The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA,Lipids and Proteins) and Induced Pathologies

Living species are continuously subjected to all extrinsic forms of reactive oxidants and others that are produced endogenously. There is extensive literature on the generation and effects of reactive oxygen species (ROS) in biological processes, both in terms of alteration and their role in cellular signaling and regulatory pathways. Cells produce ROS as a controlled physiological process, but increasing ROS becomes pathological and leads to oxidative stress and disease. The induction of oxidative stress is an imbalance between the production of radical species and the antioxidant defense systems, which can cause damage to cellular biomolecules, including lipids, proteins and DNA. Cellular and biochemical experiments have been complemented in various ways to explain the biological chemistry of ROS oxidants. However, it is often unclear how this translates into chemical reactions involving redox changes. This review addresses this question and includes a robust mechanistic explanation of the chemical reactions of ROS and oxidative stress.     

Mitochondrial Oxidative Stress—A Causative Factor and Therapeutic Target in Many Diseases

The excessive formation of reactive oxygen species (ROS) and impairment of defensive antioxidant systems leads to a condition known as oxidative stress. The main source of free radicals responsible for oxidative stress is mitochondrial respiration. The deleterious effects of ROS on cellular biomolecules, including DNA, is a well-known phenomenon that can disrupt mitochondrial function and contribute to cellular damage and death, and the subsequent development of various disease processes. In this review, we summarize the most important findings that implicated mitochondrial oxidative stress in a wide variety of pathologies from Alzheimer disease (AD) to autoimmune type 1 diabetes. This review also discusses attempts to affect oxidative stress as a therapeutic avenue.     

Friend or Foe: The Relativity of (Anti)oxidative Agents and Pathways

An element, iron, a process, the generation of reactive oxygen species (ROS), and a molecule, ascorbate, were chosen in our study to show their dual functions and their role in cell fate decision. Iron is a critical component of numerous proteins involved in metabolism and detoxification. On the other hand, excessive amounts of free iron in the presence of oxygen can promote the production of potentially toxic ROS. They can result in persistent oxidative stress, which in turn can lead to damage and cell death. At the same time, ROS—at strictly regulated levels—are essential to maintaining the redox homeostasis, and they are engaged in many cellular signaling pathways, so their total elimination is not expedient. Ascorbate establishes a special link between ROS generation/elimination and cell death. At low concentrations, it behaves as an excellent antioxidant and has an important role in ROS elimination. However, at high concentrations, in the presence of transition metals such as iron, it drives the generation of ROS. In the term of the dual function of these molecules and oxidative stress, ascorbate/ROS-driven cell deaths are not necessarily harmful processes—they can be live-savers too.     

Physico-Chemical Characterization and Oxidative Reactivity Evaluation of Aged Brake Wear Particles

Brake wear dust is a significant component of traffic emissions and has been linked to adverse health effects. Previous research found a strong oxidative stress response in cells exposed to freshly generated brake wear dust. We characterized aged dust collected from passenger vehicles, using microscopy and elemental analyses. Reactive oxygen species (ROS) generation was measured with acellular and cellular assays using 2′7-dichlorodihydrofluorescein dye. Microscopy analyses revealed samples to be heterogeneous particle mixtures with few nanoparticles detected. Several metals, primarily iron and copper, were identified. High oxygen concentrations suggested that the elements were oxidized. ROS were detected in the cell-free fluorescent test, while exposed cells were not dramatically activated by the concentrations used. The fact that aged brake wear samples have lower oxidative stress potential than fresh ones may relate to the highly oxidized or aged state of these particles, as well as their larger size and smaller reactive surface area.

