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Multiple biological barriers in solid tumors severely restrict the penetration of nanomedicines, which is a main cause for therapeutic failure in traditional tumor treatment. Here, a tumor-specific nanogenerator of peroxynitrite (ONOO - ), prepared by loading cisplatin and sodium nitroprusside into poly(d,l-lactide- co -glycolide) polymersomes, was designed to improve drug delivery and enhance tumor chemotherapy. After a cascade of nicotinamide adenine dinucleotide phosphate oxidases catalysis and glutathione reduction, the nanogenerator, namely, PMCS, could selectively induce the generation of ONOO - in tumor. The generated ONOO - could not only strengthen vascular permeability significantly but also improve the accumulation and penetration of PMCS in tumor by activating matrix metalloproteinases-mediated degradation of extracellular matrix. Along with endocytosis, PMCS released cisplatin to induce tumor cell apoptosis. Moreover, free cisplatin liberated from dead cells infected neighboring tumor cells quickly via ONOO - -mediated up-regulated copper transporter 1, further amplifying chemotherapeutic efficacy. This study advances ONOO - as a potent modality to address the main issues of therapeutic delivery, including but not limited to chemotherapy.

The interaction between cytochrome c and cardiolipin is a relevant process in the mitochondrial redox homeostasis, playing roles in the mechanism of electron transfer to cytochrome c oxidase and also modulating cytochrome c conformation, reactivity and function. Peroxynitrite is a widespread nitrating agent formed in mitochondria under oxidative stress conditions, and can result in the formation of tyrosine nitrated cytochrome c. Some of the nitro-cytochrome c species undergo conformational changes at physiological pH and increase its peroxidase activity. In this work we evaluated the influence of cardiolipin on peroxynitrite-mediated cytochrome c nitration yields and site-specificity. Our results show that cardiolipin enhances cytochrome c nitration by peroxynitrite and targets it to heme-adjacent Tyr67. Cytochrome c nitration also modifies the affinity of protein with cardiolipin. Using a combination of experimental techniques and computer modeling, it is concluded that structural modifications in the Tyr67 region are responsible for the observed changes in protein-derived radical and tyrosine nitration levels, distribution of nitrated proteoforms and affinity to cardiolipin. Increased nitration of cytochrome c in presence of cardiolipin within mitochondria and the gain of peroxidatic activity could then impact events such as the onset of apoptosis and other processes related to the disruption of mitochondrial redox homeostasis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Accumulation of collagen 4 (COL4) and thickened basement membrane are features of diabetic cardiac microvascular fibrosis that may be induced by oxidative stress. The ketone body β-hydroxybutyrate exhibits various cardiovascular protective effects, however its mechanism remains to be clarified. In the current study, the effects of β-hydroxybutyrate on cardiac microvascular fibrosis and COL4 accumulation were evaluated in streptozotocin-induced diabetic rats and in high glucose (HG) treated human cardiac microvascular endothelial cells (HCMECs). Generations of inducible nitric oxide synthase (iNOS) and copper-zinc superoxide dismutase (Cu/Zn-SOD), and the amount of nitrotyrosine (NT) were measured in vivo and in vitro. Ten weeks of β-hydroxybutyrate treatment (160, 200 and 240 mg/kg/d) attenuated cardiac microvascular fibrosis and inhibited cardiac COL4 generation and microvascular distribution in diabetic rats. Furthermore, β-hydroxybutyrate promoted cardiac Cu/Zn-SOD generation and reduced NT content, without reducing iNOS generation in diabetic rats. In HCMECs, stimulation with HG induced excess generation of COL4 via peroxynitrite. β-Hydroxybutyrate treatment (2, 4, 6 mM) attenuated HG-stimulated COL4 accumulation in a concentration-dependent manner. Similarly, 4 mM β-hydroxybutyrate promoted Cu/Zn-SOD generation and reduced NT content, without affecting excess iNOS generation in HG-stimulated HCMECs. In conclusion, this study showed that β-hydroxybutyrate promoted Cu/Zn-SOD generation, reduced peroxynitrite and inhibited cardiac microvascular COL4 accumulation in diabetes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A high level of reactive oxygen species (ROS) is widely considered one of the major causes of oocyte quality decline. Peroxynitrite is known as a powerful oxidant, which could induce multiple physical diseases. Recently, emerging pieces of evidences indicate that melatonin effectively promotes the development of oocytes, although the specific work mechanism remains to be further clarified. In this study, it was shown that peroxynitrite increased the level of ROS in porcine oocytes, which induced the apoptosis of oocytes, thereby leading to the obstruction of spindle assembly, depolymerization of actin, and decrease of polar body expulsion. These negative effects contributed to the failure of meiosis and ultimately blocked the maturation of porcine oocytes. As expected, it was found that melatonin effectively removed the accumulated ROS in oocytes, preventing oocytes from peroxynitrite-induced oocyte maturation failure, which might provide a novel approach to improve female livestock reproduction and cure female infertility in clinical practice.

