Reactive Oxygen Species Components Research Articles

Development of a Physiologically Relevant Online Chemical Assay To Quantify Aerosol Oxidative Potential.

Large-scale epidemiological studies have shown a close correlation between adverse human health effects and exposure to ambient particulate matter (PM). The oxidative potential (OP) of ambient PM has been implicated in inducing toxic effects associated with PM exposure. In particular, reactive oxygen species (ROS), either bound to PM or generated by particulate components in vivo, substantially contribute to the OP and therefore toxicity of PM by lowering antioxidant concentrations in the lung, which can subsequently lead to oxidative stress, inflammation, and disease. Traditional methods for measuring aerosol OP are labor intensive and have poor time resolution, with significant delays between aerosol collection and ROS analysis. These methods may underestimate ROS concentrations in PM because of the potentially short lifetime of some ROS species; therefore, continuous online, highly time-resolved measurement of ROS components in PM is highly advantageous. In this work, we develop a novel online method for measuring aerosol OP based on ascorbic acid chemistry, an antioxidant prevalent in the lung, thus combining the advantages of continuous online measurement with a physiologically relevant assay. The method limit of detection is estimated for a range of atmospherically important chemical components such as Cu(II) 0.22 ± 0.03 μg m-3, Fe(II) 47.8 ± 5.5 μg m-3, Fe(III) 0.63 ± 0.05 μg m-3, and secondary organic aerosol 41.2 ± 6.9 μg m-3, demonstrating that even at this early stage of development, the online method is capable of measuring the OP of PM in polluted urban environments and smog chamber studies.

Oxidative Stress induced by Over-Expressed NADPH Oxidase (NOX) Initiates Breast Cancer, Promotes Metastasis via Epithelial to Mesenchymal Transition (EMT), Remodels Chromatin and Confers Drug Resistance.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) are the known enzymes in the human body that are exclusively involved in the production of reactive oxygen species (ROS). The trans-membrane protein NADPH oxidase is known to contain seven isoforms, NOX1-5 and DUOX1-2, which catalyze the generation of reactive oxygen species (ROS) needed for a variety of cellular functions. Strong anti-oxidant networks are necessary for cells to scavenge ROS and shield biological components from oxidative damage because too much ROS causes oxidative stress. The overexpression of NOX isoforms is typically linked to the carcinogenesis of several types of carcinomas, including breast cancer (BC). Because NOX isoforms are overexpressed in BC, they cause oxidative stress, damage mtDNA, and promote angiogenesis and EMT, which in turn aid in BC spreading. With the exception of BC, a number of NOX inhibitors are being used to treat different diseases. Although numerous researchers have suggested that blocking NOX activity is a viable therapeutic approach for individuals with various types of cancer, the therapeutic utility of this approach has not yet been confirmed by clinical trials. Given that breast cancer is the most frequent cancer globally, it’s critical to comprehend how ROS and NOX isoforms contribute to the pathobiology of the disease and investigate the possible therapeutic applications of NOX inhibitors in the treatment of breast cancer. In an effort to validate the role of NOX inhibitors as a successful therapeutic approach to controlling BC, this review paper attempts to give a thorough explanation of the involvement of NOX isoforms in the promotion of metastasis associated with BC.

Evidence that hydrogen peroxide, a component of oxidative stress, induces high-anxiety-related behaviour in mice

