Role of redox imbalance and cytokines in mediating oxidative damage and disease progression of patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disorder wherein the contributory role of oxidative stress has been established in the synovial fluid. As availability of synovial fluid is limited, this study aimed to evaluate in the peripheral blood of patients with RA, the relationship if any, between the extent of oxidative stress in terms of generation of reactive oxygen species (ROS) in neutrophils, plasma NADPH oxidase and myeloperoxidase activity with markers of oxidative damage, circulating cytokines and disease activity score (DAS28). In patients with RA, neutrophils in peripheral blood demonstrated an enhanced generation of ROS, coupled with depletion of free radical scavenging activity. Furthermore, the NADPH oxidase and myeloperoxidase activity was enhanced as were markers of damage. There was a positive correlation between the DAS 28 and generation of ROS, NADPH oxidase and myeloperoxidase activity as also with oxidative stress mediated protein carbonylation. Patients with RA demonstrated an increase in proinflammatory (IL-17, IL-23, and IFN-γ) and some anti-inflammatory (IL-4, IL-5, and TGF-β) cytokines. Although the levels of IL-17 correlated positively with generation of ROS, myeloperoxidase, markers of protein damage and DAS28, IL-23 correlated positively only with protein damage, and negatively with free radical scavenging activity. Importantly, incubation of neutrophils from healthy donors with plasma or SF from patients with RA translated into an enhanced generation of ROS, along with an elevation of intracellular proinflammatory cytokines. Taken together, in patients with RA, circulating neutrophils mediated a shift in the oxidant/antioxidant balance favouring the former, which translated into protein damage and contributed towards disease progression.     

Levels of oxidative damage and proinflammatory cytokines are enhanced in patients with active vitiligo

Vitiligo is an autoimmune depigmenting skin disease characterised by loss of melanocytes wherein oxidative stress is proposed to be the initial triggering factor with subsequent immune dysregulation. This study aimed to evaluate the relationship, if any, between the generation of reactive oxygen species (ROS), markers of oxidative damage and circulating cytokines in patients with active vitiligo. The generation of ROS in erythrocytes and neutrophils was significantly higher in patients with active vitiligo than healthy controls. Alongside, markers of oxidative stress-mediated damage namely lipid peroxidation, DNA damage and protein carbonylation were evaluated. Patients with active vitiligo demonstrated increased lipid and DNA damage but minimal protein damage. There was a significant decline in the free radical scavenging capacity of active vitiligo cases. A positive correlation existed between baseline levels of ROS and lipid peroxidation as also DNA damage. Patients with active vitiligo demonstrated an increase in several proinflammatory (IL-6, TNF-α, IL-1β, IFN-γ and IL-8) and some anti-inflammatory/immunoregulatory (IL-5 and IL-10) cytokines. Importantly, the levels of IFN-γ and IL-10 consistently correlated with the generation of ROS, markers of damage and their free radical scavenging capacity. Taken together, patients with active vitiligo demonstrated an enhanced generation of ROS in erythrocytes and neutrophils which mediated lipid peroxidation, DNA damage and coupled with a decline in their antioxidant capacity created a pro-oxidant milieu that favoured tissue damage and potential generation of neoantigens, accounting for disease progression.     

Intracellular oxidative activity and respiratory burst of leukocytes isolated from multiple sclerosis patients

Oxidative damage induced by free radicals and reactive oxygen species (ROS) have been suggested to play an important role in the development of autoimmune diseases such as multiple sclerosis (MS) disease and it has been hypothesised that oxidative injury could mediate demyelination and axonal injury in MS subjects. In our study, we compared intracellular oxidative activity and the respiratory burst activity in MS patients (n=20) and healthy controls (n=15) using leukocytes as cellular model. At this purpose, intracellular ROS levels were evaluated by fluorometric assay using the 2'-7'-dichlorodihydrofluorescin diacetate probe (H(2)DCFDA) in untreated or in leukocytes stimulated with phorbol-12-myristate-13-acetate (PMA). Our results demonstrate that the intracellular spontaneous ROS production in leukocytes from MS patients was higher with respect to cells from control subjects (p<0.001). PMA addition induced a higher formation of ROS both in leukocytes from MS patients and controls (p<0.001). The PMA-induced production of ROS was significantly higher in leukocytes from MS with respect to controls (p<0.001). Significant positive correlations were established between intracellular spontaneous or PMA-induced production of ROS in leukocytes isolated from MS patients and the clinical parameters used to evaluate disease disability such as expanded disability status scale (EDSS), brain lesions evaluated by MRI and visual evoked potential (VEP) (p<0.001). In conclusion, our results demonstrate higher levels of intracellular ROS in untreated or in PMA-treated leukocytes isolated from MS patients with respect to healthy subjects confirming the role of oxidative stress in multiple sclerosis.     

