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Most of the ONOO - fluorescent probes have restricted applications because of their aggregation-caused quenching (ACQ) effect, long response time and low fluorescence enhancement. Herein, we developed a novel AIEgen fluorescent probe (PE-XY) based on a benzothiazole and quinolin scaffold with high sensitivity and selectivity for imaging of ONOO - . The results indicated that probe PE-XY exhibited fast response towards ONOO - with 2000-fold enhancement of fluorescence intensity ratio in vitro . Moreover, PE-XY exhibited a relatively high sensitivity (limit of detection: 8.58 nM), rapid response (<50 s), high fluorescence quantum yield ( δ = 0.81) and excellent selectivity over other analytes towards ONOO - in vitro . Furthermore, PE-XY was successfully applied to detect endogenous ONOO - levels in living HeLa cells, C. elegans and inflammatory mice with low cytotoxicity. Overall, this work provided a novel fast-response and highly selective AIEgen fluorescent probe for real-time monitoring ONOO - fluctuations in living systems.

Peroxynitrite (ONOO - ) is a highly reactive substance, and plays an essential part in maintaining cellular homeostasis. It is crucial to monitor the ONOO - level in cells in normal and abnormal states. We introduced a p-dimethylaminophenylether-based fluorescent probe PDPE-PN, which could be synthesized readily. The new probe had prominent sensitivity and specificity, and a fast response towards ONOO - . The spectral performance of probe PDPE-PN was outstanding and the limit of detection was 69 nM. Probe PDPE-PN with low toxicity was applied to detect endogenous/exogenous ONOO - in RAW 264.7 macrophages and zebrafish. Importantly, successful application of the new receptor opens up new ideas for the design of ONOO - probes.

Photoacoustic (PA) technology can transform light energy into acoustic wave, which can be used for either imaging or therapy that depends on the power density of pulsed laser. Here, we report photosensitizer-free polymeric nanocapsules loaded with nitric oxide (NO) donors, namely NO-NCPs, formulated from NIR light-absorbable amphiphilic polymers and a NO-releasing donor, DETA NONOate. Controlled NO release and nanocapsule dissociation are achieved in acidic lysosomes of cancer cells. More importantly, upon pulsed laser irradiation, the PA cavitation can excite water to generate significant reactive oxygen species (ROS) such as superoxide radical (O 2 .- ), which further spontaneously reacts with the in situ released NO to burst highly cytotoxic peroxynitrite (ONOO - ) in cancer cells. The resultant ONOO - generation greatly promotes mitochondrial damage and DNA fragmentation to initiate programmed cancer cell death. Apart from PA imaging, PA cavitation can intrinsically amplify reactive species via photosensitization-free materials for promising disease theranostics., (© 2020 Wiley-VCH GmbH.)

Drug-induced liver injury (DILI) has been a hot issue of public health, owing to its unpredictability and serious harm to public health. Peroxynitrite (ONOO - ) is an important biomarker for the assessment and diagnosis of DILI. In this article, based on a kind of rhodamine analogue with a near-infrared (NIR) emission (610 nm-800 nm) and a two-photon absorption cross section (54 GM), a two-photon excited NIR fluorescence probe (NIR-ONOO) for ONOO - was developed. With a high selectivity and a high sensitivity to ONOO - , NIR-ONOO has a linear range for detection of ONOO - from 5.0 × 10 -8 to 1.0 × 10 -5  M, a good detection limit (15 nM) and a large fluorescence enhancement (340-fold). In addition, NIR-ONOO has been used to monitor ONOO - in cells with satisfactory results. Because of its two-photon excied NIR emission, NIR-ONOO also showed excellent performances for imaging ONOO - including low autofluorescence, stable and persistent fluorescence, and a deep penetration (204 μm). Finally, NIR-ONOO was successfully employed to image ONOO - in inflammatory mouse, drug-induced hepatotoxicity in cells and its remediation. All the results indicated that NIR-ONOO is a powerful chemical tool to image ONOO - and assay drug-induced hepatotoxicity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Environmental pollutants like fine particulate matter can cause adverse health effects through oxidative stress and inflammation. Reactive oxygen and nitrogen species (ROS/RNS) such as peroxynitrite can chemically modify proteins, but the effects of such modifications on the immune system and human health are not well understood. In the course of inflammatory processes, the Toll-like receptor 4 (TLR4) can sense damage-associated molecular patterns (DAMPs). Here, we investigate how the TLR4 response and pro-inflammatory potential of the proteinous DAMPs α-Synuclein (α-Syn), heat shock protein 60 (HSP60), and high-mobility-group box 1 protein (HMGB1), which are relevant in neurodegenerative and cardiovascular diseases, changes upon chemical modification with peroxynitrite. For the peroxynitrite-modified proteins, we found a strongly enhanced activation of TLR4 and the pro-inflammatory transcription factor NF-κB in stable reporter cell lines as well as increased mRNA expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-8 in human monocytes (THP-1). This enhanced activation of innate immunity via TLR4 is mediated by covalent chemical modifications of the studied DAMPs. Our results show that proteinous DAMPs modified by peroxynitrite more potently amplify inflammation via TLR4 activation than the native DAMPs, and provide first evidence that such modifications can directly enhance innate immune responses via a defined receptor. These findings suggest that environmental pollutants and related ROS/RNS may play a role in promoting acute and chronic inflammatory disorders by structurally modifying the body's own DAMPs. This may have important consequences for chronic neurodegenerative, cardiovascular or gastrointestinal diseases that are prevalent in modern societies, and calls for action, to improve air quality and climate in the Anthropocene., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic β-cell damage during the pathogenesis of autoimmune diabetes. While β-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in β-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of β-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that β-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 ( Prdx1 ) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 ( Prdx2 ) had no effect. Together, these results suggest that β-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.

1,4-Dihydropyridines (1,4-DHP) possess important biochemical and pharmacological properties, including antioxidant and antimutagenic activities. AV-153-Na, an antimutagenic and DNA-repair enhancing compound was shown to interact with DNA by intercalation. Here we studied DNA binding of several AV-153 salts to evaluate the impact of AV-153 modifications on its DNA binding capacity, the ability to scavenge the peroxynitrite, to protect HeLa and B-cells cells against DNA damage. Affinity of the AV-153 salts to DNA measured by a fluorescence assay was dependent on the metal ion forming a salt in position 4 of the 1,4-DHP, and it decreased as follows: Mg > Na > Ca > Li > Rb > K. AV-153-K and AV-153-Rb could not react chemically with peroxynitrite as opposed to AV-153-Mg and AV-153-Ca, the latter increased the decomposition rate of peroxynitrite. AV-153-Na and AV-153-Ca effectively reduced DNA damage induced by peroxynitrite in HeLa cells, while AV-153-K and AV-153-Rb were less effective, AV-153-Li did not protect the DNA, and AV-153-Mg even caused DNA damage itself. The Na, K, Ca and Mg AV-153 salts were also shown to reduce the level of DNA damage in human B-cells from healthy donors. Thus, metal ions modify both DNA-binding and DNA-protecting effects of the AV-153 salts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Elevated H 2 O 2 is implicated in many cardiovascular diseases. We previously demonstrated that H 2 O 2 -induced endothelial nitric oxide synthase (eNOS) activation and excessive NO production contribute to vascular cell injury and increases in microvessel permeability. However, the mechanisms of excessive NO-mediated vascular injury and hyperpermeability remain unknown. This study aims to examine the functional role of NO-derived peroxynitrite (ONOO - ) in H 2 O 2 -induced vascular barrier dysfunction by elucidating the interrelationships between H 2 O 2 -induced NO, superoxide, ONOO - , and changes in endothelial [Ca 2+ ] i and microvessel permeability. Experiments were conducted on intact rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca 2+ ] i , NO, and O 2 - were assessed with fluorescence imaging. Perfusion of vessels with H 2 O 2 (10 µmol/L) induced marked productions of NO and O 2 - , resulting in extensive protein tyrosine nitration, a biomarker of ONOO - . The formation of ONOO - was abolished by inhibition of NOS with N G -Methyl-L-arginine. Blocking NO production or scavenging ONOO - by uric acid prevented H 2 O 2 -induced increases in endothelial [Ca 2+ ] i and Lp. Additionally, the application of exogenous ONOO - to microvessels induced delayed and progressive increases in endothelial [Ca 2+ ] i and microvessel Lp, a pattern similar to that observed in H 2 O 2 -perfused vessels. Importantly, ONOO - caused further activation of eNOS with amplified NO production. We conclude that the augmentation of NO-derived ONOO - is essential for H 2 O 2 -induced endothelial Ca 2+ overload and progressively increased microvessel permeability, which is achieved by self-promoted amplifications of NO-dependent signaling cascades. This novel mechanism provides new insight into the reactive oxygen and/or reactive nitrogen species-mediated vascular dysfunction in cardiovascular diseases., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa.