The link between oxidative stress and high-anxiety-related behaviour is uncontested; but the cause-effect relationship has yet to be completely elucidated. Here, the behavioural effects of hydrogen peroxide (H2O2), given to mice (n = 10 per group) in drinking water at 1%, were assessed in the light/dark choice test, the open field, the elevated-plus maze and the hole-board test. Compared to controls (drinking only water), subacute exposure (10–15 days) of mice to H2O2, the major component of reactive oxygen species (ROS) and the precursor of potent oxidants (hydroxyl radical and hypochlorous acid), affected emotional responses by inducing an anxious behaviour associated with hyperactivity. Our findings clearly showed that H2O2-treated mice exhibited anxiogenic behaviour in the light/dark choice test and in the hole-board test. Moreover, H2O2-treated mice displayed a hyperactive behaviour, revealed by a significant increase in the number of crossings made in the open field test relative to controls. Although H2O2-exposed mice made significantly less head-dippings in the open arms than controls, H2O2-induced hyperactivity may have blurred anxiogenic-like behaviour in H2O2-treated mice in the elevated-plus maze. Our findings provide the evidence that H2O2, an oxidizing component, caused high-anxiety-related behaviour associated with hyperactivity in mice. Antioxidants may play a role in preventing or attenuating oxidative stress-related anxiety.

Neutrophil-Hepatic Stellate Cell Interactions Promote Fibrosis inExperimental Steatohepatitis.

Background & AimsHepatic infiltration of neutrophils is a hallmark of steatohepatitis; however, the role of neutrophils in the progression of steatohepatitis remains unknown.MethodsA clinically relevant mouse model of steatohepatitis induced by high-fat diet (HFD) plus binge ethanol feeding was used. Liver fibrosis was examined. In vitro cell culture was used to analyze the interaction of hepatic stellate cells (HSCs) and neutrophils.ResultsHFD plus one binge ethanol (HFD+1B) feeding induced significant hepatic neutrophil infiltration, liver injury, and fibrosis. HFD plus multiple binges of ethanol (HFD+mB) caused more pronounced liver fibrosis. Microarray analyses showed that the most highly activated signaling pathway in this HFD+1B model was related to liver fibrosis and HSC activation. Blockade of chemokine (C-X-C motif) ligand 1 or intercellular adhesion molecule-1 expression reduced hepatic neutrophil infiltration and ameliorated liver injury and fibrosis. Disruption of the p47phox gene (also called neutrophil cytosolic factor 1), a critical component of reactive oxygen species producing nicotinamide adenine dinucleotide phosphate-oxidase in neutrophils, diminished HFD+1B–induced liver injury and fibrosis. Co-culture of HSCs with neutrophils, but not with neutrophil apoptotic bodies, induced HSC activation and prolonged neutrophil survival. Mechanistic studies showed that activated HSCs produce granulocyte-macrophage colony-stimulating factor and interleukin-15 to prolong the survival of neutrophils, which may serve as a positive forward loop to promote liver damage and fibrosis.ConclusionsThe current data from a mouse model of HFD plus binge ethanol feeding suggest that obesity and binge drinking synergize to promote liver fibrosis, which is partially mediated via the interaction of neutrophils and HSCs. Microarray data in this article have been uploaded to NCBI’s Gene Expression Omnibus (GEO accession number: GSE98153).

Melatonin ameliorates intrarenal renin-angiotensin system in a 5/6 nephrectomy rat model.

Activation of the intrarenal renin-angiotensin system (RAS) plays a critical role in the pathophysiology of chronic kidney disease (CKD) and hypertension. It has been reported that reactive oxygen species (ROS) are important components of intrarenal RAS activation. Melatonin is recognized as a powerful antioxidant, and we recently reported that impaired nighttime melatonin secretion correlates negatively with urinary angiotensinogen excretion, the surrogate marker of intrarenal RAS activity in patients with CKD. However, whether melatonin supplementation ameliorates the augmentation of intrarenal RAS in CKD has remained unknown. We aimed to clarify whether exogenous melatonin ameliorates intrarenal RAS activation via the reduction of ROS production. 5/6 Nephrectomized (Nx) rats were used as a chronic progressive CKD model and compared with sham-operated control rats. The Nx rats were divided into untreated Nx rats and melatonin-treated Nx rats. The levels of intrarenal RAS, ROS components, and renal injury were evaluated after 4weeks of treatment. Compared with the control rats, the untreated Nx rats exhibited significant increases in intrarenal angiotensinogen, angiotensin II (AngII) type 1 receptors, and AngII, accompanied by elevated blood pressure, higher oxidative stress (8-hydroxy-2'-deoxyguanosine), lower antioxidant (superoxide dismutase) activity, and increased markers of interstitial fibrosis (α-smooth muscle actin, Snail, and type I collagen) in the remnant kidneys. Treatment with melatonin significantly reversed these abnormalities. Antioxidant treatment with melatonin was shown to ameliorate intrarenal RAS activation and renal injury in a 5/6 Nx rat model.