In vitro and in vivo aggregation of a fragment of huntingtin protein directly causes free radical production

Neurodegenerative diseases are characterized by intra- and/or extracellular protein aggregation and oxidative stress. Intense attention has been paid to whether protein aggregation itself contributes to abnormal production of free radicals and ensuing cellular oxidative damage. Although this question has been investigated in the context of extracellular protein aggregation, it remains unclear whether protein aggregation inside cells alters the redox homeostasis. To address this, we have used in vitro and in vivo (cellular) models of Huntington disease, one of nine polyglutamine (poly(Q)) disorders, and examined the causal relationship among intracellular protein aggregation, reactive oxygen species (ROS) production, and toxicity. Live imaging of cells expressing a fragment of huntingtin (httExon1) with a poly(Q) expansion shows increased ROS production preceding cell death. ROS production is poly(Q) length-dependent and not due to the httExon 1 flanking sequence. Aggregation inhibition by the MW7 intrabody and Pgl-135 treatment abolishes ROS production, showing that increased ROS is caused by poly(Q) aggregation itself. To examine this hypothesis further, we determined whether aggregation of poly(Q) peptides in vitro generated free radicals. Monitoring poly(Q) protein aggregation using atomic force microscopy and hydrogen peroxide (H(2)O(2)) production over time in parallel we show that oligomerization of httEx1Q53 results in early generation of H(2)O(2). Inhibition of poly(Q) oligomerization by the single chain antibody MW7 abrogates H(2)O(2) formation. These results demonstrate that intracellular protein aggregation directly causes free radical production, and targeting potentially toxic poly(Q) oligomers may constitute a therapeutic target to counteract oxidative stress in poly(Q) diseases.     

Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems

Free radicals derived from oxygen, nitrogen and sulphur molecules in the biological system are highly active to react with other molecules due to their unpaired electrons. These radicals are important part of groups of molecules called reactive oxygen/nitrogen species (ROS/RNS), which are produced during cellular metabolism and functional activities and have important roles in cell signalling, apoptosis, gene expression and ion transportation. However, excessive ROS attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins and lipids resulting in an increased risk for cardiovascular disease, cancer, autism and other diseases. Intracellular antioxidant enzymes and intake of dietary antioxidants may help to maintain an adequate antioxidant status in the body. In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS. The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes. The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants. This review focuses on the research approaches that have been used to study oxidative stress and antioxidants in lipid peroxidation, DNA damage, protein modification as well as enzyme activity, with emphasis on the chemical and cell-free biological system.     

Aging in vertebrates,and the effect of caloric restriction: a mitochondrial free radical production–DNA damage mechanism?

Oxygen is toxic to aerobic animals because it is univalently reduced inside cells to oxygen free radicals. Studies dealing with the relationship between oxidative stress and aging in different vertebrate species and in caloric-restricted rodents are discussed in this review. Healthy tissues mainly produce reactive oxygen species (ROS) at mitochondria. These ROS can damage cellular lipids, proteins and, most importantly, DNA. Although antioxidants help to control this oxidative stress in cells in general, they do not decrease the rate of aging, because their concentrations are lower in long- than in short-lived animals and because increasing antioxidant levels does not increase vertebrate maximum longevity. However, long-lived homeothermic vertebrates consistently have lower rates of mitochondrial ROS production and lower levels of steady-state oxidative damage in their mitochondrial DNA than short-lived ones. Caloric-restricted rodents also show lower levels of these two key parameters than controls fed ad libitum. The decrease in mitochondrial ROS generation of the restricted animals has been recently localized at complex I and the mechanism involved is related to the degree of electronic reduction of the complex I ROS generator. Strikingly, the same site and mechanism have been found when comparing a long- with a short-lived animal species. It is suggested that a low rate of mitochondrial ROS generation extends lifespan both in long-lived and in caloric-restricted animals by determining the rate of oxidative attack and accumulation of somatic mutations in mitochondrial DNA.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015-15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg(9) to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

