins possess special domains/motifs to maintain the reactivity of Cys and utilize them to respond to ROS. Progressively advancing identification of ROS-effector proteins reveals the pleiotropic functions of ROS in physiological and pathological cell biology.     

ROS signaling: the new wave?

Reactive oxygen species (ROS) play a multitude of signaling roles in different organisms from bacteria to mammalian cells. They were initially thought to be toxic byproducts of aerobic metabolism, but have now been acknowledged as central players in the complex signaling network of cells. In this review, we will attempt to address several key questions related to the use of ROS as signaling molecules in cells, including the dynamics and specificity of ROS signaling, networking of ROS with other signaling pathways, ROS signaling within and across different cells, ROS waves and the evolution of the ROS gene network.     

Role of mitochondria in reactive oxygen species generation and removal; relevance to signaling and programmed cell death

Reactive oxygen species (ROS) are universal products of aerobic metabolism, which can be also produced in stress conditions. In eukaryotic cells, mitochondria are the main source of ROS. The main mitochondrial sites of ROS formation are electron carriers of respiratory chain. However, there are also other enzymatic sites capable of ROS generation in different mitochondrial compartments. Reactive oxygen species can cause serious damage to many biological macromolecules, such as proteins, lipids and nucleic acids, which oxidation leads to a lost of their biological properties and eventually to a cell death. Mitochondria, which are also exposed to harmful ROS action, have a defense system that decreases ROS production (first line of defense) or removes generated ROS (second line of defense). Mitochondrial antioxidant system involves proteins that decrease ROS formation, enzymes that directly react with ROS, and non-enzymatic antioxidants that also remove ROS and other oxygen derivatives. Mitochondrial ROS can also act as signal messengers and modify operation of many routes in different cell compartments. Mitochondrial ROS are also important in execution of programmed cell death.     

Cellular redox regulation,signaling, and stress response in plants

Cellular and organellar redox states, which are characterized by the balance between oxidant and antioxidant pool sizes, play signaling roles in the regulation of gene expression and protein function in a wide variety of plant physiological processes including stress acclimation. Reactive oxygen species (ROS) and ascorbic acid (AsA) are the most abundant oxidants and antioxidants, respectively, in plant cells; therefore, the metabolism of these redox compounds must be strictly and spatiotemporally controlled. In this review, we provided an overview of our previous studies as well as recent advances in (1) the molecular mechanisms and regulation of AsA biosynthesis, (2) the molecular and genetic properties of ascorbate peroxidases, and (3) stress acclimation via ROS-derived oxidative/redox signaling pathways, and discussed future perspectives in this field.     

Oxidative stress response of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. (PCC 6803) due to exposure to microcystin-LR and cell-free cyanobacterial crude extract containing microcystin-LR

Cyanobacteria are able to produce a variety of secondary metabolites such as the microcystins. The ecological role of microcystins for the cyanobacteria themselves and in the aquatic ecosystem is not well understood. The aim of this study is to evaluate if microcystins might be used as a communication tool for interspecies cyanobacterial communication via the promotion of oxidative stress. Reactive oxygen species (ROS) are known to be used as plant signals. The main questions relate to the promotion of oxidative stress in Synechocystis sp. via exposure to the cyanobacterial toxins and the physiological effects. This study shows a few markers for oxidative stress, such as the antioxidative enzymes superoxide dismutase, peroxidases and catalases, and cell damage due to extensive generation of ROS leading to lipid peroxidation. End products of lipid peroxidation (malonaldehyde and 4-hydroxynonenal) are conjugated by glutathione S-transferase.     

A newly adapted pulsed-field gel electrophoresis technique allows to detect distinct types of DNA damage at low frequencies in human dermal fibroblasts upon exposure to non-toxic H2O2 concentrations

Reactive oxygen species (ROS) comprise several oxygen containing compounds, among them hydrogen peroxide (H₂O₂), which are generated by internal and external sources and play pleiotropic roles in physiological and pathological states. Skin cells as well as cells from other tissues have developed antioxidant defense mechanisms to protect themselves from high concentrations of ROS. Although biological and pathological roles of ROS have previously been elucidated, so far only limited knowledge exists regarding ROS-mediated generation of DNA breaks and base lesions occurring at low frequency in intact skin cells. This study was therefore designed to probe a newly adapted pulsed-field gel electrophoresis technique for the adequate measurement of high molecular weight DNA fragments as well as to investigate the protective role of the antioxidant enzyme catalase against H₂O₂-mediated damage in human dermal fibroblasts. We stably transfected and overexpressed the full-length catalase cDNA in the human dermal fibroblast cell line 1306 in culture and found that these cells are significantly more protected from cytotoxicity, overall DNA strand breaks, and 8-oxodeoxyguanine base lesions resulting from H₂O₂-triggered oxidative stress compared to vector-transfected 1306 cells or secondary dermal fibroblasts. This work has outlined the importance of catalase in the protection from H₂O₂-mediated cytotoxicity and DNA damage which — if unbalanced — even when occurring at low frequency are known to lead to genomic instability, a hallmark in carcinogenesis and premature aging.