NF-E2-Related Factor 2 Suppresses Intestinal Fibrosis by Inhibiting Reactive Oxygen Species-Dependent TGF-β1/SMADs Pathway.

This study aimed to evaluate the antifibrotic effects of NF-E2-Related Factor 2 (Nrf2) on intestinal fibrosis. Intestinal fibrosis is a common complication of Crohn's disease; however, its mechanism of intestinal fibrosis is largely unclear. BALB/c mice received 2,4,6-trinitrobenzene sulfonic acid weekly via intrarectal injections to induce chronic fibrotic colitis. They also diet containing received 1% (w/w) tert-butylhydroquinone (tBHQ), which is an agonist of Nrf2. Human intestinal fibroblasts (CCD-18Co cells) were pretreated with tBHQ or si-Nrf2 followed by stimulation with transforming growth factor-β1 (TGF-β1), which transformed the cells into myofibroblasts. The main fibrosis markers such as α-smooth muscle actin, collagen I, tissue inhibitor of metalloproteinase-1, and TGF-β1/SMADs signaling pathway were detected by quantitative real-time RT-PCR, immunohistochemical analysis, and Western blot analysis. Levels of cellular reactive oxygen species (ROS) were detected by dichlorodihydrofluorescein diacetate. tBHQ suppressed the intestinal fibrosis through the TGF-β1/SMADs signaling pathway in TNBS-induced colitis and CCD-18Co cells. Moreover, Nrf2 knockdown enhanced the TGF-β1-induced differentiation of CCD-18Co cells. ROS significantly increased in TGF-β1-stimulated CCD-18Co cells. Pretreatment with H2O2, the primary component of ROS, was demonstrated to block the effect of tBHQ on reducing the expression of TGF-β1. Moreover, scavenging ROS by N-acetyl cysteine could inhibit the increasing expression of TGF-β1 promoted by Nrf2 knockdown. The results suggested that Nrf2 suppressed intestinal fibrosis by inhibiting ROS/TGF-β1/SMADs pathway in vivo and in vitro.

Aldose reductase mediates endothelial cell dysfunction induced by high uric acid concentrations

BackgroundUric acid (UA) is an antioxidant found in human serum. However, high UA levels may also have pro-oxidant functions. According to previous research, aldose reductase (AR) plays a vital role in the oxidative stress-related complications of diabetes. We sought to determine the mechanism by which UA becomes deleterious at high concentrations as well as the effect of AR in this process.MethodEndothelial cells were divided into three groups cultured without UA or with 300 μM or 600 μM UA. The levels of total reactive oxygen species (ROS), of four ROS components, and of NO and NOX4 expression were measured. Changes in the above molecules were detected upon inhibiting NOX4 or AR, and serum H2O2 and vWF levels were measured in vivo.ResultsIncreased AR expression in high UA-treated endothelial cells enhanced ROS production by activating NADPH oxidase. These effects were blocked by the AR inhibitor epalrestat. 300 μM UA decreased the levels of the three major reactive oxygen species (ROS) components: O2•-, •OH, and 1O2. However, when the UA concentration was increased, both O2•- levels and downstream H2O2 production significantly increased. Finally, an AR inhibitor reduced H2O2 production in hyperuricemic mice and protected endothelial cell function.ConclusionsOur findings indicate that inhibiting AR or degrading H2O2 could protect endothelial function and maintain the antioxidant activities of UA. These findings provide new insight into the role of UA in chronic kidney disease.