Peroxynitrite is a reactive nitrogen species produced in the intravascular compartment from superoxide anion and nitric oxide. Peroxynitrite destroys blood plasma proteins and membranes of red blood cells and of platelets. This explains why excessive production of peroxynitrite contributes to diseases and to ageing. Therapeutics that antagonize peroxynitrite may delay ageing and the progression of disease. We developed an in vitro assay that allows the investigation of the oxidative damage caused by peroxynitrite in the intravascular compartment. This assay correlates the damage with the rate of formation of protein carbonyl groups, 3-nitrotyrosine (3-NT) and thiobarbituric acid reactive substances. Using this assay, we evaluated the ability of phenelzine, a scavenger of reactive aldehydes, to antagonize the effects of peroxynitrite. Herein, we showed that phenelzine significantly decreased the lipid peroxidative damage caused by peroxynitirite in blood plasma and platelets. Moreover, it inhibited carbonyl group and 3-NT formation in blood plasma and platelet proteins.

Peroxynitrite (ONOO - ) is a reactive oxidant involved in numerous pathological conditions. Thymoquinone (TQ) is an active constituent of Nigella sativa and is reported to have anti-disease activities, but its role on ONOO - has never been investigated. This study was undertaken to investigate the role of TQ on ONOO - -induced damage of histone-H2A. Our novel data showed TQ significantly inhibited ONOO - -induced oxidative damage in histone-H2A. ONOO - induces UV-hypochromicity of histone-H2A, whereas TQ reversed this effect to hyperchromicity. Tyrosine fluorescence was significantly reduced by ONOO - and was significantly increased upon TQ treatment. TQ reduces ONOO - -induced hydrophobicity in histone-H2A and also reduces thermal stability of ONOO - -histone H2A complex. SDS-PAGE of native histone-H2A showed a single band, which disappeared when treated with ONOO - alone. This changed was retained when protein samples were treated with TQ. Similar protective effects of TQ were found when protein carbonyl contents were estimated. In conclusion, this is the first study that shows the potential of TQ against ONOO - -induced damaged of histone-H2A. TQ inhibits oxidative modification of tyrosine, lysine, arginine, proline and threonine in histone-H2A. These results have importance for the development of novel therapeutic strategies for the treatment of disorders, where ONOO - plays a role., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cerebral ischemia-reperfusion (I/R) injury initiates a cascade of events, generating nitric oxide (NO) and superoxide(O 2 •- ) to form peroxynitrite (ONOO - ), a potent oxidant. Arctic ground squirrels (AGS; Urocitellus parryii) show high tolerance to I/R injury. However, the underlying mechanism remains elusive. We hypothesize that tolerance to I/R modeled in an acute hippocampal slice preparation in AGS is modulated by reduced oxidative and nitrative stress. Hippocampal slices (400µm) from rat and AGS were subjected to oxygen glucose deprivation (OGD) using a novel microperfusion technique. Slices were exposed to NO, O 2 .- donors with and without OGD; pretreatment with inhibitors of NO, O 2 .- and ONOO - followed by OGD. Perfusates collected every 15min were analyzed for LDH release, a marker of cell death. 3-nitrotyrosine (3NT) and 4-hydroxynonenal (4HNE) were measured to assess oxidative and nitrative stress. Results show that NO/O 2 .- alone is not sufficient to cause ischemic-like cell death, but with OGD enhances cell death more in rat than in AGS. A NOS inhibitor, SOD mimetic and ONOO - inhibitor attenuates OGD injury in rat but has no effect in AGS. Rats also show a higher level of 3NT and 4HNE with OGD than AGS suggesting the greater level of injury in rat is via formation of ONOO - ., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Our work reveals that the aerial parts of Pulmonaria officinalis L. are a new source of yunnaneic acid B. We studied antioxidant activity and cytotoxicity of this compound (1-50 μg/mL) and its contents in various plant extracts. This is the first study confirming the presence of yunnaneic acid B in P. officinalis L. and Pulmonaria obscura Dumort and hence in the Boraginaceae family. Determination of 1,1-diphenyl-2-picrylhydrazyl radical reduction and peroxynitrite-scavenging efficacy in inorganic experimental systems provided EC 50 values of 7.14 and 50.45 μg/mL, respectively. Then we examined the antioxidant action of yunnaneic acid B in blood plasma under peroxynitrite-induced oxidative stress in vitro. Yunnaneic acid B effectively diminished oxidative damage to blood plasma proteins and lipids. Furthermore, it was able to prevent the peroxynitrite-induced decrease in nonenzymatic antioxidant capacity of blood plasma. Additionally, cytotoxicity of yunnaneic acid B (at concentrations ≤50 μg/mL) toward peripheral blood mononuclear cells was excluded.

The most toxic species in live systems include reactive nitrogen species such as peroxynitrite, which at high levels induces nitrosative stress. In human spermatozoa, the negative effect of peroxynitrite on motility and mitochondrial membrane potential was recently demonstrated, and the hypothesis of this work is that impairment of ATP production could be one cause of the effect on motility. Therefore, the aim here was to evaluate ATP production by both glycolysis and oxidative phosphorylation (OXPHOS) in spermatozoa exposed to peroxynitrite in vitro. Human spermatozoa were incubated with SIN-1, a molecule which generates peroxynitrite, and the ATP level was evaluated. Then, to inactivate glycolysis or OXPHOS, spermatozoa were incubated with pharmacological inhibitors of these pathways. Spermatozoa treated for inactivating one or the other pathway were exposed to SIN-1, and the ATP level was compared to the control without SIN-1 in each condition. The ATP level fell after peroxynitrite exposure. The ATP in spermatozoa treated for inactivating one or the other metabolic pathway and subsequently exposed to peroxynitrite was reduced compared with the control. These results show for the first time that an important mechanism by which peroxynitrite reduces sperm function is the inhibition of ATP production, affecting both glycolysis and OXPHOS., (© 2016 Blackwell Verlag GmbH.)

