Elevated Intracellular Reactive Oxygen Species Levels Research Articles

Increased Intracellular Oxygen Radical Production in Neutrophils During Febrile Episodes of Periodic Fever, Aphthous Stomatitis, Pharyngitis, and Cervical Adenitis Syndrome

Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is an autoinflammatory disease of unknown etiology that primarily affects preschool-aged children. PFAPA syndrome is characterized by recurrent attacks of fever and symptoms of inflammation consistent with the disease acronym. Since autoinflammatory diseases are, by definition, mediated by cells of the innate immune system, the aim of this study was to evaluate the functional features of neutrophils, the most abundant innate immune cell in the circulation, in children with PFAPA syndrome. Blood polymorphonuclear leukocytes (PMNs), obtained from patients with PFAPA syndrome during both febrile and asymptomatic, afebrile phases of the disease, as well as from healthy children (afebrile controls) and children with fever and abdominal pain (febrile controls), were analyzed for 3 key neutrophil characteristics: 1) apoptosis (measured by annexin V/7-aminoactinomycin D staining), 2) production of reactive oxygen species (ROS) (measured by luminol/isoluminol-amplified chemiluminescence), and 3) priming status (measured as responsiveness to galectin-3 and up-regulation of CD11b). Compared to PMNs obtained from patients with PFAPA syndrome during an afebrile interval and those from febrile controls, PMNs obtained from patients during a PFAPA syndrome flare produced elevated levels of intracellular NADPH oxidase-derived ROS, had significantly diminished rates of spontaneous apoptosis, and displayed signatures of priming. In contrast, PMNs from afebrile patients with PFAPA syndrome had a significantly elevated rate of spontaneous apoptosis compared to PMNs from afebrile controls. These findings demonstrate that 3 key aspects of neutrophil innate immune function, namely, apoptosis, priming, and generation of an intracellular oxidative burst, are altered, most prominently during febrile attacks, in children with PFAPA syndrome.

Antiproliferation and anti-migration induced by gypenosides in human colon cancer SW620 and esophageal cancer Eca-109 cells

Gypenosides (Gyp), found in Gynostemma pentaphyllum Makino, has attracted more attention owing to its wide bioactivities. However, the effects of Gyp on esophageal cancer cells and colon cancer cells are still unknown. The present study was to investigate the possible anti-proliferative and anti-migration activity of Gyp on human colon cancer cells SW620 and esophageal cancer cells Eca-109. Cell viability was evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell membrane integrity was evaluated using flow cytometry following propidium iodide staining. Apoptotic cell death was determined by nuclear 4'-6-diamidino-2-phenylindole staining. Generation of intracellular reactive oxygen species (ROS) and changes in mitochondrial membrane potential (Δψ m) was analyzed by flow cytometry using 2',7'-dichlorofluorescein-diacetate and rhodamine 123 staining, respectively. Wound healing assay was carried out to investigate Gyp-inhibited migration of SW620 and Eca-109 cells. The results indicated that Gyp inhibited cell proliferation and migration in SW620 and Eca-109 cells in dose- and time-dependent manner. Gyp elevated intracellular ROS level, decreased the Δψ m, and induced apoptotic morphology such as cell shrinkage and chromatin condensation, suggesting oxidative stress and mitochondria-dependent cell apoptosis that might be involved in Gyp-induced cell viability loss in SW620 and Eca-109 cells. The findings indicate Gyp may have valuable application in clinical colon cancer and esophageal cancer treatments.