Recent studies have focused on the contribution of capillary endothelial TRPV4 channels to pulmonary pathologies, including lung edema and lung injury. However, in pulmonary hypertension (PH), small pulmonary arteries are the focus of the pathology, and endothelial TRPV4 channels in this crucial anatomy remain unexplored in PH. Here, we provide evidence that TRPV4 channels in endothelial cell caveolae maintain a low pulmonary arterial pressure under normal conditions. Moreover, the activity of caveolar TRPV4 channels is impaired in pulmonary arteries from mouse models of PH and PH patients. In PH, up-regulation of iNOS and NOX1 enzymes at endothelial cell caveolae results in the formation of the oxidant molecule peroxynitrite. Peroxynitrite, in turn, targets the structural protein caveolin-1 to reduce the activity of TRPV4 channels. These results suggest that endothelial caveolin-1-TRPV4 channel signaling lowers pulmonary arterial pressure, and impairment of endothelial caveolin-1-TRPV4 channel signaling contributes to elevated pulmonary arterial pressure in PH. Thus, inhibiting NOX1 or iNOS activity, or lowering endothelial peroxynitrite levels, may represent strategies for restoring vasodilation and pulmonary arterial pressure in PH., Competing Interests: The authors declare no competing interest.

Phosphorylase is one of the most carefully studied proteins in history, but knowledge of its regulation during intense muscle contraction is incomplete. Tyrosine nitration of purified preparations of skeletal muscle phosphorylase results in inactivation of the enzyme and this is prevented by antioxidants. Whether an altered redox state affects phosphorylase activity and glycogenolysis in contracting muscle is not known. Here, we investigate the role of the redox state in control of phosphorylase and glycogenolysis in isolated mouse fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscle preparations during repeated contractions. Exposure of crude muscle extracts to H 2 O 2 had little effect on phosphorylase activity. However, exposure of extracts to peroxynitrite (ONOO - ), a nitrating/oxidizing agent, resulted in complete inactivation of phosphorylase (half-maximal inhibition at ∼200 µM ONOO - ), which was fully reversed by the presence of an ONOO - scavanger, dithiothreitol (DTT). Incubation of isolated muscles with ONOO - resulted in nitration of phosphorylase and marked inhibition of glycogenolysis during repeated contractions. ONOO - also resulted in large decreases in high-energy phosphates (ATP and phosphocreatine) in the rested state and following repeated contractions. These metabolic changes were associated with decreased force production during repeated contractions (to ∼60% of control). In contrast, repeated contractions did not result in nitration of phosphorylase, nor did DTT or the general antioxidant N -acetylcysteine alter glycogenolysis during repeated contractions. These findings demonstrate that ONOO - inhibits phosphorylase and glycogenolysis in living muscle under extreme conditions. However, nitration does not play a significant role in control of phosphorylase and glycogenolysis during repeated contractions. NEW & NOTEWORTHY Here we show that exogenous peroxynitrite results in nitration of phosphorylase as well as inhibition of glycogenolysis in isolated intact mouse skeletal muscle during short-term repeated contractions. However, repeated contractions in the absence of exogenous peroxynitrite do not result in nitration of phosphorylase or affect glycogenolysis, nor does the addition of antioxidants alter glycogenolysis during repeated contractions. Thus phosphorylase is not subject to redox control during repeated contractions.