An automated online instrument to quantify aerosol-bound reactive oxygen species (ROS) for ambient measurement and health-relevant aerosol studies

Abstract. The adverse health effects associated with ambient aerosol particles have been well documented, but it is still unclear which aerosol properties are most important for their negative health impact. Some studies suggest the oxidative effects of particle-bound reactive oxygen species (ROS) are potential major contributors to the toxicity of particles. Traditional ROS measurement techniques are labour-intensive, give poor temporal resolution and generally have significant delays between aerosol sampling and ROS analysis. However, many oxidising particle components are reactive and thus potentially short-lived. Thus, a technique to quantify particle-bound ROS online would be beneficial to quantify also the short-lived ROS components. We introduce a new portable instrument to allow online, continuous measurement of particle-bound ROS using a chemical assay of 2′7′-dichlorofluorescein (DCFH) with horseradish peroxidase (HRP), via fluorescence spectroscopy. All components of the new instrument are attached to a containing shell, resulting in a compact system capable of automated continuous field deployment over many hours or days. From laboratory measurements, the instrument was found to have a detection limit of ∼ 4 nmol [H2O2] equivalents per cubic metre (m3) air, a dynamic range up to at least ∼ 2000 nmol [H2O2] equivalents per m3 air and a time resolution of ≤ 12 min. The instrument allows for ∼ 16 h automated measurement if unattended and shows a fast response to changes in concentrations of laboratory-generated oxidised organic aerosol. The instrument was deployed at an urban site in London, and particulate ROS levels of up to 24 nmol [H2O2] equivalents per m3 air were detected with PM2.5 concentrations up to 28 µg m−3. The new and portable Online Particle-bound ROS Instrument (OPROSI) allows fast-response quantification; this is important due to the potentially short-lived nature of particle-bound ROS as well as fast-changing atmospheric conditions, especially in urban environments. The instrument design allows for automated operation and extended field operation with twice-daily presence of an operator. As well as having sensitivity suitable for ambient level measurement, the instrument is also suitable at concentrations such as those required for laboratory and chamber toxicological studies.

A superoxide dismutase/catalase mimetic nanomedicine for targeted therapy of inflammatory bowel disease.

Oxidative stress, resulting from excessive generation of reactive oxygen species (ROS), plays a pivotal role in the initiation and progression of inflammatory bowel disease (IBD). To develop an efficacious and safe nanotherapy against IBD, we designed and developed a superoxide dismutase/catalase mimetic nanomedicine comprising a hydrogen peroxide-eliminating nanomatrix and a free radical scavenger Tempol (Tpl). To this end, an oxidation-responsive β-cyclodextrin material (OxbCD) was synthesized, and a Tpl-loaded OxbCD nanoparticle (Tpl/OxbCD NP) was produced. Hydrolysis of OxbCD NP could be triggered by hydrogen peroxide, leading to on-demand release of loaded Tpl molecules from Tpl/OxbCD NP. OxbCD NP was able to efficiently accumulate in the inflamed colon in mice, thereby dramatically reducing nonspecific distribution after oral delivery. In three mouse colitis models, oral administration of Tpl/OxbCD NP notably mitigated manifestations relevant to colitis, and significantly suppressed expression of proinflammatory mediators, with the efficacy superior over free Tpl or a control nanomedicine based on poly(lactide-co-glycolide) (PLGA). Accordingly, by scavenging multiple components of ROS, Tpl/OxbCD NP may effectively reduce ulcerative colitis in mice, and it can be intensively developed as a translational nanomedicine for the management of IBD and other inflammatory diseases.