Copyright 2015 American Association for Aerosol Research     

Micro RNAs in Regulation of Cellular Redox Homeostasis

In living cells Reactive Oxygen Species (ROS) participate in intra- and inter-cellular signaling and all cells contain specific systems that guard redox homeostasis. These systems contain both enzymes which may produce ROS such as NADPH-dependent and other oxidases or nitric oxide synthases, and ROS-neutralizing enzymes such as catalase, peroxiredoxins, thioredoxins, thioredoxin reductases, glutathione reductases, and many others. Most of the genes coding for these enzymes contain sequences targeted by micro RNAs (miRNAs), which are components of RNA-induced silencing complexes and play important roles in inhibiting translation of their targeted messenger RNAs (mRNAs). In this review we describe miRNAs that directly target and can influence enzymes responsible for scavenging of ROS and their possible role in cellular redox homeostasis. Regulation of antioxidant enzymes aims to adjust cells to survive in unstable oxidative environments; however, sometimes seemingly paradoxical phenomena appear where oxidative stress induces an increase in the levels of miRNAs which target genes which are supposed to neutralize ROS and therefore would be expected to decrease antioxidant levels. Here we show examples of such cellular behaviors and discuss the possible roles of miRNAs in redox regulatory circuits and further cell responses to stress.     

Oxidative Stress as an Underlying Contributor in the Development of Chronic Complications in Diabetes Mellitus

The high prevalence of diabetes mellitus and its increasing incidence worldwide, coupled with several complications observed in its carriers, have become a public health issue of great relevance. Chronic hyperglycemia is the main feature of such a disease, being considered the responsible for the establishment of micro and macrovascular complications observed in diabetes. Several efforts have been directed in order to better comprehend the pathophysiological mechanisms involved in the course of this endocrine disease. Recently, numerous authors have suggested that excess generation of highly reactive oxygen and nitrogen species is a key component in the development of complications invoked by hyperglycemia. Overproduction and/or insufficient removal of these reactive species result in vascular dysfunction, damage to cellular proteins, membrane lipids and nucleic acids, leading different research groups to search for biomarkers which would be capable of a proper and accurate measurement of the oxidative stress (OS) in diabetic patients, especially in the presence of chronic complications. In the face of this scenario, the present review briefly addresses the role of hyperglycemia in OS, considering basic mechanisms and their effects in diabetes mellitus, describes some of the more commonly used biomarkers of oxidative/nitrosative damage and includes selected examples of studies which evaluated OS biomarkers in patients with diabetes, pointing to the relevance of such biological components in general oxidative stress status of diabetes mellitus carriers.     

Targeting the Redox Balance in Inflammatory Skin Conditions

Reactive oxygen species (ROS) can be both beneficial and deleterious. Under normal physiological conditions, ROS production is tightly regulated, and ROS participate in both pathogen defense and cellular signaling. However, insufficient ROS detoxification or ROS overproduction generates oxidative stress, resulting in cellular damage. Oxidative stress has been linked to various inflammatory diseases. Inflammation is an essential response in the protection against injurious insults and thus important at the onset of wound healing. However, hampered resolution of inflammation can result in a chronic, exaggerated response with additional tissue damage. In the pathogenesis of several inflammatory skin conditions, e.g., sunburn and psoriasis, inflammatory-mediated tissue damage is central. The prolonged release of excess ROS in the skin can aggravate inflammatory injury and promote chronic inflammation. The cellular redox balance is therefore tightly regulated by several (enzymatic) antioxidants and pro-oxidants; however, in case of chronic inflammation, the antioxidant system may be depleted, and prolonged oxidative stress occurs. Due to the central role of ROS in inflammatory pathologies, restoring the redox balance forms an innovative therapeutic target in the development of new strategies for treating inflammatory skin conditions. Nevertheless, the clinical use of antioxidant-related therapies is still in its infancy.     

Chromophore of an Enhanced Green Fluorescent Protein Can Play a Photoprotective Role Due to Photobleaching

Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging mechanisms by the enhanced green fluorescent protein, EGFP. To check if this protein could fulfill a photoprotective function, we employed electron spin resonance (ESR) in combination with spin-trapping. Two organic photosensitizers, rose bengal and methylene blue, as well as an inorganic photocatalyst, nano-TiO₂, were used to photogenerate ROS. Spin-traps, TMP-OH and DMPO, and a nitroxide radical, TEMPOL, served as molecular targets for ROS. Our results show that EGFP quenches various forms of ROS, including superoxide radicals and singlet oxygen. Compared to the three proteins PNP, papain, and BSA, EGFP revealed high ROS quenching ability, which suggests its photoprotective role in living systems. Damage to the EGFP chromophore was also observed under strong photo-oxidative conditions. This study contributes to the discussion on the protective function of fluorescent proteins homologous to the green fluorescent protein (GFP). It also draws attention to the possible interactions of GFP-like proteins with ROS in systems where such proteins are used as biological markers.     