Background: Diabetes-induced vascular dysfunction may arise from reduced nitric oxide (NO) availability, following interaction with superoxide to form peroxynitrite. Peroxynitrite can induce formation of 3-nitrotyrosine-modified proteins. RhoA/ROCK signaling is also involved in diabetes-induced vascular dysfunction. The study aimed to investigate possible links between Rho/ROCK signaling, hyperglycemia, and peroxynitrite in small coronary arteries., Methods: Rat small coronary arteries were exposed to normal (NG; 5.5 mM) or high (HG; 23 mM) D-glucose. Vascular ring constriction to 3 mM 4-aminopyridine and dilation to 1 μM forskolin were measured. Protein expression (immunohistochemistry and western blot), mRNA expression (real-time PCR), and protein activity (luminescence-based G-LISA and kinase activity spectroscopy assays) of RhoA, ROCK1, and ROCK2 were determined., Results: Vascular ring constriction and dilation were smaller in the HG group than in the NG group (P < 0.05); inhibition of RhoA or ROCK partially reversed the effects of HG. Peroxynitrite impaired vascular ring constriction/dilation; this was partially reversed by inhibition of RhoA or ROCK. Protein and mRNA expressions of RhoA, ROCK1, and ROCK2 were higher under HG than NG (P < 0.05). This HG-induced upregulation was attenuated by inhibition of RhoA or ROCK (P < 0.05). HG increased RhoA, ROCK1, and ROCK2 activity (P < 0.05). Peroxynitrite also enhanced RhoA, ROCK1, and ROCK2 activity; these actions were partially inhibited by 100 μM urate (peroxynitrite scavenger). Exogenous peroxynitrite had no effect on the expression of the voltage-dependent K + channels 1.2 and 1.5., Conclusions: Peroxynitrite-induced coronary vascular dysfunction may be mediated, at least in part, through increased expressions and activities of RhoA, ROCK1, and ROCK2.

Nitrative stress is considered as an important pathological process of hepatic ischemia and reperfusion injury but its regulating mechanisms are largely unknown. In this study, we tested the hypothesis that caveolin-1 (Cav-1), a plasma membrane scaffolding protein, could be an important cellular signaling against hepatic I/R injury through inhibiting peroxynitrite (ONOO(-))-induced cellular damage. Male wild-type mice and Cav-1 knockout (Cav-1(-/-)) were subjected to 1h hepatic ischemia following 1, 6 and 12h of reperfusion by clipping and releasing portal vessels respectively. Immortalized human hepatocyte cell line (L02) was subjected to 1h hypoxia and 6h reoxygenation and treated with Cav-1 scaffolding domain peptide. The major discoveries included: (1) the expression of Cav-1 in serum and liver tissues of wild-type mice was time-dependently elevated during hepatic ischemia-reperfusion injury. (2) Cav-1 scaffolding domain peptide treatment inhibited cleaved caspase-3 expression in the hypoxia-reoxygenated L02 cells; (3) Cav-1 knockout (Cav-1(-/-)) mice had significantly higher levels of serum transaminases (ALT&AST) and TNF-α, and higher rates of apoptotic cell death in liver tissues than wild-type mice after subjected to 1h hepatic ischemia and 6hour reperfusion; (4) Cav-1(-/-) mice revealed higher expression levels of iNOS, ONOO(-) and 3-nitrotyrosine (3-NT) in the liver than wild-type mice, and Fe-TMPyP, a representative peroxynitrite decomposition catalyst (PDC), remarkably reduced level of ONOO(-) and 3-NT and ameliorated the serum ALT, AST and TNF-α levels in both wild-type and Cav-1(-/-) mice. Taken together, we conclude that Cav-1 could play a critical role in preventing nitrative stress-induced liver damage during hepatic ischemia-reperfusion injury., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Identification of factors contributing to the development of chronic obstructive pulmonary disease (COPD) is crucial for developing new treatments. An increase in the levels of protein-disulfide isomerase (PDI), a multifaceted endoplasmic reticulum resident chaperone, has been demonstrated in human smokers, presumably as a protective adaptation to cigarette smoke (CS) exposure. We found a similar increase in the levels of PDI in the murine model of COPD. We also found abnormally high levels (4-6 times) of oxidized and sulfenilated forms of PDI in the lungs of murine smokers compared with non-smokers. PDI oxidation progressively increases with age. We begin to delineate the possible role of an increased ratio of oxidized PDI in the age-related onset of COPD by investigating the impact of exposure to CS radicals, such as acrolein (AC), hydroxyquinones (HQ), peroxynitrites (PN), and hydrogen peroxide, on their ability to induce unfolded protein response (UPR) and their effects on the structure and function of PDIs. Exposure to AC, HQ, PN, and CS resulted in cysteine and tyrosine nitrosylation leading to an altered three-dimensional structure of the PDI due to a decrease in helical content and formation of a more random coil structure, resulting in protein unfolding, inhibition of PDI reductase and isomerase activity in vitro and in vivo, and subsequent induction of endoplasmic reticulum stress response. Addition of glutathione prevented the induction of UPR, and AC and HQ induced structural changes in PDI. Exposure to PN and glutathione resulted in conjugation of PDI possibly at active site tyrosine residues. The findings presented here propose a new role of PDI in the pathogenesis of COPD and its age-dependent onset., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: Atrial fibrillation (AF) is the most common cardiac arrhythmia. However, the current drug interference of antiarrhythmia has limited efficacy and off-target effects. Accumulating evidence has implicated a potential role of nitration stress in the pathogenesis of AF. The aim of the study was to determine whether TPEN provided antinitration effects on atrial myocytes during AF, especially under circumstances of nitration stress., Methods: We utilized a rapid paced HL-1 cells model for AF. The changes of electrophysiological characteristics and structure of paced HL-1 cells were determined by a patch clamp and a TEM method. The effects of TPEN on pacing and ONOO(-) pretreated HL-1 cells were examined using MTT assay, TUNEL technique, confocal microscope experiment, and Western blot analysis., Results: The results revealed that ONOO(-) reduced the viability of HL-1 cells in a dose-dependent manner, and 1 μmol/L TPEN significantly ameliorated the damage caused by 50 μmol/L ONOO(-) (P < 0.05). Pacing and/or ONOO(-) -induced marked shortening of APD, myolysis, and nuclear condensation. TPEN inhibited the Ca(2+) overload induced by rapid pacing (P < 0.05) and ONOO(-) stimulation (P < 0.05). The application of TPEN significantly prevented the protein nitration caused by pacing or pacing plus ONOO(-) (P < 0.05). Additionally, pacing in combination with ONOO(-) treatment led to increase in apoptosis in HL-1 cells (P < 0.01), which could be reduced by pretreatment with TPEN (P < 0.05)., Conclusions: TPEN prevents Ca(2+) overload and nitration stress in HL-1 atrial myocytes during rapid pacing and circumstances of nitration stress., (© 2015 John Wiley & Sons Ltd.)

Background: Oxidative and nitrative processes have an important role in the pathogenesis of glaucomatous neurodegeneration. Oxidative stress occurs when cellular production of reactive oxygen species outweighs the protective capacity of antioxidant defences. Reactive oxygen species are generated as by-products of cellular metabolism, primarily in the mitochondria. Herein, we present a novel investigation of the effects of molecular hydrogen (H2 ) on retinal cells exposed to oxidative stress., Methods: We cultured adult rat retinal tissues in an organotypic culture system with a nitric oxide donor, S-nitroso-N-acetylpenicillamine, in the presence or absence of H2 . Loss of mitochondrial membrane potential and apoptosis of retinal cells were analysed using a MitoTMRE detection kit and TdT-mediated dUTP nick end labeling (TUNEL) assay, respectively. Tyrosine nitration levels and oxidative stress damage in the retina were evaluated using immunohistochemical staining. Retinal damage was quantified by measuring the numbers of cells in the ganglion cell and inner nuclear layers and the thickness of the retina., Results: H2 suppressed loss of mitochondrial membrane potential and apoptosis in retinal cells. Moreover, H2 decreased the tyrosine nitration level and suppressed oxidative stress damage in retinal cells. S-nitroso-N-acetylpenicillamine treatment decreased the cell numbers in the ganglion cell layer and inner nuclear layer, but the presence of H2 inhibited this reduction. These findings suggest that H2 has a neuroprotective effect against retinal cell oxidative damage, presumably by scavenging peroxynitrite., Conclusions: H2 reduces cellular peroxynitrite, a highly toxic reactive nitrogen species. Thus, H2 may be an effective and novel clinical tool for treating glaucoma and other oxidative stress-related diseases., (© 2015 Royal Australian and New Zealand College of Ophthalmologists.)