A novel sensor to estimate the prevalence of hypochlorous (HOCl) toxicity in individuals with type 2 diabetes and dyslipidemia

BackgroundOxidative stress is common in type 2 diabetes. It is characterised by increased levels of reactive oxygen species (ROS), of which hypochlorous acid (HOCl) is an important component. Type 2 diabetes is characterised not only by hyperglycemia, but also by dyslipidemia. It probably underlies both the development of diabetes and also resulting complications, like cardiovascular disease. We applied a novel fluoroprobe RHQ to estimate endogenous HOCl and myeloperoxidase (MPO) activity in diabetes and dyslipidemia. MethodsOur newly designed probe, RHQ (rhodamine–quinoline based chemodosimeter) is capable of estimating endogenous HOCl selectively out of the ROS components. Isolated leukocytes from study subjects were treated with DCFDA and monocytes and neutrophils with RHQ for estimating endogenous ROS and HOCl respectively. Plasma AOPP, an indicator of HOCl was also measured. We attempted to find out the key reasons of higher HOCl content in diabetic dyslipidemic subjects by quantitating endogenous hydrogen peroxide (H2O2) and myeloperoxidase (MPO) activity. ResultsIsolated PBMCs from diabetic dyslipidemic subjects indicated enhanced ROS and HOCl generation followed by diabetic subjects without dyslipidemia and healthy controls. We explored increased production of H2O2 and enhanced enzymatic activity of myeloperoxidase (MPO) among diabetic dyslipidemic subjects (p<0.0001) resulting in higher HOCl content. ConclusionThe hyperglycemic and hyperlipidemic challenges together enhance the production of HOCl and the fluoroprobe RHQ may be used as a novel diagnostic marker to evaluate the extent of this toxicity.

Plant Aquaporin AtPIP1;4 Links Apoplastic H2O2 Induction to Disease Immunity Pathways.

Hydrogen peroxide (H2O2) is a stable component of reactive oxygen species, and its production in plants represents the successful recognition of pathogen infection and pathogen-associated molecular patterns (PAMPs). This production of H2O2 is typically apoplastic but is subsequently associated with intracellular immunity pathways that regulate disease resistance, such as systemic acquired resistance and PAMP-triggered immunity. Here, we elucidate that an Arabidopsis (Arabidopsis thaliana) aquaporin (i.e. the plasma membrane intrinsic protein AtPIP1;4) acts to close the cytological distance between H2O2 production and functional performance. Expression of the AtPIP1;4 gene in plant leaves is inducible by a bacterial pathogen, and the expression accompanies H2O2 accumulation in the cytoplasm. Under de novo expression conditions, AtPIP1;4 is able to mediate the translocation of externally applied H2O2 into the cytoplasm of yeast (Saccharomyces cerevisiae) cells. In plant cells treated with H2O2, AtPIP1;4 functions as an effective facilitator of H2O2 transport across plasma membranes and mediates the translocation of externally applied H2O2 from the apoplast to the cytoplasm. The H2O2-transport role of AtPIP1;4 is essentially required for the cytoplasmic import of apoplastic H2O2 induced by the bacterial pathogen and two typical PAMPs in the absence of induced production of intracellular H2O2 As a consequence, cytoplasmic H2O2 quantities increase substantially while systemic acquired resistance and PAMP-triggered immunity are activated to repress the bacterial pathogenicity. By contrast, loss-of-function mutation at the AtPIP1;4 gene locus not only nullifies the cytoplasmic import of pathogen- and PAMP-induced apoplastic H2O2 but also cancels the subsequent immune responses, suggesting a pivotal role of AtPIP1;4 in apocytoplastic signal transduction in immunity pathways.

Global Plant Stress Signaling: Reactive Oxygen Species at the Cross-Road.

Current technologies have changed biology into a data-intensive field and significantly increased our understanding of signal transduction pathways in plants. However, global defense signaling networks in plants have not been established yet. Considering the apparent intricate nature of signaling mechanisms in plants (due to their sessile nature), studying the points at which different signaling pathways converge, rather than the branches, represents a good start to unravel global plant signaling networks. In this regard, growing evidence shows that the generation of reactive oxygen species (ROS) is one of the most common plant responses to different stresses, representing a point at which various signaling pathways come together. In this review, the complex nature of plant stress signaling networks will be discussed. An emphasis on different signaling players with a specific attention to ROS as the primary source of the signaling battery in plants will be presented. The interactions between ROS and other signaling components, e.g., calcium, redox homeostasis, membranes, G-proteins, MAPKs, plant hormones, and transcription factors will be assessed. A better understanding of the vital roles ROS are playing in plant signaling would help innovate new strategies to improve plant productivity under the circumstances of the increasing severity of environmental conditions and the high demand of food and energy worldwide.