NADPH Oxidase 4 (NOX4) in Cancer: Linking Redox Signals to Oncogenic Metabolic Adaptation

Cancer cells can survive and maintain their high proliferation rate in spite of their hypoxic environment by deploying a variety of adaptative mechanisms, one of them being the reorientation of cellular metabolism. A key aspect of this metabolic rewiring is the promotion of the synthesis of antioxidant molecules in order to counter-balance the hypoxia-related elevation of reactive oxygen species (ROS) production and thus combat the onset of cellular oxidative stress. However, opposite to their negative role in the inception of oxidative stress, ROS are also key modulatory components of physiological cellular metabolism. One of the major physiological cellular ROS sources is the NADPH oxidase enzymes (NOX-es). Indeed, NOX-es produce ROS in a tightly regulated manner and control a variety of cellular processes. By contrast, pathologically elevated and unbridled NOX-derived ROS production is linked to diverse cancerogenic processes. In this respect, NOX4, one of the members of the NOX family enzymes, is of particular interest. In fact, NOX4 is closely linked to hypoxia-related signaling and is a regulator of diverse metabolic processes. Furthermore, NOX4 expression and function are altered in a variety of malignancies. The aim of this review is to provide a synopsis of our current knowledge concerning NOX4-related processes in the oncogenic metabolic adaptation of cancer cells.     

Bioactive Compounds and Their Impact on Protein Modification in Human Cells

Reactive oxygen species (ROS) represent a group of molecules with a signaling role that are involved in regulating human cell proliferation and differentiation. Increased ROS concentrations are often associated with the local nonspecific oxidation of biological macromolecules, especially proteins and lipids. Free radicals, in general, may randomly damage protein molecules through the formation of protein-centered radicals as intermediates that, in turn, decay into several end oxidation products. Malondialdehyde (MDA), a marker of free-radical-mediated lipid oxidation and cell membrane damage, forms adducts with proteins in a nonspecific manner, leading to the loss of their function. In our study, we utilized U-937 cells as a model system to unveil the effect of four selected bioactive compounds (chlorogenic acid, oleuropein, tomatine, and tyrosol) to reduce oxidative stress associated with adduct formation in differentiating cells. The purity of the compounds under study was confirmed by an HPLC analysis. The cellular integrity and changes in the morphology of differentiated U-937 cells were confirmed with confocal microscopy, and no significant toxicity was found in the presence of bioactive compounds. From the Western blot analysis, a reduction in the MDA adduct formation was observed in cells treated with compounds that underlaid the beneficial effects of the compounds tested.     

Oxidative Stress Response in Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells

Reactive oxygen species (ROS) can irreversibly damage biological molecules, a process known as oxidative stress. Elevated ROS levels are associated with immune cell activation. Sustained immune system activation can affect many different cells in the environment. One cell type that has been detected in almost all tissues of the body is mesenchymal stem/stromal cells (MSC). MSC possess proliferation and differentiation potential, thus facilitating regeneration processes. However, the regenerative capacity of MSC might be impaired by oxidative stress, and the effects of long-term oxidative stress on MSC functions are sparsely described. The examination of oxidative stress is often performed by exposure to H₂O₂. Since H₂O₂ is rapidly degraded, we additionally exposed the cell cultures to glucose oxidase (GOx), resulting in sustained exposure to H₂O₂. Using these model systems, we have focused on the effects of short- and long-term oxidative stress on viability, migration, differentiation, and signaling. All cellular functions examined were affected by the applied oxidative stress. The differences that occur between pulsed and sustained oxidative stress indicated higher oxidative stress in MSC upon direct H₂O₂ exposure, whereas the GOx-induced prolonged exposure to H₂O₂ seems to allow for better cellular adaptation. The mechanisms underlying these different responses are currently unknown.     