Peroxynitrite as one crucial metabolite of NO-derived agents has been well multi-investigated to inspect its potential role and sought to define its concrete mechanism underlying the memory loss and impaired cognition involved in pathological processes. In this investigation, the cell viability was assessed by the MTT assay. The neurotoxicity of peroxynitrite was analyzed by using immunohistochemical measurements in cultured PC12 cells to explore the underlying mechanisms. The generation of ROS was evaluated by a fluorometry assay by a fluorometry assay. Apoptosis was assayed by annexin V-FITC and PI staining with flow cytometry. [Ca2+]i was examined by using the microspectrofluorometer. Hsp70 was detected by western blot assay. The results revealed that PC12 cells were inhibited by peroxynitrite both in a dose-dependent and time-dependent manner. The level of ROS in PC12 cells exposed to SIN-1 was increased in a dose-dependent manner. The result indicated that the SIN-1 induced apoptosis of PC12 cells in a dose-dependent manner. Quercetin inhibited the viability of PC12 cells in a concentration-dependent manner. [Ca2+]i was increased gradually when cells treated with quercetin alone and also increased with treatment of dantrolene-containing. Hsp70 was significantly decreased in SIN-1-treated group compared with that of control group (P<0.01). In conclusion, Ca2+ homeostasis and chaperone Hsp70 were critically involved in peroxynitrite induced nitrosative stress as protective. Peroxynitrite acts as the pathological agent in learning and memory defects in CNS disorders associated with challenge.

Molecular hydrogen (H2) can scavenge hydroxyl radical and diminish the toxicity of peroxynitrite; hence, it has interesting potential for antioxidant protection. Recently, a number of studies have explored the utility of inhaled hydrogen gas, or of hydrogen-saturated water, administered parenterally or orally, in rodent models of pathology and in clinical trials, oftentimes with very positive outcomes. The efficacy of orally ingested hydrogen-rich water (HW) has been particularly surprising, given that only transient and rather small increments in plasma hydrogen can be achieved by this method. A recent study in mice has discovered that orally administered HW provokes increased gastric production of the orexic hormone ghrelin, and that this ghrelin mediates the favorable impact of HW on a mouse model of Parkinson's disease. The possibility that most of the benefits observed with HW in experimental studies are mediated by ghrelin merits consideration. Ghrelin is well known to function as an appetite stimulant and secretagogue for growth hormone, but it influences physiological function throughout the body via interaction with the widely express GHS-R1a receptor. Rodent and, to a more limited extent, clinical studies establish that ghrelin has versatile neuroprotective and cognitive enhancing activity, favorably impacts vascular health, exerts anti-inflammatory activity useful in autoimmune disorders, and is markedly hepatoprotective. The stimulatory impact of ghrelin on GH-IGF-I activity, while potentially beneficial in sarcopenia or cachectic disorders, does raise concerns regarding the long-term impact of ghrelin up-regulation on cancer risk. The impact of ingesting HW water on ghrelin production in humans needs to be evaluated; if HW does up-regulate ghrelin in humans, it may have versatile potential for prevention and control of a number of health disorders., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Increased production of reactive nitrogen (RNS) and oxygen (ROS) species and its detrimental effect to mitochondria are associated with endothelial dysfunction. This study was designed to determine the effect of a peroxynitrite flux, promoted by 1,3-morpholinosydnonimine (SIN-1), in mitochondrial function and some redox homeostasis parameters in bovine aortic endothelial cells (BAEC). Moreover, the effect of diphenyl diselenide (PhSe)2, a simple organic selenium compound, in preventing peroxynitrite-mediated cytotoxicity was also investigated. Our results showed that overnight exposure to SIN-1 (250 μM) caused a profound impairment of oxygen consumption, energy generation and reserve capacity in mitochondria of BAEC. Mitochondrial dysfunction resulted in an additional intracellular production of peroxynitrite, amplifying the phenomenon and leading to changes in redox homeostasis. Moreover, we observed an extensive decline in mitochondrial membrane potential (ΔΨm) induced by peroxynitrite and this event was associated with apoptotic-type cell death. Alternatively, the pretreatment of BAEC with (PhSe)2, hindered peroxynitrite-mediated cell damage by preserving mitochondrial and endothelial function and consequently preventing apoptosis. The protective effect of (PhSe)2 was related to its ability to improve the intracellular redox state by increasing the expression of different isoforms of peroxiredoxins (Prx-1-3), efficient enzymes in peroxynitrite detoxification.

Oxidative stress is characterized by excessive production of various free radicals and reactive species among which, peroxynitrite is most frequently produced in several pathological conditions. Peroxynitrite is the product of the superoxide anion reaction with nitric oxide, which is reported to take place in the intravascular compartment. Several studies have reported that peroxynitrite targets red blood cells, platelets and plasma proteins, and induces various forms of oxidative damage. This in vitro study was designed to further characterize the types of oxidative damage induced in platelets and plasma proteins by peroxynitrite. This study also determined the ability of tempol to protect blood plasma and platelets against peroxynitrite-induced oxidative damage. The ability of various concentrations of tempol (25, 50, 75, and 100 µM) to antagonize peroxynitrite-induced oxidation was evaluated by measuring the levels of protein carbonyl groups and thiobarbituric-acid-reactive substances in experimental groups. Exposure of platelets and plasma to 100 µM peroxynitrite resulted in an increased levels of carbonyl groups and lipid peroxidation (P < 0.05). Tempol significantly inhibited carbonyl group formation in plasma and platelet proteins (P < 0.05). In addition, tempol significantly reduced the levels of lipid peroxidation in both plasma and platelet samples (P < 0.05). Thus, tempol has antioxidative properties against peroxynitrite-induced oxidative damage in blood plasma and platelets., (© 2014 by the Society for Experimental Biology and Medicine.)

Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons-a form of cell death called "phagoptosis." UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y6 receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y6 signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y6 receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1-42, 5 μM peroxynitrite, or 50 μM 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y6 receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y6 signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons., (© 2014 The Authors. Glia Published by Wiley Periodicals, Inc.)