The mitochondrial thioredoxin/peroxiredoxin system encompasses NADPH, thioredoxin reductase 2 (TrxR2), thioredoxin 2, and peroxiredoxins 3 and 5 (Prx3 and Prx5) and is crucial to regulate cell redox homeostasis via the efficient catabolism of peroxides (TrxR2 and Trxrd2 refer to the mitochondrial thioredoxin reductase protein and gene, respectively). Here, we report that endothelial TrxR2 controls both the steady-state concentration of peroxynitrite, the product of the reaction of superoxide radical and nitric oxide, and the integrity of the vascular system. Mice with endothelial deletion of the Trxrd2 gene develop increased vascular stiffness and hypertrophy of the vascular wall. Furthermore, they suffer from renal abnormalities, including thickening of the Bowman's capsule, glomerulosclerosis, and functional alterations. Mechanistically, we show that loss of Trxrd2 results in enhanced peroxynitrite steady-state levels in both vascular endothelial cells and vessels by using a highly sensitive redox probe, fluorescein-boronate. High steady-state peroxynitrite levels were further found to coincide with elevated protein tyrosine nitration in renal tissue and a substantial change of the redox state of Prx3 toward the oxidized protein, even though glutaredoxin 2 (Grx2) expression increased in parallel. Additional studies using a mitochondria-specific fluorescence probe (MitoPY1) in vessels revealed that enhanced peroxynitrite levels are indeed generated in mitochondria. Treatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin [Mn(III)TMPyP], a peroxynitrite-decomposition catalyst, blunted intravascular formation of peroxynitrite. Our data provide compelling evidence for a yet-unrecognized role of TrxR2 in balancing the nitric oxide/peroxynitrite ratio in endothelial cells in vivo and thus establish a link between enhanced mitochondrial peroxynitrite and disruption of vascular integrity., Competing Interests: The authors declare no competing interest.

Memantine has demonstrated beneficial effects on several types of brain insults via therapeutic mechanisms mainly related to its activity as a receptor antagonist of N-methyl-d-aspartate. However, the influences of memantine on intracerebral hemorrhage (ICH) remain obscure. This research probed into the neurovascular protective mechanisms of memantine after ICH and its impacts on neuronal nitric oxide synthase (nNOS) ser1412 phosphorylation. ICH model was established by employing intrastriatal collagenase injection in rats. After modeling, rats were then allocated randomly into sham-operated (sham), vehicle-treated (ICH+V), and memantine-administrated (ICH+M) groups. Memantine (20 mg/kg/day) was intraperitoneally administered 30 min after ICH and thenceforth once daily. Rats were dedicated at 0.25, 6, 12, 24 h, 3 and 7 d post-ICH for measurement of corresponding indexes. Behavioral changes, brain edema, levels of nNOS ser1412 phosphorylation, peroxynitrite, matrix metalloproteinase (MMP)-9, NLRP3, IL-1β and numbers of dying neurons, as well as the cellular localization of gelatinolytic activity, were detected among the groups. Memantine improved the neurologic deficits and mitigated brain water content, levels of MMP-9, NLRP3, IL-1β and dying neurons. Additionally, treatment with memantine also reduced nNOS ser1412 phosphorylation and peroxynitrite formation compared with the ICH+V group at 24 h after ICH. In situ zymography simultaneously revealed that gelatinase activity was primarily colocalized with vessel walls and neurons. We concluded that memantine ameliorated blood-brain barrier disruption and neurologic dysfunction in an ICH rat model. The underlying mechanism might involve repression of nNOS ser1412 phosphorylation, as well as peroxynitrite-related MMP-9 and NLRP3 inflammasome activation., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Epilepsy is a chronic neurodegenerative disease that has seriously threatened human health. Accumulating evidence reveals that the pathological progression of epilepsy is closely related to peroxynitrite (ONOO - ). Unfortunately, understanding the physiological roles of ONOO - in epilepsy is still challenging due to the lack of powerful imaging probes for the determination of the level of fluctuations of ONOO - in the epileptic brain. Herein, a near-infrared (NIR) two-photon (TP) fluorescent probe [dicyanomethylene-4 H -pyran (DCM)-ONOO] is presented to trace ONOO - in living cells and in kainate (KA)-induced rat epilepsy models with satisfactory sensitivity and selectivity. The probe is composed of a NIR TP DCM fluorophore and a recognition moiety diphenylphosphinamide. The phosphoramide bond of the probe is interrupted after reacting with ONOO - for 10 min, and then, the released amino groups emit strong fluorescence due to the restoration of the intramolecular charge transfer process. The probe can effectively detect the changes of endogenous ONOO - with excellent temporal and spatial resolution in living cells and in rat epileptic brain. The imaging results demonstrate that the increasing level of ONOO - is closely associated with epilepsy and severe neuronal damage in the brain under KA stimulation. In addition, the low-dose resveratrol can effectively inhibit ONOO - overexpression and further relieve neuronal damage. With the assistance of TP fluorescence imaging in the epileptic brain tissue, we hypothesize that the abnormal levels of ONOO - may serve as a potential indicator for the diagnosis of epilepsy. The TP fluorescence imaging based on DCM-ONOO provides a great potential approach for understanding the epilepsy pathology and diagnosis.

Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Freshly isolated brain nonsynaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knockout (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 µmol/L) or a PN donor (SIN-1; 50 µmol/L) or a PN scavenger (FeTMPyP; 2.5 µmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by Western blots. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but were unaffected by PN or SIN-1. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency. NEW & NOTEWORTHY Mitochondria generate superoxide and nitric oxide that could potentially react with each other to produce PN. We observed eNOS and nNOS immunoreactivity in isolated brain and heart mitochondria with pharmacological inhibition of nNOS found to modulate the mitochondrial respiratory function. This study provides evidence of generation of functionally significant PN in isolated brain mitochondria that affects respiratory function under physiological conditions. Importantly, the mitochondrial PN levels and activity were exaggerated in the eNOS-deficient mice, suggesting its pathological significance.

Nitric oxide (NO) donors are promising therapeutic candidates for treating intraocular hypertension (IOP) and glaucoma. This study aims to investigate the effect of prolonged use of NO donor sodium nitroprusside (SNP) on IOP. Since SNP has a short biological half-life, a nanoparticle drug delivery system (mesoporous silica nanoparticles) has been used to deliver SNP to the target tissues (trabecular meshwork and Schlemm's canal). We find that the sustained use of NO donor initially reduced IOP followed, surprisingly, by IOP elevation, which could not recover by drug withdraw but could be reversed by the antioxidant MnTMPyP application. The IOP elevation and normalization coincide with increased and reduced protein nitration in the mouse conventional outflow tissue. These findings suggest that the prolonged use of NO donor SNP may be problematic as it can cause outflow tissue damage by protein nitration. MnTMPyP is protective of the nitrative damage which could be considered to be co-applied with NO donors., (Copyright © 2020. Published by Elsevier Ltd.)

Peroxynitrite is known to react with biomolecules leading to their structural and function alteration. Structural alteration in DNA induced by peroxynitrite is not clearly known. The current study was carried out to decipher the changes induced by peroxynitrite using UV-Vis spectra, circular dichrometry, molecular dynamics simulation followed by restriction digestion. Apoptotic markers Bax, Bcl-2 and caspase genes were also studied by FACS in peroxynitrite induced PC12 cells. The results obtained showed that PXN binds to DNA leading to hyperchromicity of DNA in the presence of PXN over a period of time and the same was established by In silico studies where PXN modifies the DNA to accommodate itself into the stacking and brings about the significant structural alterations. Further, FACS studies reveal that Bcl-2 gene expression was down regulated whereas BAXand caspase genes were up regulated compared to control concluding that PXN induces apoptotic cell death in PC12 cells.

Cold physical plasmas modulate cellular redox signaling processes, leading to the evolution of a number of clinical applications in recent years. They are a source of small reactive species, including reactive nitrogen species (RNS). Wound healing is a major application and, as its physiology involves RNS signaling, a correlation between clinical effectiveness and the activity of plasma-derived RNS seems evident. To investigate the type and reactivity of plasma-derived RNS in aqueous systems, a model with tyrosine as a tracer was utilized. By high-resolution mass spectrometry, 26 different tyrosine derivatives including the physiologic nitrotyrosine were identified. The product pattern was distinctive in terms of plasma parameters, especially gas phase composition. By scavenger experiments and isotopic labelling, gaseous nitric dioxide radicals and liquid phase peroxynitrite ions were determined as dominant RNS. The presence of water molecules in the active plasma favored the generation of peroxynitrite. A pilot study, identifying RNS driven post-translational modifications of proteins in healing human wounds after the treatment with cold plasma (kINPen), demonstrated the presence of in vitro determined chemical pathways. The plasma-driven nitration and nitrosylation of tyrosine allows the conclusion that covalent modification of biomolecules by RNS contributes to the clinically observed impact of cold plasmas.