Reverse function of ROS-induced CBL10 during salt and drought stress responses

Cellular levels of Ca2+ and reactive oxygen species (ROS) are maintained at low levels in the cytosol but fluctuate greatly when acting as second messengers to decode environmental and developmental signals. Phytohormones are primary signals leading to various changes in ROS or Ca2+ signaling during synergistic and antagonistic cross-talk. In this study, we found that brassinosteroids (BRs), hormones involved in diverse plant developmental processes, promote ROS production. To identify downstream signaling components of ROS during BR-mediated plant development, we searched for genes whose expression remained unchanged by ROS only in BR- signaling mutants and found calcineurin B-like (CBL) 10, which encodes a CBL should be changed to CBL10. protein that senses calcium. ROS-induced CBL10 expression was nullified and endogenous CBL10 expression in the shoot was low in the BR-signaling mutant. Using a cbl10 mutant and a transgenic plant overexpressing CBL10, we showed that BR sensitivity during hypocotyl growth decreased in the cbl10 mutant under salt stress, providing an additional mechanism for positive regulation of salt stress by CBL10. We also demonstrated that CBL10 negatively affects tolerance to drought and is not mediated by abscisic acid-induced signaling. Our results suggest that Ca2+ signaling through CBL10 differently affects the response to abiotic stresses, partly by regulating BR sensitivity of plant tissues.

Hyperthermia, dehydration, and osmotic stress: unconventional sources of exercise-induced reactive oxygen species.

Evidence of increased reactive oxygen species (ROS) production is observed in the circulation during exercise in humans. This is exacerbated at elevated body temperatures and attenuated when normal exercise-induced body temperature elevations are suppressed. Why ROS production during exercise is temperature dependent is entirely unknown. This review covers the human exercise studies to date that provide evidence that oxidant and antioxidant changes observed in the blood during exercise are dependent on temperature and fluid balance. We then address possible mechanisms linking exercise with these variables that include shear stress, effects of hemoconcentration, and signaling pathways involving muscle osmoregulation. Since pathways of muscle osmoregulation are rarely discussed in this context, we provide a brief review of what is currently known and unknown about muscle osmoregulation and how it may be linked to oxidant production in exercise and hyperthermia. Both the circulation and the exercising muscle fibers become concentrated with osmolytes during exercise in the heat, resulting in a competition for available water across the muscle sarcolemma and other tissues. We conclude that though multiple mechanisms may be responsible for the changes in oxidant/antioxidant balance in the blood during exercise, a strong case can be made that a significant component of ROS produced during some forms of exercise reflect requirements of adapting to osmotic challenges, hyperthermia challenges, and loss of circulating fluid volume.

Cytotoxicity and genotoxicity of urban particulate matter in mammalian cells.

Ambient air particulate matter (PM)-associated reactive oxygen species (ROS) have been linked to a variety of altered cellular outcomes. In this study, three different PM samples from diesel exhaust particles (DEPs), urban dust standard reference material SRM1649a and air collected in Manchester have been tested for their ability to oxidise DNA in a cell-free assay, to increase intracellular ROS levels and to induce CYP1A1 gene expression in mammalian cells. In addition, the cytotoxicity and genotoxicity of PM were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and alkaline comet assay, respectively. All PM samples catalysed the Fenton reaction in a cell-free assay, but only DEP resulted in the generation of ROS as measured by dichlorodihydrofluorescein diacetate oxidation in mammalian cells. However, there was no evidence that increased ROS was a consequence of polycyclic aromatic hydrocarbon metabolism via CYP1A1 induction as urban dust, the Manchester dust samples but not DEP-induced CYP1A1 expression. Urban dust was more cytotoxic in murine embryonic fibroblasts (MEFs) than the other PM samples and also induced expression of GADD45a in the GreenScreen Human Cell assay without S9 activation suggesting the presence of a direct-acting genotoxicant. Urban dust and DEP produced comparable levels of DNA damage, as assessed by the alkaline comet assay, in MEFs at higher levels than those induced by Manchester PM. In conclusion, results from the cytotoxic and genotoxic assays are not consistent with ROS production being the sole determinant of PM-induced toxicity. This suggests that the organic component can contribute significantly to this toxicity and that further work is required to better characterise the extent to which ROS and organic components contribute to PM-induced toxicity.