The physiological role of myoglobin (Mb) within the heart depends on its oxygenation state. The myocardium exhibits a broad oxygen partial pressure (pO2) spectrum with a transmural gradient from the epicardial to the subendocardial layer, ranging from arterial values to an average of 19.3 mm Hg down to 0 mm Hg. The function of Mb as an O2 storage depot is well appreciated, especially during systolic compression. In addition, Mb controls myocardial nitric oxide (NO) homeostasis and thus modulates mitochondrial respiration under physiological and pathological conditions. We recently discovered the role of Mb as a myocardial O2 sensor; in its oxygenated state Mb scavenges NO, protecting the heart from the deleterious effects of excessive NO. Under hypoxia, however, deoxygenated Mb changes its role from an NO scavenger to an NO producer. The NO produced protects the cell from short phases of hypoxia and from myocardial ischemia/reperfusion injury. In this review we summarize the traditional and novel aspects of Mb and its (patho)physiological role in the heart., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Peroxynitrite, a potent oxidising and nitrating species, has been implicated in the pathogenesis of neurodegenerative diseases. This study was undertaken to investigate the protective effect of myricitrin on peroxynitrite-mediated toxicity and the underlying mechanism. Our results showed that the presence of myricitrin was found to significantly inhibit peroxynitrite-mediated DNA damage. EPR spectroscopy demonstrated that myricitrin potently diminished the DMPO-hydroxyl radical adduct signal from peroxynitrite. Further study showed that glutathione (GSH) depletion caused by peroxynitrite can be effectively prevented by pre-incubation of astrocytes with myricitrin. Moreover, co-incubation of astrocytes with myricitrin and buthionine sulfoximine (BSO) eliminated the myricitrin-induced GSH increase. In contrast, co-incubation of myricitrin with BSO slightly protected astrocytes against cytotoxicity and DNA damage mediated by peroxynitrite. These results revealed that myricitrin can protect against peroxynitrite-induced DNA damage and cytotoxicity, which might have implications for myricitrin-mediated neuroprotection., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objectives: Although the most accepted mechanisms of action of amphotericin B and azoles are related to ergosterol, it is possible that these drugs have other effects on the fungal cell. In the present study, the role of endogenous reactive oxygen species (ROS) and peroxynitrite produced by azoles and amphotericin B in the fungus Cryptococcus gattii were examined., Methods: We studied distinct parameters to evaluate the effect of oxidative and nitrosative stresses induced by these drugs in C. gattii cells: lipid peroxidation, ergosterol content, ROS and peroxynitrite production, enzymatic activity of the antioxidant system and the in vitro interaction of antifungal drugs with a peroxidase inhibitor, a superoxide dismutase inhibitor and a peroxynitrite scavenger., Results: The data demonstrated that itraconazole led to ROS formation and lipid peroxidation in C. gattii cells in the early stages of the treatment; this did not occur with fluconazole. This phenomenon strongly increased the activities of enzymes of the antioxidant system. These results were confirmed by synergism observed between the catalase inhibitor and itraconazole. Amphotericin B caused lipid peroxidation in C. gattii cells through a greatly enhanced production of oxidative and nitrosative radicals with increased peroxidase activity. These data were confirmed by the synergism between the catalase/superoxide dismutase inhibitors and amphotericin B. In addition, the effect of this antifungal was antagonized by the peroxynitrite scavenger., Conclusions: Oxidative and nitrosative bursts play an important role in the antifungal activity of itraconazole and amphotericin B against C. gattii.

Excess production of superoxide (O₂(-)) and nitric oxide (NO) in blood vessel walls may occur early in atherogenesis leading to the formation of peroxynitrite, a strong oxidant and nitrating agent. This study was designed to determine the effect of diphenyl diselenide (PhSe)₂, a synthetic organoselenium compound, in comparison with ebselen, on peroxynitrite-mediated endothelial damage. Experimental results showed that pre-incubation of BAEC (24 h) with low concentrations of (PhSe)₂ (0.5 and 1 μM) protected the cells from peroxynitrite-dependent apoptosis and protein tyrosine nitration. The intracellular levels of GSH were almost completely depleted by peroxynitrite and, although the compounds did not restore its normal levels, (PhSe)₂ per se significantly increased GSH in a concentration-dependent manner. Moreover, (PhSe)₂, which was about two times more active as a GPx mimic than ebselen, induced a significantly higher increase in both cellular GPx expression and activity. Taking into account the kinetics of the reaction between peroxynitrite and (PhSe)₂, our data indicate that (PhSe)₂ protects BAEC against peroxynitrite-mediated cell damage not by a direct reaction, but rather by increasing cellular GPx expression as a consequence of enhanced nuclear translocation of Nrf-2, which together with the increase in intracellular GSH, may work catalytically to reduce peroxynitrite to nitrite., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The intense systemic inflammatory response characterizing septic shock is associated with an increased generation of free radicals by multiple cell types in cardiovascular and non cardiovascular tissues. The oxygen-centered radical superoxide anion (O2 .-) rapidly reacts with the nitrogen-centered radical nitric oxide (NO.) to form the potent oxidant species peroxynitrite. Peroxynitrite oxidizes multiple targets molecules, either directly or via the secondary generation of highly reactive radicals, resulting in significant alterations in lipids, proteins and nucleic acids, with significant cytotoxic consequences. The formation of peroxynitrite is a key pathophysiological mechanism contributing to the cardiovascular collapse of septic shock, promoting vascular contractile failure, endothelial and myocardial dysfunction, and is also implicated in the occurrence of multiple organ dysfunction in this setting. The recent development of various porphyrin-based pharmacological compounds accelerating the degradation of peroxynitrite has allowed to specifically address these pathophysiological roles of peroxynitrite in experimental septic shock. Such agents, including 5,10,15,20-tetrakis(4- sulfonatophenyl)porphyrinato iron III chloride (FeTTPs), manganese tetrakis(4-N-methylpyridyl)porphyrin (MnTMPyP), Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether)pyridyl porphyrin) (FP-15) and WW-85, have been shown to improve the cardiovascular and multiple organ failure in small and large animal models of septic shock. Therefore, these findings support the development of peroxynitrite decomposition catalysts as potentially useful novel therapeutic agents to restore cardiovascular function in sepsis.

Oxidative damage to DNA has many origins, including irradiation, inflammation, and oxidative stress, but the chemistries are not the same. The most oxidizable base in DNA is 2-deoxyguanosine (dG), and the primary oxidation products are 8-oxodG and 2-amino-imidazolone. The latter rapidly converts to 2,2-diamino-oxazolone (Ox), and 8-oxodG is further oxidized to spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). In this study, we have examined the dose-response relationship for the formation of the above four products arising in calf thymus DNA exposed to gamma irradiation, photoactivated rose bengal, and two sources of peroxynitrite. In order to carry out these experiments, we developed a chromatographic system and synthesized isotopomeric internal standards to enable accurate and precise analysis based upon selected reaction monitoring mass spectrometry. 8-OxodG was the most abundant products in all cases, but its accumulation was highly dependent on the nature of the oxidizing agent and the subsequent conversion to Sp and Gh. Among the other oxidation products, Ox was the most abundant, and Sp was formed in significantly greater yield than Gh.

Oxidative stress interferes with nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) signalling pathway through reduction of endogenous NO and formation of the strong intermediate oxidant peroxynitrite and leads to vascular dysfunction. We evaluated the effects of oral treatment with NO- and heme-independent sGC activator cinaciguat on peroxynitrite-induced vascular dysfunction in rat aorta. Sprague-Dawley rats were treated orally 2 times at an interval of 17 hours with vehicle or with cinaciguat (10 mg/kg). One hour after the last treatment, the animals were anesthetized, the thoracic aorta was removed, and the aortic segment preparations were incubated with and without the reactive oxidant peroxynitrite (200 µmol/L, 30 minutes). Endothelium-dependent (acetylcholine), -independent (sodium nitroprusside) vasorelaxations were investigated, and histopathological examination was performed. Incubation of aortic rings with peroxynitrite significantly attenuated the maximal endothelium-dependent relaxation (R (max)) to acetylcholine (peroxynitrite, 44.5% ± 5.9% vs control, 93.2% ± 2.0%, P < .05) and decreased pD(2) values (-logEC(50), EC(50) being the concentration of acetylcholine that elicited 50% of the maximal response) for the concentration-response curves as compared to control segments. Treatment of rats with cinaciguat significantly improved the decreased acetylcholine-induced vasorelaxation after exposure of aortic rings to peroxynitrite (cinaciguat + peroxynitrite, 67.1% ± 3.5% vs peroxynitrite, 44.5% ± 5.9%, P < .05). Incubation of aortic segments with peroxynitrite caused a significant shift of the sodium nitroprusside concentration-response curves to the right without any alterations in the R (max). Moreover, exposure of aortic rings to peroxynitrite resulted in increased nitro-oxidative stress and DNA breakage which were improved by cinaciguat. Treatment of rats with cinaciguat significantly increased intracellular cGMP levels in the aortic wall. Our results show under conditions of nitro-oxidative stress when signalling in the NO/sGC/cGMP pathway is impaired, acute activation of sGC by cinaciguat might be advantageous in the treatment of endothelial dysfunction in cardiovascular disease.