Ferric nitrobindins (Nbs) selectively bind NO and catalyze the conversion of peroxynitrite to nitrate. In this study, we show that NO scavenging occurs through the reductive nitrosylation of ferric Mycobacterium tuberculosis and Homo sapiens nitrobindins ( Mt -Nb(III) and Hs -Nb(III), respectively). The conversion of Mt -Nb(III) and Hs -Nb(III) to Mt -Nb(II)-NO and Hs -Nb(II)-NO, respectively, is a monophasic process, suggesting that over the explored NO concentration range (between 2.5 × 10 -5 and 1.0 × 10 -3 M), NO binding is lost in the mixing time (i.e., NO k on ≥ 1.0 × 10 6 M -1 s -1 ). The pseudo-first-order rate constant for the reductive nitrosylation of Mt -Nb(III) and Hs -Nb(III) (i.e., k ) is not linearly dependent on the NO concentration but tends to level off, with a rate-limiting step (i.e., k lim ) whose values increase linearly with [OH - ]. This indicates that the conversion of Mt -Nb(III) and Hs -Nb(III) to Mt -Nb(II)-NO and Hs -Nb(II)-NO, respectively, is limited by the OH - -based catalysis. From the dependence of k lim on [OH - ], the values of the second-order rate constant k OH- for the reductive nitrosylation of Mt -Nb(III)-NO and Hs -Nb(III)-NO were obtained (4.9 (±0.5) × 10 3 M -1 s -1 and 6.9 (±0.8) × 10 3 M -1 s -1 , respectively). This process leads to the inactivation of two NO molecules: one being converted to HNO 2 and another being tightly bound to the ferrous heme-Fe(II) atom.

We hypothesized that NO • is generated in isolated cardiac mitochondria as the source for ONOO - production during oxidative stress. We monitored generation of ONOO - from guinea pig isolated cardiac mitochondria subjected to excess Ca 2+ uptake before adding succinate and determined if ONOO - production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl 2 and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O 2 •- . L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO - to form dityrosine (diTyr) in proportion to the ONOO - present. We found that exposing mitochondria to excess CaCl 2 before succinate resulted in an increase in diTyr and amplex red fluorescence (H 2 O 2 ) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa 2+ and succinate, increased mitochondrial ONOO - production via NO • and O 2 •- . Changes in mitochondrial ONOO - production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO • scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO - production, PTIO decreased production of ONOO - , and TEMPOL decreased ONOO - levels by converting more O 2 •- to H 2 O 2 . Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO - into the buffer is dependent both on O 2 •- released from mitochondria and NO • derived from a mtCa 2+ -inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible., Competing Interests: Declaration of competing interest The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cerebral venous sinus thrombosis (CVST) is a rare type of stroke, which is life-threatening in severe cases. However, considerably less attention has been concentrated on the mechanism of neural cell damage after CVST. This study aims to investigate the role of endoplasmic reticulum stress, oxidative stress, and pyroptosis in a well-established rodent model of CVST. Rat brains were harvested at 0 h, 6 h, days 1, days 3, days 7, and days 14 post-CVST for measurement of corresponding indexes. Endoplasmic reticulum stress sensors (including protein kinase RNA-like ER kinase (PERK) and inositol-requiring enzyme-1α (IRE1α)), oxidative stress markers (thioredoxin-interacting protein (TXNIP) and peroxynitrite), NLRP3, caspase p20, IL-1β, and gasdermin D (GSDMD, an indicator of pyroptosis) were separately evaluated by Western-blot and Immunohistochemistry/Immunofluorescence. Co-immunoprecipitation and Fluorescent double-labeling were employed to probe into the relationship between TXNIP/peroxynitrite and NLRP3 inflammasome. In the damaged cortex region, profuse p-PERK, p-IRE1α, TXNIP were produced and predominantly localized in neurons accompanied by a small amount expressed in microglia and astrocytes. The levels of 3-nitrotyrosine (3-NT, as a footprint of peroxynitrite), NLRP3, caspase p20, IL-1β, and GSDMD were distinctly elevated post-CVST and cellular localization of peroxynitrite, NLRP3, caspase p20, and IL-1β was largely observed in neurons and/or microglia. Importantly, sites of enhanced TXNIP and 3-NT immunoreactivity were colocalized with increased NLRP3 staining, indicating the involvement of TXNIP and peroxynitrite in NLRP3 inflammasome activation and subsequent pyroptosis. Besides, co-immunoprecipitation also hinted that there might be an interaction or causality between TXNIP/peroxynitrite and NLRP3 inflammasome. We concluded that endoplasmic reticulum stress and oxidative stress may jointly lead to neuronal NLRP3 inflammasome activation and pyroptosis after CVST., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