Endogenous hydrogen peroxide in the hypothalamic paraventricular nucleus regulates neurohormonal excitation in high salt-induced hypertension

Reactive oxygen species (ROS) in the brain plays an important role in the progression of hypertension and hydrogen peroxide (H2O2) is a major component of ROS. The aim of this study is to explore whether endogenous H2O2 changed by polyethylene glycol-catalase (PEG-CAT) and aminotriazole (ATZ) in the hypothalamic paraventricular nucleus (PVN) regulates neurotransmitters, renin-angiotensin system (RAS), and cytokines, and whether subsequently affects the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in high salt-induced hypertension. Male Sprague–Dawley rats received a high-salt diet (HS, 8% NaCl) or a normal-salt diet (NS, 0.3% NaCl) for 10 weeks. Then rats were treated with bilateral PVN microinjection of PEG-CAT (0.2 i.u./50nl), an analog of endogenous catalase, the catalase inhibitor ATZ (10nmol/50nl) or vehicle. High salt-fed rats had significantly increased MAP, RSNA, plasma norepinephrine (NE) and pro-inflammatory cytokines (PICs). In addition, rats with high-salt diet had higher levels of NOX-2, NOX-4 (subunits of NAD(P)H oxidase), angiotensin-converting enzyme (ACE), interleukin-1beta (IL-1β), glutamate and NE, and lower levels of gamma-aminobutyric acid (GABA) and interleukin-10 (IL-10) in the PVN than normal diet rats. Bilateral PVN microinjection of PEG-CAT attenuated the levels of RAS and restored the balance of neurotransmitters and cytokines, while microinjection of ATZ into the PVN augmented those changes occurring in hypertensive rats. Our findings demonstrate that ROS component H2O2 in the PVN regulating MAP and RSNA are partly due to modulate neurotransmitters, renin-angiotensin system, and cytokines within the PVN in salt-induced hypertension.

Effects of UV-A LED light irradiation on growth of cultured RAW 264.7 cells

The aim of this study was to examine the effects of ultraviolet A (UV-A) irradiation-induced damage on cultured macrophage RAW 264.7 cells and determine which components produced these manifestations. RAW 264.7 cells were irradiated with 365 nm UV-A using a light-emitting diode (LED). Cell viability and damage were determined using a calcein-AM and propidium iodide dual-staining assay and lactate dehydrogenase leakage, respectively. Intracellular reactive oxygen species (ROS) were measured by H2DCF-DA. The components of ROS in each medium were measured using an electron paramagnetic resonance (EPR) spectrometer in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC). While UV-A irradiation for 2 min significantly suppressed cell growth, LDH leakage did not occur. Addition of N-acetyl cysteine restored inhibition of cell proliferation, and reduced intracellular ROS levels. The EPR signal in the presence of TPC increased with time but was decreased by sodium azide. In addition, a typical EPR spectrum was obtained in the presence of DMPO, indicating the presence of a hydroxyradical. The spectrum was diminished by L-histidine. Data suggest that ROS generated in cells or culture medium by UV-A irradiation is predominantly singlet oxygen, and this singlet oxygen suppressed cell proliferation.