Reactive oxygen species and Ca2+are involved in sodium arsenite-induced cell killing in yeast cells

Arsenic is a toxic metalloid that is widely distributed in the environment, and its toxicity has been demonstrated in several models. However, the mechanism of arsenic toxicity still remains unclear. In this study, the toxic effects of sodium arsenite (1-7 mM) on yeast cells were investigated. The experimental results showed that sodium arsenite inhibited yeast cell growth, and the inhibitory effect of cell growth (OD600 nm values) was positively correlated with arsenite concentrations. Sodium arsenite caused loss of cell viability in a concentration- and duration-dependent manner in yeast cells. However, arsenite-caused cell viability loss was blocked by either antioxidants (200 U mL(-1) CAT and 0.5 mM AsA) or Ca(2+) antagonists (0.5 mM LaCl3 and 0.5 mM EGTA). We also found intracellular reactive oxygen species (ROS) and Ca(2+) levels increased significantly in yeast cells after exposure to 3 mM and 7 mM sodium arsenite for 6 h compared with the control. These results indicated that high concentrations of arsenite-induced yeast cell killing was associated with elevated levels of intracellular ROS and Ca(2+).

Porphyrin-nanosensor conjugates. New tools for the measurement of intracellular response to reactive oxygen species

Reactive oxygen species (ROS) have for some time been implicated in the onset and progression of medical conditions including cancer, ageing, heart disease and Alzheimer's disease. Recently, it has been postulated that ROS play a much more subtle role in intracellular signalling mechanisms as second messengers. Given the importance of these species in influencing cellular processes, it is surprising that tools for studying intracellular levels of ROS are extremely limited and devices for studying the cells' response to internally generated ROS are virtually non-existent. In order to study the response of cells to intracellular ROS we have designed a nano-scale device that can both generate ROS and simultaneously monitor the cells' reaction as a function of changes in the important signalling ion, calcium. Here we report the synthesis, characterisation, and calibration of a new ROS nano-probe and demonstrate its ability to detect cellular response to elevated levels of intracellular ROS.

Common and distinct mechanisms of different redox‐active carcinogens involved in the transformation of mouse JB6P+ cells

We transformed JB6P+ cells with prolonged intermittent low-dose UVB radiation or prolonged exposure to low-dose H(2)O(2) or CdCl(2). Stable transformation was confirmed by an anchorage-independence assay. The JB6P+ transformants formed more colonies (approximately six folds) in soft agar as compared to their JB6P+ parent cells and were associated with increased intracellular reactive oxygen species (ROS) levels. Activating protein-1 (AP-1) is a family of transcription factors that are rapidly activated by elevated intracellular ROS levels, and their composition is important in the process of cellular transformation and/or tumor progression. To investigate if carcinogenesis induced by distinct carcinogens was via similar molecular mechanisms in these transformants, gel mobility shift and immunoblot analyses were utilized to determine the distinct AP-1 compositions. Compared to parent JB6P+ cells, the gain of JunB and Fra-1 in AP-1 DNA binding complexes was markedly increased in all transformed cells, which might contribute to a more proliferative phenotype, while loss of Fra-2 occurred in JB6P+/H(2)O(2) and JB6P+/Cd cells. Differential AP-1 components in the transformants suggested that their transformations might be mediated by distinct transcription signalings with distinct AP-1 dimer compositions. However, all three transformants exhibited increased activation of pathways involved in cell proliferation (ERK/Fra-1/AP-1 and JNK/c-jun/AP-1) and anti-apoptosis (Bcl-xl). The development of the JB6P+ transformants (JB6P+/UVB; JB6P+/H(2)O(2); JB6P+/Cd) provides a unique tool to study the mechanisms that contribute to different redox-active carcinogens in a single model.