P-MAPA is a complex compound, derived from Aspergillus oryzae cultures, that has shown immunomodulatory properties in infection and cancer animal models. Despite promising results in these models, the mechanisms of cellular activation by P-MAPA, suggested to be Toll-like receptor- (TLR-) dependent, and its effect on human immune cells, remain unclear. Using an ex vivo model of human whole blood, the effects of P-MAPA on complement system activation, production of cytokines, and the expression of complement receptors (CD11b, C5aR, and C3aR), TLR2, TLR4, and the coreceptor CD14 were analyzed in neutrophils and monocytes. P-MAPA induced complement activation in human blood, detected by increased levels of C3a, C5a, and SC5b-9 in plasma. As a consequence, CD11b expression increased and C5aR decreased upon activation, while C3aR expression remained unchanged in leukocytes. TLR2 and TLR4 expressions were not modulated by P-MAPA treatment on neutrophils, but TLR4 expression was reduced in monocytes, while CD14 expression increased in both cell types. P-MAPA also induced the production of TNF- α , IL-8, and IL-12 and oxidative burst, measured by peroxynitrite levels, in human leukocytes. Complement inhibition with compstatin showed that P-MAPA-induced complement activation drives modulation of C5aR, but not of CD11b, suggesting that P-MAPA acts through both complement-dependent and complement-independent mechanisms. Compstatin also significantly reduced the peroxynitrite generation. Altogether, our results show that P-MAPA induced proinflammatory response in human leukocytes, which is partially mediated by complement activation. Our data contribute to elucidate the complement-dependent and complement-independent mechanisms of P-MAPA, which ultimately result in immune cell activation and in its immunomodulatory properties in infection and cancer animal models., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2020 Mariana Torrente Gonçalves et al.)

Hydroxycinnamic acids represent a versatile group of dietary plant antioxidants. Oxidation of methyl- p -coumarate ( pcm ) and methyl caffeate ( cm ) was previously found to yield potent antitumor metabolites. Here, we report the formation of potentially bioactive products of pcm and cm oxidized with peroxynitrite (ONOO¯), a biologically relevant reactive nitrogen species (RNS), or with α,α'-azodiisobutyramidine dihydrochloride (AAPH) as a chemical model for reactive oxygen species (ROS). A continuous flow system was developed to achieve reproducible in situ ONOO¯ formation. Reaction mixtures were tested for their cytotoxic effect on HeLa, SiHa, MCF-7 and MDA-MB-231 cells. The reaction of pcm with ONOO¯ produced two fragments, an o-nitrophenol derivative, and a new chlorinated compound. Bioactivity-guided isolation from the reaction mixture of cm with AAPH produced two dimerization products, including a dihydrobenzofuran lignan that exerted strong antitumor activity in vitro, and has potent in vivo antimetastatic activity which was previously reported. This compound was also detected from the reaction between cm and ONOO¯. Our results demonstrate the ROS/RNS dependent formation of chemically stable metabolites, including a potent antitumor agent ( 5 ), from hydroxycinnamic acids. This suggests that diversity-oriented synthesis using ROS/RNS to obtain oxidized antioxidant metabolite mixtures may serve as a valid natural product-based drug discovery strategy.