Comparison of on-line and off-line methods to quantify reactive oxygen species (ROS) in atmospheric aerosols

Atmospheric aerosol particle concentrations have been linked with a wide range of pulmonary and cardio-vascular diseases but the particle properties responsible for these negative health effects are largely unknown. It is often speculated that reactive oxygen species (ROS) present in atmospheric particles lead to oxidative stress in, and ultimately disease of, the human lung. The quantification of ROS is highly challenging because some ROS components such as radicals are highly reactive and therefore short-lived. Thus, fast analysis methods are likely advantageous over methods with a long delay between aerosol sampling and ROS analysis. We present for the first time a detailed comparison of conventional off-line and fast on-line methods to quantify ROS in organic aerosols. For this comparison a new and fast on-line instrument was built and characterized to quantify ROS in aerosol particles with high sensitivity and a limit of detection of 4 nmol H2O2 equivalents per m3 air. ROS concentrations are measured with a time resolution of approximately 15 min, which allows the tracking of fast changing atmospheric conditions. The comparison of the off-line and on-line method shows that, in oxidized organic model aerosol particles, the majority of ROS have a very short lifetime of a few minutes whereas a small fraction is stable for a day or longer. This indicates that off-line techniques, where there is often a delay of hours to days between particle collection and ROS analysis, may severely underestimate true ROS concentrations and that fast on-line techniques are necessary for a reliable ROS quantification in atmospheric aerosol particles and a meaningful correlation with health outcomes.

Neuroprotection by genipin against reactive oxygen and reactive nitrogen species-mediated injury in organotypic hippocampal slice cultures

Genipin, the multipotent ingredient in Gardenia jasmenoides fruit extract (GFE), may be an effective candidate for treatment following stroke or traumatic brain injury (TBI). Secondary injury includes damage mediated by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can alter the biological function of key cellular structures and eventually lead to cell death. In this work, we studied the neuroprotective potential of genipin against damage stemming from ROS and RNS production in organotypic hippocampal slice cultures (OHSC), as well as its potential as a direct free radical scavenger. A 50µM dose of genipin provided significant protection against tert-butyl hydroperoxide (tBHP), a damaging organic peroxide. This dosage of genipin significantly reduced cell death at 48h compared to vehicle control (0.1% DMSO) when administered 0, 1, 6, and 24h after addition of tBHP. Similarly, genipin significantly reduced cell death at 48h when administered 0, 1, 2, and 6h after addition of rotenone, which generates reactive oxygen species via a more physiologically relevant mechanism. Furthermore, genipin significantly reduced both cell death and nitrite levels at 24h caused by S-nitroso-N-acetylpenicillamine (SNAP), a direct nitric oxide (NO) donor, and successfully quenched 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, suggesting that genipin may act as a direct free radical scavenger. Our encouraging findings suggest that genipin should be tested in animal models of CNS injury with a significant component of ROS- and RNS-mediated damage, such as TBI and stroke, to assess its in vivo efficacy.

Development and testing of an online method to measure ambient fine particulate reactive oxygen species (ROS) based on the 2',7'-dichlorofluorescin (DCFH) assay

Abstract. An online, semi-continuous instrument to measure fine particle (PM2.5) reactive oxygen species (ROS) was developed based on the fluorescent probe 2'7'-dichlorofluorescin (DCFH). Parameters that influence probe response were first characterized to develop an optimal method for use in a field instrument. The online method used a mist chamber scrubber to collect total (gas plus particle) ROS components (ROSt) alternating with gas phase ROS (ROSg) by means of an inline filter. Particle phase ROS (ROSp) was determined by the difference between ROSt and ROSg. The instrument was deployed in urban Atlanta, Georgia, USA, and at a rural site during various seasons. Concentrations from the online instrument generally agreed well with those from an intensive filter measurement of ROSp. Concentrations of the ROSp measurements made with this instrument were lower than reported in other studies, often below the instrument's average limit of detection (0.15 nmol H2O2 equivalents m−3). Mean ROSp concentrations were 0.26 nmol H2O2 equivalents m−3 at the Atlanta urban sites compared to 0.14 nmol H2O2 equivalents m−3 at the rural site.