Norepinephrine treatment and aging lead to systemic and intracellular oxidative stress in rats

Reactive oxygen species (ROS) play important roles in cellular senescence and organismic aging. Furthermore, they have been implicated in some of the adverse effects of chronic stress due to elevated peripheral levels of catecholamines. Here, we applied three different techniques to individually compare the systemic and intracellular oxidative stress in aged (23 months) and young (5 months) Sprague–Dawley rats, and in young rats treated for 12 or 24 h with norepinephrine (NE). Thiol groups of blood serum proteins (RSH) were determined by means of Ellman’s reaction. Intracellular ROS were assessed in spleen cells and peripheral blood lymphocytes (PBL) by carbonylation of cellular (spleen) proteins as determined by immunoblotting (Oxyblot) and/or by means of 2′,7′-dichlorofluorescein (DCF) fluorescence. As compared to the young, untreated controls, both old rats and NE treated young rats showed similarly lowered RSH values paralleled by elevated intracellular ROS levels or enhanced Oxyblot signals. Individual RSH values were highly significantly, negatively correlated with respective Oxyblot data as well as with DCF fluorescence. The results confirm the roles of ROS in aging and adrenergic stress in the rat model, and suggest that the decrease in RSH of blood serum may be taken as a valid indicator for the enhanced oxidative stress in lymphocytes.

Diallyl disulphide depletes glutathione in Candida albicans: oxidative stress‐mediated cell death studied by two‐photon microscopy

Using two-photon scanning laser microscopy, we investigated the effect of an Allium sativum (garlic) constituent, diallyl disulphide (DADS), on key physiological functions of the opportunistic pathogen Candida albicans. A short 30 min exposure to 0.5 mM DADS followed by removal induced 70% cell death (50% necrotic, 20% apoptotic) within 2 h, increasing to 75% after 4 h. The early intracellular events associated with DADS-induced cell death were monitored with two-photon fluorescence microscopy to track mitochondrial membrane potential (Deltapsi(m)), reactive oxygen species (ROS) and NADH or reduced glutathione (GSH) under aerobic conditions. DADS treatment decreased intracellular GSH and elevated intracellular ROS levels. Additionally, DADS induced a marked decrease of Deltapsi(m) and lowered respiration in cell suspensions and isolated mitochondria. In vitro kinetic experiments in cell-free extracts suggest that glutathione-S-transferase (GST) is one of the intracellular targets of DADS. Additional targets were also identified, including inhibition of a site or sites between complexes II-IV in the electron transport chain, as well as the mitochondrial ATP-synthase. The results indicate that DADS is an effective antifungal agent able to trigger cell death in Candida, most probably by eliciting oxidative stress as a consequence of thiol depletion and impaired mitochondrial function.

Implication of PTEN in production of reactive oxygen species and neuronal death in in vitro models of stroke and Parkinson's disease

Oxidative stress plays crucial role in the pathogenesis of neurodegenerative diseases. However, the precise mechanism for an increased production of reactive oxygen species (ROS) under pathological conditions is not yet fully understood. We have recently demonstrated an implication of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a tumor suppressor, in ROS generation and neuronal apoptosis induced by staurosporine. These findings raised further interest whether PTEN functions as a common mediator of oxidative stress in neurodegenerative processes. To address this issue, neural cells were exposed to oxygen-glucose deprivation (OGD) and to the neurotoxin 1-methyl-4-phenylpyridinium iodide (MPP +), which mimic cerebral ischemia and Parkinson's disease, respectively. OGD for 4 h followed by 16 h of reoxygenation or incubation with MPP + (250 μM) for 48 h induced 33% and 45% neuronal death in rat hippocampal and in human dopaminergic SH-SY5Y neurons, respectively, accompanied by a gradual increase in the intracellular level of ROS. The increase in ROS by OGD and by MPP + did not cause oxidative inactivation of PTEN and thus, PTEN remains constitutively active. In support, the protein level of PTEN was not reduced in both cell cultures after challenging with OGD or MPP +. Importantly, the elevated intracellular ROS levels and the neuronal death caused by OGD or by MPP + toxicity were significantly inhibited when PTEN was downregulated by a specific antisense oligonucleotide or by siRNA. Because SOD2 protein level is not altered either by knockdown of PTEN nor by an inhibition of the PI3K/Akt signalling, we suggest that SOD2 do not contribute to the pathomechanism of oxidative stress induced by PTEN or by inhibiting the related Akt signalling. The present study highlights PTEN as a crucial and common mediator of ROS generation and neuronal death and suggests that PTEN could become a potential therapeutic target for interfering with neurodegeneration.