Dolichol: A Component of the Cellular Antioxidant Machinery

Dolichol, an end product of the mevalonate pathway, has been proposed as a biomarker of aging, but its biological role, not to mention its catabolism, has not been fully understood. UV‐B radiation was used to induce oxidative stress in isolated rat hepatocytes by the collagenase method. Effects on dolichol, phospholipid‐bound polyunsaturated fatty acids (PL‐PUFA) and known lipid soluble antioxidants [coenzyme Q (CoQ) and α‐tocopherol] were studied. The increase in oxidative stress was detected by a probe sensitive to reactive oxygen species (ROS). Peroxidation of lipids was assessed by measuring the release of thiobarbituric acid reactive substances (TBARS). Dolichol, CoQ, and α‐tocopherol were assessed by high‐pressure liquid chromatography (HPLC), PL‐PUFA by gas–liquid chromatography (GC). UV‐B radiation caused an immediate increase in ROS as well as lipid peroxidation and a simultaneous decrease in the levels of dolichol and lipid soluble antioxidants. Decrease in dolichol paralleled changes in CoQ levels and was smaller to that in α‐tocopherol. The addition of mevinolin, a competitive inhibitor of the enzyme 3‐hydroxy‐3‐methylglutaryl CoA reductase (HMG‐CoAR), magnified the loss of dolichol and was associated with an increase in TBARS production. Changes in PL‐PUFA were minor. These findings highlight that oxidative stress has very early and similar effects on dolichol and lipid soluble antioxidants. Lower levels of dolichol are associated with enhanced peroxidation of lipids, which suggest that dolichol may have a protective role in the antioxidant machinery of cell membranes and perhaps be a key to understanding some adverse effects of statin therapy.     

Association of total cholesterol/apoB100 with lipid and protein oxidation products in stable angina patients treated with statins

Oxidative modification of lipids contained in lipoproteins may contribute to initiation of local inflammation in the vascular endothelium and ultimately to the atherosclerotic plaque formation. Therefore, in patients with high cardiovascular risk in primary as well as in secondary prevention, it is recommended that the serum low‐density lipoprotein (LDL) cholesterol be reduced. However, the management of patients with LDL level at goal is still a matter of debate. Various parameters have been proposed for predicting increased cardiovascular risk. It was found that among known indicators of lipid metabolism deregulation the ratio of total cholesterol/apoB100 associates with the severity of coronary arteriosclerosis in population of individuals with LDL levels < 100 mg/dL. There are several possible hypotheses for this phenomenon (impaired reduction of apoB100 expression by statins or increased triglyceride content of apoB100 containing lipoproteins compatible with oxidized LDL phenotype in subjects with high cardiovascular burden). In this paper, we discuss the increased susceptibility of plasma lipoproteins to oxidation. To verify this hypothesis, we undertook to determine, in the susceptible population described previously, whether there is a relation of total cholesterol (TC) ratio to apoB100 with known oxidative stress exponents such as protein and lipid oxidation products (LPP). TC/apoB100 was found to have a significant association with the level of LPPs in most examined subgroups (except subjects on statins with LDL > 100 mg/dL). See commentary by Grabowski http://dx.doi.org/10.1002/ejlt.201200295     

Lipid profiles of plasma lipoproteins of fasted and fed normal and choline-deficient rats

Three major density classes of lipoproteins and a residual protein (d>1.21) were isolated by ultracentrifugation from plasma of fasted, fed normal, and choline-deficient rats. Lipid extracts were obtained from total plasma and the various density classes of lipoproteins, and each extract was examined in detail by thin layer and gas chromatographies. The results indicated essentially identical compositions of molecular species of phosphatidyl choline, which suggested their rapid equilibration among the different plasma lipoprotein classes. In contrast, the molecular species of the triacylglycerols and cholesteryl esters showed significant differences among the chylomicrons, very low and low, and high density lipoproteins, which excluded the possibility of their ready equilibration in vivo. Omission of choline from diet resulted in a sharp and statistically significant decrease in all lipid components of the very low and low density lipoproteins within 2 days. After 10 days of choline deficiency, the lipid levels of chylomicrons and very low and low density lipoproteins were ca. one-half the levels found in the choline supplemented animals, and there were discernible distortions in their lipid composition. Reintroduction of choline led to a prompt return to normal levels and lipid composition of both chylomicron and very low and low density lipoprotein fractions. The lack of equilibration of the triacylglycerols among the lipoprotein classes under normal conditions and in choline deficiency demonstrates an as yet unrecognized source of compartmentation of plasma lipids.     