Protective Role of Quercetin on PCBs-Induced Oxidative Stress and Apoptosis in Hippocampus of Adult Rats

Polychlorinated biphenyls (PCBs) exposure produces neurodegeneration and induces oxidative stress. Neuroprotective role of quercetin, on PCBs induced apoptosis in hippocampus has not yet been studied. The present study is focused to see whether quercetin supplementation precludes against PCBs induced oxidative stress and hippocampal apoptosis. The results have shown that quercetin at 50 mg/kg bwt/30 days has protected oxidative stress in hippocampus of adult male rats. Quercetin, a free radical scavenger decreased the levels of oxidative stress markers in the hippocampus of simultaneous PCB+quercetin treated rats. The pro-apoptotic and anti-apoptotic molecules such as Bad, Bid, Bax and Bcl2 were altered in the hippocampus of experimental animals. PCBs increased the DNA damage and induced neurodegeneration were assessed by histological studies. PCB induced ROS may be linked to increased hippocampal neuronal apoptosis. Quercetin supplementation decreased the neuronal damage and scavenged the free radicals induced by PCBs and protects PCBs induced apoptosis and oxidative stress.     

A Meta-Analysis of Oxidative Stress Markers in Depression

ObjectStudies have suggested that depression was accompanied by oxidative stress dysregulation, including abnormal total antioxidant capacity (TAC), antioxidants, free radicals, oxidative damage and autoimmune response products. This meta-analysis aims to analyse the clinical data quantitatively by comparing the oxidative stress markers between depressed patients and healthy controls.MethodsA search was conducted to collect the studies that measured the oxidative stress markers in depressed patients. Studies were searched in Embase, Medline, PsychINFO, Science direct, CBMDisc, CNKI and VIP from 1990 to May 2015. Data were subjected to meta-analysis by using a random effects model for examining the effect sizes of the results. Bias assessments, heterogeneity assessments and sensitivity analyses were also conducted.Results115 articles met the inclusion criteria. Lower TAC was noted in acute episodes (AEs) of depressed patients (p<0.05). Antioxidants, including serum paraoxonase, uric acid, albumin, high-density lipoprotein cholesterol and zinc levels were lower than controls (p<0.05); the serum uric acid, albumin and vitamin C levels were increased after antidepressant therapy (p<0.05). Oxidative damage products, including red blood cell (RBC) malondialdehyde (MDA), serum MDA and 8-F₂-isoprostanes levels were higher than controls (p<0.05). After antidepressant medication, RBC and serum MDA levels were decreased (p<0.05). Moreover, serum peroxide in free radicals levels were higher than controls (p<0.05). There were no differences between the depressed patients and controls for other oxidative stress markers.ConclusionThis meta-analysis supports the facts that the serum TAC, paraoxonase and antioxidant levels are lower, and the serum free radical and oxidative damage product levels are higher than controls in depressed patients. Meanwhile, the antioxidant levels are increased and the oxidative damage product levels are decreased after antidepressant medication. The pathophysiological relationships between oxidative stress and depression, and the potential benefits of antioxidant supplementation deserve further research.     

Occurrence of oxidative stress during myocardial reperfusion

Reperfusion, without doubt, is the most effective way to treat the ischaemic myocardium. Late reperfusion may however cause further damage. Myocardial production of oxygen free radicals above the neutralizing capacity of the myocytes is an important cause of this reperfusion damage. There is evidence that prolonged ischaemia reduces the naturally occurring defence mechanisms of the heart against oxygen free radicals, particularly mitochondrial manganese superoxide dismutase, and intracellular pool of reduced glutathione. Consequently, reperfusion results in a severe oxidative damage, as evidenced by tissue accumulation and release of oxidized glutathione.An oxygen free radical-mediated impairment of mechanical function also occurs during reperfusion of human heart. In fact we observed during surgical reperfusion of coronary artery disease (CAD) patients, a prolonged and sustained release of oxidized glutathione;the degree of oxidative stress was inversely correlated with recovery of mechanical and haemodynamic function. These findings represent the rationale for therapeutic interventions which increase the cellular antioxidant capacities and improve the efficacy of myocardial reperfusion.     