Silica-Induced Apoptosis in Alveolar Macrophages: Evidence of in Vivo Thiol Depletion and the Activation of Mitochondrial Pathway

Studies have shown that silica induces apoptosis through mechanisms that also regulate the inflammatory responses of lung cells to silica exposure. Although implicated in cell culture studies, the major in vivo pathway through which silica induces apoptosis has not been characterized. The present study is to study the role of mitochondria in silica-induced oxidative stress and apoptosis in vivo. Rats were intratracheally instilled with saline or silica (20 mg/kg) and sacrificed at 3 days post-exposure unless otherwise specified. Alveolar macrophages (AM) were harvested by bronchoalveolar lavage and measured for apoptosis and secretion of inflammatory mediators in the presence or absence of appropriate inhibitors. Concurrent studies were carried out to determine the presence of intracellular reactive oxygen species (ROS) via confocal microscopy, mitochondrial trans-membrane potential by flow cytometry, mitochondrial release of cytochrome c, and the activation of caspase activities in AM by Western blot analysis. Silica was shown to induce elevated levels of intracellular ROS, resulting in a marked decrease in intracellular glutathione (GSH) and cysteine and a sustained presence of apoptotic AM in silica-exposed rats up to two weeks post-exposure. The apoptotic AM were characterized by decreased mitochondrial trans-membrane potential, increased mitochondrial release of cytochrome c, activated caspase 9 (but not caspase 8) and caspase 3 activities, and PARP degradation, comparing to cells from the saline control. Silica induced AM production of IL-1 and TNF-α, which may be inhibited by ex vivo treatment of cells with N-acetylcysteine (NAC) or microtubule modifiers such as tetrandrine and taxol. NAC was shown to prevent intracellular GSH depletion and silica-induced production of IL-1β and TNF-α but not apoptosis in AM from silica-exposed rats. These results show that silica-induced apoptosis is mediated through the mitochondrial pathway but not through cellular production of inflammatory cytokines, ROS generation, however, induces both apoptosis and cellular secretion of inflammatory mediators.

Vulnerability to ROS-induced cell death in ageing articular cartilage: The role of antioxidant enzyme activity

To test the hypothesis that age-related loss of chondrocytes in cartilage is associated with impaired reactive oxygen species (ROS) homeostasis resulting from reduced antioxidant defence. Cell numbers: The total number of chondrocytes in the articular cartilage of the femoral head of young, mature and old rats was estimated using an unbiased stereological method. ROS quantification: Fluorescence intensity in chondrocytes was quantified using the oxygen free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal laser scanning microscopy and densitometric image analysis. In order to delineate the reactive species, explants were pre-treated with N-acetylcysteine (NAC) or N(G)-nitro-l-arginine methyl ester (l-NAME) prior to ROS quantification. Induction of intracellular ROS: Explants were incubated in the redox-cycling drug menadione after which they underwent ROS quantification and cell-viability assay. Antioxidant enzyme activity: The activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX) was measured. Chondrocyte numbers: A significant and progressive loss of chondrocytes was observed with ageing. Cellular ROS levels: A significant age-related increase in cellular ROS-induced fluorescence was demonstrated. NAC significantly reduced ROS levels in old chondrocytes only. Induction of intracellular ROS: Menadione increased cellular ROS levels dose-dependently in young and old chondrocytes, with a greater effect in the latter. Old chondrocytes were more vulnerable to menadione-induced cytotoxicity. Antioxidant enzymes: Catalase activity declined significantly in aged cartilage whilst SOD and GPX activities were unaltered. Substantial loss of chondrocytes occurs in rat articular cartilage which may result from increased vulnerability to elevated intracellular ROS levels, consequent upon a decline in antioxidant defence.