Drug-induced liver injury (DILI) is considered gradually as a serious public health issue, and hepatotoxicity has been regarded as the main clinical problem caused by it. We suspected that both the intracellular viscosity and peroxynitrite (ONOO - ) levels in drug-induced hepatotoxicity tissue are higher than those in a healthy liver. For this reason, we have presented a fluorescent probe VO for multichannel imaging viscosity and ONOO - simultaneously. Experimental results showed that VO has satisfactory detection performance for both viscosity and ONOO - , and based on the advantages of its lower cytotoxicity and pH-stabilities, VO was successfully employed to image viscosity and ONOO - in living cells and animals. More importantly, we use the probe to successfully showcase drug-induced hepatotoxicity by imaging viscosity and ONOO - induced by acetaminophen (APAP). All the results indicate that VO has great potential for the detection of viscosity and ONOO - and to assay drug-induced hepatotoxicity. Overall, this work offers a new detection tool/method for a deeper understanding of drug-induced organism injury.

Background/aim: To evaluate the protective effect of melatonin on ovarian ischemia reperfusion injury in a rat model., Materials and Methods: Forty-eight rats were separated equally into 6 groups. Group 1: sham; Group 2: surgical control with 3-h bilateral ovarian torsion and detorsion; Group 3: intraperitoneal 5% ethanol (1 mL) just after detorsion (as melatonin was dissolved in ethanol); Group 4: 10 mg/kg intraperitoneal melatonin 30 min before 3-h torsion; Group 5:10 mg/kg intraperitoneal melatonin just after detorsion; Group 6:10 mg/kg intraperitoneal melatonin 30 min before torsion and just after detorsion. Both ovaries and blood samples were obtained 7 days after detorsion for histopathological and biochemical analysis., Results: In Group 1, serum levels of total oxidant status (TOS) (μmol H2O2 equivalent/g wet tissue)were significantly lower than in Group2 (P = 0.0023), while tissue TOS levels were lower than in Group 3 (P = 0.0030). Similarly, serum and tissue levels of peroxynitrite in Group 6were significantly lower than those ofGroup 2 (P = 0.0023 and P = 0.040, respectively). Moreover, serum oxidative stress index (OSI) (arbitrary unit) levels were significantly increased in Group 2 when compared to groups 1 and 6 (P = 0.0023 and P= 0.0016, respectively) and in Group 3 with respect to groups 1, 4, 5, and 6 (P = 0.0023, P = 0.0026, P = 0.0008, and P = 0.0011, respectively). Furthermore, there was a significant decrease in histopathological scores including follicular degeneration, vascular congestion, hemorrhage, and inflammation in the melatonin and sham groups in comparison with control groups. Additionally, primordial follicle count was significantly higher in Group 6 than in Group 2 (P = 0.0002)., Conclusion: Melatonin attenuates ischemia reperfusion damage in a rat torsion/detorsion model by improving histopathological and biochemical findings including OSI and peroxynitrite., Competing Interests: The authors report no conflict of interest with any funding agency or commercial sectors., (This work is licensed under a Creative Commons Attribution 4.0 International License.)

Peroxynitrite (ONOO-) is a potent bio-oxidant involved in many physiological and pathological processes; however, most of the pathological effects associated with ONOO-in vivo are still ambiguous. Herein, we designed and synthesized two near-infrared ratiometric fluorescent probes, Ratio-A and Ratio-B, for the detection and biological evaluation of ONOO-. The recognition unit diene in the probes could be specifically cleaved by ONOO- with a 94-fold enhancement in the ratiometric fluorescence signal. By imaging ONOO- in immune stimulated cells and acute inflammation mice model using Ratio-A, we investigated the fluctuations of ONOO- levels in a rheumatoid arthritis (RA) model of mice. Ratio-A could be applied for the effective imaging of RA and could rapidly evaluate the response of the RA treatment with methotrexate (MTX). Thus, Ratio-A can be considered as a promising tool for pathological diagnosis and the therapeutic assessment of a wide range of diseases including RA.

Cold atmospheric plasma (CAP) is a promising new agent for (selective) cancer treatment, but the underlying cause of the anti-cancer effect of CAP is not well understood yet. Among different theories and observations, one theory in particular has been postulated in great detail and consists of a very complex network of reactions that are claimed to account for the anti-cancer effect of CAP. Here, the key concept is a reactivation of two specific apoptotic cell signaling pathways through catalase inactivation caused by CAP. Thus, it is postulated that the anti-cancer effect of CAP is due to its ability to inactivate catalase, either directly or indirectly. A theoretical investigation of the proposed theory, especially the role of catalase inactivation, can contribute to the understanding of the underlying cause of the anti-cancer effect of CAP. In the present study, we develop a mathematical model to analyze the proposed catalase-dependent anti-cancer effect of CAP. Our results show that a catalase-dependent reactivation of the two apoptotic pathways of interest is unlikely to contribute to the observed anti-cancer effect of CAP. Thus, we believe that other theories of the underlying cause should be considered and evaluated to gain knowledge about the principles of CAP-induced cancer cell death.