Antioxidants prevent oxidative DNA damage and cellular transformation elicited by the over-expression of c-MYC

Reactive oxygen species (ROS)-induced genomic damage may have important consequences in the initiation and progression of cancer. Deregulated expression of the proto-oncogene c-MYC is associated with intracellular oxidative stress and increased DNA damage. However, the protective role of antioxidants such as Vitamin C against MYC-induced genomic damage has not been fully investigated. In a variety of cell lines, we show that ectopic MYC over-expression results in the elevation of intracellular ROS levels and a concomitant increase in oxidative DNA damage, as assessed by levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in the genomic DNA. Loading cells with ascorbic acid (AA) relieved MYC-elicited intracellular oxidative stress and conferred genomic protection. A mitochondrially targeted Vitamin E analog, TPPB, also protected cells from MYC-elicited oxidative DNA damage, suggesting the involvement of mitochondria in increased ROS production. We found that deregulated MYC expression resulted in the attenuation of intracellular glutathione levels, which was reversed by loading cells with Vitamin C. Additionally, cells over-expressing MYC had elevated levels of intracellular superoxide, which was significantly quenched by Vitamin C or the selective superoxide quencher, Tiron. Consequently, Vitamin C and other antioxidants protected cells from MYC-induced cellular transformation. Our studies implicate a role for ROS, and superoxide in particular, in MYC-elicited oxidative DNA damage and cellular transformation, and point to a pharmacological role of antioxidants in cancer chemoprevention.     

Impaired mitochondrial function protects against free radical-mediated cell death

Free radical damage can have fatal consequences. Mitochondria carry out essential cellular functions and produce high levels of reactive oxygen species (ROS). Many agents also generate ROS. Using the yeast Saccharomyces cerevisiae as a eukaryotic model, the role of functional mitochondria in surviving free radical damage was investigated. Respiratory-deficient cells lacking mitochondrial DNA (rho(0)) were up to 100-fold more resistant than isogenic rho(+) cells to killing by ROS generated by the bleomycin-phleomycin family of oxidative agents. Up to approximately 90% of the survivors of high oxidative stress lost mitochondrial function and became "petites." The selective advantage of respiratory deficiency was studied in several strains, including DNA repair-deficient rad52/rad52 and blm5/blm5 diploid strains. These mutant strains are hypersensitive to lethal effects of free radicals and accumulate more DNA damage than related wild-type strains. Losses in mitochondrial function were dose-dependent, and mutational alteration of the RAD52 or BLM5 gene did not affect the resistance of surviving cells lacking mitochondrial function. The results indicate that inactivation of mitochondrial function protects cells against lethal effects of oxygen free radicals.     

The effects of whole-body cryotherapy on oxidative stress in multiple sclerosis patients

There is strong evidence that multiple sclerosis (MS) is characterized not only by immune mediated inflammatory reactions but also by neurodegenerative processes. Accumulated data indicate that oxidative stress (OS) plays a major role in this process. Generated in excess, reactive oxygen species (ROS) lead to oxidative stress and are involved in demyelination and axonal damage in MS. ROS generation may be inhibited partly by hypothermia, which is known as a potent putative neuroprotectant and may inhibit generating free radicals and oxidative stress. Whole-body cryotherapy (WBCT) treatment may improve both survival and neurological outcome in MS patients.     

Free radicals and antioxidants in normal physiological functions and human disease