Promoted Proliferation of Hematopoietic Stem Cells Enabled by a Hyaluronic Acid/Carbon Nanotubes Antioxidant Hydrogel

Reactive oxygen species (ROS) level is closely associated with the physiological function of hematopoietic stem cells (HSCs) in ex vivo culture systems. Previously developed hydrogels in use of HSCs culture have generally failed in considering their antioxidant property. In the present work, antioxidant hydrogels are constructed to evaluate the biological effect of ROS on HSCs proliferation by suppressing oxidative stress damage. Functionalized carbon nanotubes (CNTs), which act as potential antioxidants, are physically encapsulated in biocompatible hyaluronic acid (HA) hydrogels to achieve long‐term antioxidant activity. Consequently, these hybrid hydrogels exhibit enhanced physical properties and superior scavenging capability on oxides and peroxides compared to the pure HA hydrogel. The results indicate that ROS significantly inhibit the biological characteristics of HSCs. Nevertheless, the proliferation ability and pluripotency are dramatically improved when the culture system is supplied with the antioxidant hydrogel, revealing that the CNT‐incorporated hydrogel can effectively relieve oxidative stress response in HSCs and reduce apoptosis from ROS. Therefore, the HA/CNT hydrogel can provide a novel strategy to establish an artificial microenvironment with a low ROS level for HSCs proliferation.     

Oxidative stress: the role of cytochromes P450 in oxygen activation

Oxidative stress is associated with a number of degenerative disease states, such as cancer and AIDS. Fundamental to oxidative stress is the generation of superoxide, peroxide and other reactive oxygen species (ROS). This review focuses on the importance of cytochrome P450 (CYP) enzymes in the activation of oxygen and ROS generation, together with a discussion of defence mechanisms which can offer protection against oxidative stress. © 2002 Society of Chemical Industry.     

Periodic Exposure of Plasma-Activated Medium Alters Fibroblast Cellular Homoeostasis

Excess amounts of redox stress and failure to regulate homeostatic levels of reactive species are associated with several skin pathophysiologic conditions. Nonmalignant cells are assumed to cope better with higher reactive oxygen and nitrogen species (RONS) levels. However, the effect of periodic stress on this balance has not been investigated in fibroblasts in the field of plasma medicine. In this study, we aimed to investigate intrinsic changes with respect to cellular proliferation, cell cycle, and ability to neutralize the redox stress inside fibroblast cells following periodic redox stress in vitro. Soft jet plasma with air as feeding gas was used to generate plasma-activated medium (PAM) for inducing redox stress conditions. We assessed cellular viability, energetics, and cell cycle machinery under oxidative stress conditions at weeks 3, 6, 9, and 12. Fibroblasts retained their usual physiological properties until 6 weeks. Fibroblasts failed to overcome the redox stress induced by periodic PAM exposure after 6 weeks, indicating its threshold potential. Periodic stress above the threshold level led to alterations in fibroblast cellular processes. These include consistent increases in apoptosis, while RONS accumulation and cell cycle arrest were observed at the final stages. Currently, the use of NTP in clinical settings is limited due to a lack of knowledge about fibroblasts’ behavior in wound healing, scar formation, and other fibrotic disorders. Understanding fibroblasts’ physiology could help to utilize nonthermal plasma in redox-related skin diseases. Furthermore, these results provide new information about the threshold capacity of fibroblasts and an insight into the adaptation mechanism against periodic oxidative stress conditions in fibroblasts.