Objectives: The present study was designed to determine whether or not the caspase-independent apoptotic pathway participated in the cellular death of spiral ganglion neurons (SGNs) after exposure to peroxynitrite (ONOO(-)), with particular attention given to the intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in this process., Methods: The rat SGNs were isolated and primary cultured in vitro and were exposed to ONOO(-) with pre-treatment of pan-caspase inhibitor. Morphological changes of SGNs were observed by acridine orange cytochemistry staining, and apoptosis was examined by flow cytometry. The translocation of mitochondrial AIF and Endo G was detected by immunocytochemistry and Western blot. The protein expressions of Bcl-2 family in SGNs exposed to ONOO(-) were determined by Western blot., Results: Treatment of SGNs with ONOO(-) resulted in the occurrence of caspase-independent apoptosis as evidenced by acridine orange staining and flow cytometry analysis. The immunocytochemical analysis showed that AIF and Endo G labeling were marked in neuronal nuclei, while the Western blot demonstrated the intranuclear localization of AIF and Endo G in SGNs treated with ONOO(-). Western blot analysis demonstrated that ONOO(-) increased the Bax expression while reducing Bcl-2 expression, which was not prevented by pre-treatment with caspase inhibitor., Conclusion: These data indicate that ONOO(-) can trigger caspase-independent apoptosis in SGNs associated with mitochondrial AIF and Endo G intranuclear localization.

Anthocyanins are the most abundant flavonoid constituents of fruits and vegetables and several epidemiological studies suggest that the consumption of these compounds protect against several diseases, including vascular disorders. Previously, we have reported that anthocyanins are able to counteract peroxynitrite-induced apoptotic effects in endothelial cells through inhibition of several crucial signaling cascades, upstream and downstream of mitochondria. Following these studies, here we investigated possible effects of malvidin-3-glucoside, one of the main dietary anthocyanins, on NO bioavailability and on peroxynitrite-induced NF-kB activation in the same cell model. Our results show that treatment of bovine arterial endothelial cells with malvidin-3-glucoside up-regulated eNOS mRNA, leading to the enhancement of eNOS activity and NO production, an effect even greater when cells were further stimulated with peroxynitrite. On the other hand, in these activated endothelial cells, malvidin-3-glucoside suppressed pro-inflammatory mediators, namely iNOS expression/NO biosynthesis, COX-2 expression and IL-6 production, through inhibition of NF-kB activation. These findings suggest a potential role of malvidin-3-glucoside in NO balance and in inhibition of pro-inflammatory signaling pathways, supporting its benefits in cardiovascular health and pointing to anthocyanins as a promising tool for development of functional foods and nutraceuticals to improve endothelial function., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Oxidative stress plays an important role in the progression of many chronic diseases including cardiovascular diseases, diabetes, cancer and neurodegenerative disorders. One such mediator of oxidative stress is peroxynitrite, which is highly toxic to cultured neurons and astrocytes, and has been reported to be involved in the pathogenesis of various types of neuronal diseases. Therefore, searching for natural compounds with peroxynitrite-scavenging activity might be an effective therapy for peroxynitrite-mediated cytotoxicity. Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong antioxidant, anticancer, and antidiabetic properties. However, the astrocyte protective efficacy of hispidin has been not examined. This study was undertaken to investigate whether the astrocyte protective effect of hispidin is associated with inhibition of peroxynitrite-induced DNA damage, a critical event leading to peroxynitrite-mediated cytotoxicity. Our results showed that peroxynitrite can cause DNA damage in φX-174 plasmid DNA and rat primary astrocytes. The presence of hispidin (10-20 μg/ml) was found to significantly inhibit peroxynitrite-induced DNA damage and cytotoxicity. EPR spectroscopy demonstrated that the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from peroxynitrite, and that hispidin potently diminished the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that hispidin can protect against peroxynitrite-mediated cytotoxicity, DNA damage and hydroxyl radical formation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Boronates, a group of organic compounds, are emerging as one of the most effective probes for detecting and quantifying peroxynitrite, hypochlorous acid, and hydrogen peroxide. Boronates react with peroxynitrite nearly a million times faster than with hydrogen peroxide. Boronate-containing fluorogenic compounds have been used to monitor real time generation of peroxynitrite in cells and for imaging hydrogen peroxide in living animals. This perspective highlights potential applications of boronates and other fluorescent probes to high-throughput analyses of peroxynitrite and hydroperoxides in toxicological studies.

Methionine sulfoxide reductases (Msrs) in lens cells are important for the maintenance of lens cell viability and resistance to oxidative stress damage. Peroxynitrite (ONOO(-)), as a strong oxidizing and nitrating agent, occurred in diabetic retinopathy patients and diabetic model animal. In an attempt to shed light on the roles of MsrB1, known as selenoprotein R, in protecting human lens epithelial (HLE) cells against peroxynitrite damage, and contribution of loss of its normal activity to cataract, the influences of MsrB1 gene silencing on peroxynitrite-induced apoptosis in HLE cells were studied. The results showed that both exogenous peroxynitrite and MsrB1 gene silencing by short interfering RNA (siRNA) independently resulted in oxidative stress, endoplasmic reticulum (ER) stress, activation of caspase-3 as well as an increase of apoptosis in HLE cells; moreover, when MsrB1-gene-silenced cells were exposed to 300 μM peroxynitrite, these indexes were further aggravated at the same conditions and DNA strand breaks occurred. The results demonstrate that in HLE cells MsrB1 may play important roles in regulating redox balance and mitigating ER stress as induced by oxidative stress under physiological conditions; MsrB1 may also protect HLE cells against peroxynitrite-induced apoptosis by inhibiting the activation of caspase-3 and oxidative damage of DNA under pathological conditions. Our results imply that loss of its normal activity is likely to contribute to cataract., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.

The health-promoted benefits of anthocyanins, including vascular protective effects and antiatherogenic properties, have now been recognized, but the involved molecular mechanisms have not been well elucidated. Following our previous work on cytoprotective mechanisms of some anthocyanins against apoptosis triggered by peroxynitrite in endothelial cells, here we investigated the protective role of malvidin-3-glucoside, a major dietary anthocyanin, on such deleterious process, by exploring the interference on cellular reactive species formation and on apoptotic mitochondrial pathway. Preincubation of cells with 25 μM malvidin-3-glucoside protected efficiently endothelial cells from peroxynitrite-promoted apoptotic death, an effect which may be partially mediated by its ability to decrease the formation of reactive species after cell aggression, as assessed by the dichlorodihydrofluorescein diacetate assay and by carbonyl groups formation. Moreover, malvidin-3-glucoside inhibited mitochondrial apoptotic signaling pathways induced by peroxynitrite, by counteracting mitochondrial membrane depolarization, the activation of caspase-3 and -9, and the increase in the expression of the proapoptotic Bax protein. Altogether, our data expands our knowledge about the molecular mechanisms underlying the vascular protection afforded by malvidin-3-glucoside, and anthocyanins in general, in the context of prevention of endothelial dysfunction and atherosclerosis.

Inflammation associated reactive oxygen and nitrogen species (RONs), including peroxynitrite (ONOO(-)) and nitric oxide (NO), create base lesions that potentially play a role in the toxicity and large genomic rearrangements associated with many malignancies. Little is known about the role of base excision repair (BER) in removing these endogenous DNA lesions. Here, we explore the role of X-ray repair cross-complementing group 1 (XRCC1) in attenuating RONs-induced genotoxicity. XRCC1 is a scaffold protein critical for BER for which polymorphisms modulate the risk of cancer. We exploited CHO and human glioblastoma cell lines engineered to express varied levels of BER proteins to study XRCC1. Cytotoxicity and the levels of DNA repair intermediates (single-strand breaks; SSB) were evaluated following exposure of the cells to the ONOO(-) donor, SIN-1, and to gaseous NO. XRCC1 null cells were slightly more sensitive to SIN-1 than wild-type cells. We used small-scale bioreactors to expose cells to NO and found that XRCC1-deficient CHO cells were not sensitive. However, using a molecular beacon assay to test lesion removal in vitro, we found that XRCC1 facilitates AAG-initiated excision of two key NO-induced DNA lesions: 1,N(6)-ethenoadenine and hypoxanthine. Furthermore, overexpression of AAG rendered XRCC1-deficient cells sensitive to NO-induced DNA damage. These results show that AAG is a key glycosylase for BER of NO-induced DNA damage and that XRCC1's role in modulating sensitivity to RONs is dependent upon the cellular level of AAG. This demonstrates the importance of considering the expression of other components of the BER pathway when evaluating the impact of XRCC1 polymorphisms on cancer risk., (Published by Elsevier B.V.)