Reactive oxygen species (ROS) and reactive nitrogen species (RNS, e.g. nitric oxide, NO(*)) are well recognised for playing a dual role as both deleterious and beneficial species. ROS and RNS are normally generated by tightly regulated enzymes, such as NO synthase (NOS) and NAD(P)H oxidase isoforms, respectively. Overproduction of ROS (arising either from mitochondrial electron-transport chain or excessive stimulation of NAD(P)H) results in oxidative stress, a deleterious process that can be an important mediator of damage to cell structures, including lipids and membranes, proteins, and DNA. In contrast, beneficial effects of ROS/RNS (e.g. superoxide radical and nitric oxide) occur at low/moderate concentrations and involve physiological roles in cellular responses to noxia, as for example in defence against infectious agents, in the function of a number of cellular signalling pathways, and the induction of a mitogenic response. Ironically, various ROS-mediated actions in fact protect cells against ROS-induced oxidative stress and re-establish or maintain "redox balance" termed also "redox homeostasis". The "two-faced" character of ROS is clearly substantiated. For example, a growing body of evidence shows that ROS within cells act as secondary messengers in intracellular signalling cascades which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. This review will describe the: (i) chemistry and biochemistry of ROS/RNS and sources of free radical generation; (ii) damage to DNA, to proteins, and to lipids by free radicals; (iii) role of antioxidants (e.g. glutathione) in the maintenance of cellular "redox homeostasis"; (iv) overview of ROS-induced signaling pathways; (v) role of ROS in redox regulation of normal physiological functions, as well as (vi) role of ROS in pathophysiological implications of altered redox regulation (human diseases and ageing). Attention is focussed on the ROS/RNS-linked pathogenesis of cancer, cardiovascular disease, atherosclerosis, hypertension, ischemia/reperfusion injury, diabetes mellitus, neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), rheumatoid arthritis, and ageing. Topics of current debate are also reviewed such as the question whether excessive formation of free radicals is a primary cause or a downstream consequence of tissue injury.     

The heart-protective mechanism of nitronyl nitroxide radicals on murine viral myocarditis induced by CVB3

Our previous researches showed that nitronyl nitroxyl derivatives, NNP and NNVP were good anti-oxidants and provided radioprotective effects in C6 cells. The objective of the present study is to investigate the possible antiviral effects and underlying pharmacological of the two nitronyl nitroxide radicals against CVB3 in vitro and in vivo. The results showed that NNP and NNVP were some of the most potent compounds in terms of their antiviral effects by protecting myocardial cells against oxidative damage of free radicals. Treatment with NNP or NNVP could decrease the intracellular ROS level in vitro. They could lead to a significant decrease in activities of biochemical markers AST, CK and LDH in infected murine serum and could increase SOD and CAT activities and decreased MDA activities compared with infected control in vivo. NNP and NNVP could reduce NO production in infected mice by reacting with NO to produce the imino nitroxides which was confirmed by ESR spectrometry. In addition, NNP and NNVP could both decrease the mRNA expression of proinflammatory cytokines, TNF-α, IL-2 and IL-6. In conclusion, nitronyl nitroxide radicals NNP and NNVP were shown to have antiviral activities against CVB3 and they may represent potential therapeutic agents for viral myocarditis.     

The visualization of oxidant stress in tissues and isolated cells

Many studies have implicated the role of oxidant stress in a wide range of human diseases and have led to the rapid expansion of research in this area. With many experimental approaches a direct detection of the production of reactive oxygen species (ROS) and free radicals is not possible. Free radicals are very reactive, short-lived and react in a non-specific way, so that ongoing oxidative damage is generally analyzed by measurement of secondary products e.g. H2O2, "oxidized" proteins, peroxidized lipids and their break-down products, "oxidized" DNA or by fluorographic analysis in combination with fluorescent dyes e.g. dichlorofluorescin (DCFH). The histochemical visualization of selected molecular markers for oxidative phenomena can often provide valuable information concerning the distribution of oxidative processes in vivo. A number of biochemical methods are available for the monitoring of almost all oxidant stress-related processes, although their applicability in vivo is limited. This review summarizes the biochemical methods currently available for histochemical detection and indirect visualization of an excess of free radicals and ROS. The cited methods are discussed and the results obtained from their application are critically evaluated.     

Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells

Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high-level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.     

Reactive oxygen species regulation of autophagy in cancer: Implications for cancer treatment

Reactive oxygen species (ROS) are important in regulating normal cellular processes, but deregulated ROS contribute to the development of various human diseases including cancers. Autophagy is one of the first lines of defense against oxidative stress damage. The autophagy pathway can be induced and upregulated in response to intracellular ROS or extracellular oxidative stress. This leads to selective lysosomal self-digestion of intracellular components to maintain cellular homeostasis. Hence, autophagy is the survival pathway, conferring stress adaptation and promoting viability under oxidative stress. However, increasing evidence has demonstrated that autophagy can also lead to cell death under oxidative stress conditions. In addition, altered autophagic signaling pathways that lead to decreased autophagy are frequently found in many human cancers. This review discusses the advances in understanding of the mechanisms of ROS-induced autophagy and how this process relates to tumorigenesis and cancer therapy.