Peroxynitrite is widely reported as highly cytotoxic; yet recent evidence indicates that at certain concentrations, it can induce pulmonary cell hyper-proliferation and tissue remodelling. This study aimed to establish the threshold concentration of peroxynitrite to induce functional impairment of bovine pulmonary artery endothelial (PAEC) and smooth muscle cells (PASMC). PAEC or PASMC were exposed to solution of peroxynitrite or 3-morpholinosydnonimine (SIN-1). Twenty-four hour cell viability, DNA synthesis, and protein biochemistry were assessed by trypan blue dye exclusion, [3H] thymidine incorporation and western blot analysis, respectively. Threshold concentration of peroxynitrite to significantly impair viability of PAEC and PASMC was 2 μM peroxynitrite. In PASMC and PAEC, low concentrations of peroxynitrite (2 nM-0.2 μM) increased cell proliferation and did not activate p38 MAP kinase. The decrease in DNA synthesis and cell viability caused by 2 μM peroxynitrite was associated with caspase-3 cleavage but not p38 activation. Also, 2-20 μM peroxynitrite significantly activated poly ADP ribose polymerase and stress activated kinase JNK in PAEC. However, the higher concentration of 20 μM peroxynitrite did cause a threefold increase in p38 activation. In conclusion, the threshold for the cytotoxic effects of peroxynitrite was 2 μM; which caused apoptotic cell death independent of p38 MAP kinase activation in pulmonary artery cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: The purpose of this work was to investigate whether L-cysteine was able to protect spiral ganglion neurons (SGNs) against peroxynitrite (ONOO(-))-elicited toxicity., Methods: The rat SGNs were isolated and cultured in this work. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The morphological changes were examined under inverted phase contrast microscope. Cells underwent apoptosis were determined by TdT-mediated dUTP nick end labeling (TUNEL) assay. Intracellular glutathione (GSH) content, superoxide dismutase (SOD) activity and malonaldehyde (MDA) level were detected by biochemical methods. Laser scanning confocal microscope was employed to analyze cytosolic Ca(2+) concentration., Results: Results showed that ONOO(-) reduced the cell viability of SGNs in a time- and dose-dependent manner. ONOO(-)-triggered cell damage was further confirmed via apoptotic pathway rather than necrosis. Pretreatment with L-cysteine (5 mM) for 12 hours could almost completely rescue SGNs from ONOO(-)-induced damage. The decrease in intracellular GSH content and SOD activity, as well as the increase in MDA level induced by ONOO(-) were correspondingly antagonized by the administration of L-cysteine. Furthermore, L-cysteine can significantly inhibit elevation of Ca(2+) concentration induced by ONOO(-)., Discussion: Our findings indicate that L-cysteine protects SGNs from ONOO(-)-induced damage via enhancing the antioxidative activity and, suppressing the lipid peroxidation as well as the release of cytosolic Ca(2+), thereby indicating that oxidation resistance was useful to prevent audiological diseases initiated by oxidative stress.

Peroxynitrite has been suggested to be the potent oxidant causing toxicity to neurons and oligodendrocytes (OLs). Our previous studies have illustrated that intracellular zinc liberation contributes to peroxynitrite toxicity to mature OLs. In this study, we further investigated the signaling pathways involved in this event and identified protein kinase C (PKC) as an important early signaling molecule. We found that a non-selective PKC inhibitor bisindolylmaleimide-1 blocked OL toxicity induced by a peroxynitrite generator SIN-1 and exogenous zinc. The protective effects were due to its inhibition on ERK1/2 phosphorylation and ROS generation. The same phenomenon was also observed in OLs following prolonged treatment with phorbol 12 myristate 13 acetate (PMA), which downregulates the conventional and the novel PKC isoforms (cPKCs and nPKCs). To determine the role of specific PKC isoforms, we found that a specific nPKC inhibitor rottlerin significantly reduced SIN-1- or zinc-induced toxicity, whereas Go6976, a cPKC inhibitor, reduced OL toxicity triggered by zinc, but not by SIN-1 at high concentrations. Rottlerin was more potent than Go6976 to attenuate ERK1/2 phosphorylation and ROS generation induced by SIN-1 or zinc. Surprisingly, zinc only induced phosphorylation of PKCθ, but not PKCδ. Knockdown of PKCθ using lentiviral shRNA attenuated SIN-1- or zinc-induced toxicity. These results suggest that PKCθ might be the major PKC isoform involved in peroxynitrite and zinc toxicity to mature OLs, and provide a rationale for development of specific inhibitors of PKCθ in the treatment of multiple sclerosis and other neurodegenerative diseases, in which peroxynitrite formation plays a pathogenic role., (Published by Elsevier Inc.)

The study was designed to reveal the pathogenic mechanism of peroxynitrite in hepatic encephalopathy (HE), assess oxidative/nitrative stress in relation to HE induced by thiacetamide (TAA) and provide new ideas and scientific basis for the etiology and treatment of HE. Male Wistar rats were divided into four groups randomly: A (control), B (model), C (ebselen) and D (solvent). All the groups were treated with TAA by intraperitoneal (i.p.) except group A (treated with saline i.p.) to manufacture the model of HE. When rats treated with TAA came to the second stage of HE the four groups were administered intragastrically (i.g.) with saline (A, B), ebselen (C) and dimethyl sulfoxide (DMSO) (D), respectively. Plasma was collected to detect the levels of 3-nitrotyrosine (3-NT), NO, T-SOD and MDA. The results showed that the levels of 3-NT, NO, MDA significantly increased and T-SOD decreased obviously in rats suffering from HE. With the development and progression of HE the extent of oxidative/nitrative stress increased. When treated with ebselen the symptoms of HE mitigated and the levels of biochemical indicators ameliorated significantly. This indicates that oxidative/nitrative stress is involved in the mechanisms of hepatic encephalopathy.

Some polycyclic aromatic hydrocarbons (PAHs) are typical promutagens that require metabolic activation to exhibit their mutagenicities and carcinogenicities. The metabolites of three PAHs, pyrene (PY), fluoranthene (FLU), and benzo[a]pyrene (BaP), produced from the peroxynitrite/T(p-Cl)PPFeCl(peroxynitrite/(chloride)iron(III)tetrakis(p-chlorophenyl)porphyrin) system, have been identified with high-performance liquid chromatography coupled with electron spray ionization tandem mass spectrometry. The results demonstrated that three major metabolites were the quinone group, OH group, and nitro group. In the Ames test, all three PAH metabolites became mutagenic without using the enzymatic activating system, whereas their parents did not show positive results. Cell transformation assay indicated that 1,3-nitro-BaP and BaP metabolites produced from this biomimetic system have more serious effects in inducing cancer than the BaP parent., (Copyright © 2010 SETAC.)

During acute lung injury, nitric oxide (NO) exerts cytotoxic effects by reacting with superoxide radicals, yielding the reactive nitrogen species peroxynitrite (ONOO(-)). ONOO(-) exerts cytotoxic effects, among others, by nitrating/nitrosating proteins and lipids, by activating the nuclear repair enzyme poly(ADP-ribose) polymerase and inducing VEGF. Here we tested the effect of the ONOO(-) decomposition catalyst INO-4885 on the development of lung injury in chronically instrumented sheep with combined burn and smoke inhalation injury. The animals were randomized to a sham-injured group (n = 7), an injured control group [48 breaths of cotton smoke, 3rd-degree burn of 40% total body surface area (n = 7)], or an injured group treated with INO-4885 (n = 6). All sheep were mechanically ventilated and fluid-resuscitated according to the Parkland formula. The injury-related increases in the abundance of 3-nitrotyrosine, a marker of protein nitration by ONOO(-), were prevented by INO-4885, providing evidence for the neutralization of ONOO(-) action by the compound. Burn and smoke injury induced a significant drop in arterial Po(2)-to-inspired O(2) fraction ratio and significant increases in pulmonary shunt fraction, lung lymph flow, lung wet-to-dry weight ratio, and ventilatory pressures; all these changes were significantly attenuated by INO-4885 treatment. In addition, the increases in IL-8, VEGF, and poly(ADP-ribose) in lung tissue were significantly attenuated by the ONOO(-) decomposition catalyst. In conclusion, the current study suggests that ONOO(-) plays a crucial role in the pathogenesis of pulmonary microvascular hyperpermeability and pulmonary dysfunction following burn and smoke inhalation injury in sheep. Administration of an ONOO(-) decomposition catalyst may represent a potential treatment option for this injury.

To reveal the role of aging in the changes of immune system cells response to proinflammatory (oxidizing) agent we have studied peroxynitrite (30 microM) influence on the cell surface layer structure of different age rats' thymocytes. In the absence of proinflammatory agent, there were no significant changes in morphological and topological parameters of thymocytes from the rats at 3 and 8 months of age. According to atomic force microscopy data, peroxynitrite stimulates actin cytoskeleton structures assembly in thymocytes from 3-month rats but inhibits it in cells from 8-month rats. Obtained results make clear the difference between the immune system responses in inflammatory processes of young and old organisms.

The present study was designed to investigate the effect of curcumin on peroxynitrite (ONOO(-))-induced damage in rat spiral ganglion neurons (SGNs). The primary cultured rat SGNs were exposed to ONOO(-) with or without curcumin pretreatment. Cell viability was measured by MTT assay. Apoptosis was determined by Ho.33342 and propidium iodide (PI) double staining and flow cytometry. The cellular glutathione (GSH) content, superoxide dismutase (SOD) activity and malonaldehyde (MDA) levels were evaluated by spectrophotometer. The mRNA expressions of Apaf-1, Caspase-9, Caspase-3, Bcl-2, and Bax were examined by RT-PCR, while, the protein expressions of mitochondrial and cytosolic cytochrome c, Caspase-9, Caspase-3, Bcl-2 and Bax proteins were determined by Western blot respectively. The cell viability was markedly reduced, while, the apoptotic rate increased significantly after exposure of ONOO(-) (100μM) to SGNs. The activity of SOD and level of GSH were notably reduced, whereas, the MDA level was significantly increased. Pretreatment with curcumin protected SGNs against ONOO(-)-induced cell damage, declined the apoptotic rate, and improved the levels of SOD and GSH, decreased the elevation of MDA. ONOO(-) induced cytochrome c release from the mitochondria of SGNs and subsequently activated Caspase-9, Caspase-3 and cell apoptosis. Meanwhile, pretreatment with curcumin abrogated cytochrome c release, blocked activation of Caspase-3, and altered the expression of Bcl-2 family triggered by ONOO(-). Our data indicate that curcumin can attenuate ONOO(-)-induced damage in SGNs by the anti-oxidative activity as well as protect mitochondria from oxidative stress., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Oxidative/nitrosative stress plays a crucial role in Parkinson's disease (PD) by triggering mitochondrial dysfunction. Nitrosative stress is mediated by reactive species such as peroxynitrite (PN) which could damage biomolecules thereby impinging on the cellular machinery. We observed that PN (0-1000 μM) inhibited brain mitochondrial complex I (CI) activity in a dose-dependent manner with concomitant tyrosine nitration of proteins. We also observed that exposure to PN at low concentrations (62.5-125 μM) significantly decreased the mitochondrial membrane potential and affected the mitochondrial integrity at higher doses (500-750 μM) as indicated by the mitochondrial swelling experiment. Therefore, it could be surmised that compounds that prevent such mitochondrial damage might have therapeutic value in neurological conditions such as PD. We previously showed that curcumin could detoxify PN and protect against CI inhibition and protein nitration. However, the therapeutic potential of curcumin is constrained by limited bioavailability. To address this issue and obtain improved antioxidants, three bioconjugates of curcumin (Di-demethylenated piperoyl, di-valinoyl and di-glutamoyl esters) were generated and tested against PN-mediated nitrosative stress and mitochondrial damage. We found that among the bioconjugates, the glutamoyl diester of curcumin showed improved protection against PN-dependent CI inhibition and protein nitration compared to other conjugates. Di-glutamoyl curcumin protected dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP(+))-mediated neuronal death. These effects were improved compared to curcumin alone suggesting that di-glutamoyl curcumin could be a better neuroprotective agent in neurodegenerative diseases such as PD.

Paraquat, a cationic herbicide, produces degenerative lesions in the lung and in the nervous system after systemic administration to man and animals. Many cases of acute poisoning and death have been reported over the past few decades. Although a definitive mechanism of toxicity of paraquat has not been delineated, a cyclic single electron reduction/oxidation is a critical mechanistic event. The redox cycling of paraquat has two potentially important consequences relevant to the development of toxicity: the generation of the superoxide anion, which can lead to the formation of more toxic reactive oxygen species which are highly reactive to cellular macromolecules; and the oxidation of reducing equivalents (e.g., NADPH, reduced glutathione), which results in the disruption of important NADPH-requiring biochemical processes necessary for normal cell function. Nitric oxide is an important signaling molecule that reacts with superoxide derived from the paraquat redox cycle, to form the potent oxidant peroxynitrite, which causes serious cell damage. Although nitric oxide has been involved in the mechanism of paraquat-mediated toxicity, the role of nitric oxide has been controversial as both protective and harmful effects have been described. The present review summarizes recent findings in the field and describes new knowledge on the role of nitric oxide in the paraquat-mediated toxicity., (Copyright © 2010 Wiley Periodicals, Inc.)

Oxidant damage from reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major contributor to the cellular damage seen in numerous types of renal injury. Resveratrol (trans-3,4',5-trihydroxystilbene) is a phytoalexin found naturally in many common food sources. The anti-oxidant properties of resveratrol are of particular interest because of the fundamental role that oxidant damage plays in numerous forms of kidney injury. To examine whether resveratrol could block damage to the renal epithelial cell line, mIMCD-3, cells were exposed to the peroxynitrite donor 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1). Resveratrol produced a concentration-dependent inhibition of cytotoxicity induced by SIN-1. To examine the mechanism of protection, resveratrol was incubated with authentic peroxynitrite and found to block nitration of bovine serum albumin with an EC(50) value of 22.7 microM, in contrast to the known RNS scavenger, N-acetyl-l-cysteine, which inhibited nitration with an EC(50) value of 439 microM. These data suggested that resveratrol could provide functional protection by directly scavenging peroxynitrite. To examine whether resveratrol was a substrate for peroxynitrite oxidation, resveratrol was reacted with authentic peroxynitrite. Resveratrol nitration products and dimers were detected using liquid chromatograph with tandem electrospray mass spectrometry. Similar products were detected in the media of cells treated with SIN-1 and resveratrol. Taken collectively, the data suggest that resveratrol is able to provide functional protection of renal tubular cells, at least in part, by directly scavenging the RNS peroxynitrite. This property of resveratrol may contribute to the understanding of its anti-oxidant activities., (Copyright 2010 Elsevier Inc. All rights reserved.)