The chemokine fractalkine is synthesized as a membrane-bound protein, but studies have shown that serum levels of soluble fractalkine are elevated in inflammatory and autoimmune diseases. Patients with autoimmune diseases also have increased serum levels of neuropeptide substance P (SP). The shedding activity of the ADAM family is induced by peroxynitrite, but that of SP is unclear. Treatment of human macrophages with SP upregulated levels of membrane-bound fractalkine. Interestingly, small interfering RNA (siRNA) for DUOX2 further increased membrane-bound fractalkine but decreased soluble fractalkine compared with cells treated with SP alone. SP induced nitric oxide 2/inducible nitric oxide synthase (NOS2/iNOS) mRNA and increased levels of nitrotyrosine, a biomarker of peroxynitrite, whereas transfection with DUOX2 siRNA blunted upregulation of nitrotyrosine. Most importantly, N(ω)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) decreased protein levels of nitrotyrosine and concomitantly increased expression of membrane-bound fractalkine after exposure to SP. As for the signaling pathway of TGFβ1 (an inhibitor of iNOS mRNA expression), silencing of RNA for TAK-1 upregulated membrane-bound fractalkine, but silencing of RNA for the Smad family did not. Interfering RNA of transcription factor specificity protein 1 (Sp1) upregulated protein levels of TGFβ1/LAP. Most importantly, double transfection with siRNA for Sp1 and TRIM28/TIF1βor Fli-1 led to a significant increase in TGFβ1/LAP levels and a corresponding reduction of NOS2/iNOS, which inhibited the shedding of membrane-bound fractalkine. In conclusion, TRIM28/TIF1β and Fli-1 negatively regulate TGFβ1 expression to upregulate the generation of peroxynitrite, leading to increased shedding of membrane-bound fractalkine induced by SP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Peroxynitrite (ONOO - ) and high mobility group box 1 protein (HMGB1) are important cytotoxic factors contributing to cerebral ischemia-reperfusion injury. However, the roles of ONOO - in mediating HMGB1 expression and its impacts on hemorrhagic transformation (HT) in ischemic brain injury with delayed t-PA treatment remain unclear. In the present study, we tested the hypothesis that ONOO - could directly mediate the activation and release of HMGB1 in ischemic brains with delayed t-PA treatment. With clinical studies, we found that plasma nitrotyrosine (NT, a surrogate marker of ONOO - ) was positively correlated with HMGB1 level in acute ischemic stroke patients. Hemorrhagic transformation and t-PA-treated ischemic stroke patients had increased levels of nitrotyrosine and HMGB1 in plasma. In animal experiments, we found that FeTmPyP, a representative ONOO - decomposition catalyst (PDC), significantly reduced the expression of HMGB1 and its receptor TLR2, and inhibited MMP-9 activation, preserved collagen IV and tight junction claudin-5 in ischemic rat brains with delayed t-PA treatment. ONOO - donor SIN-1 directly induced expression of HMGB1 and its receptor TLR2 in naive rat brains in vivo and induced HMGB1 in brain microvascular endothelial b.End3 cells in vitro. Those results suggest that ONOO - could activate HMGB1/TLR2/MMP-9 signaling. We then addressed whether glycyrrhizin, a natural HMGB1 inhibitor, could inhibit ONOO - production and the antioxidant properties of glycyrrhizin contribute to the inhibition of HMGB1 and the neuroprotective effects on attenuating hemorrhagic transformation in ischemic stroke with delayed t-PA treatment. Glycyrrhizin treatment downregulated the expressions of NADPH oxidase p47 phox and p67 phox and iNOS, inhibited superoxide and ONOO - production, reduced the expression of HMGB1, TLR2, MMP-9, preserved type IV collagen and claudin-5 in ischemic brains. Furthermore, glycyrrhizin significantly decreased the mortality rate, attenuated hemorrhagic transformation, brain swelling, blood-brain barrier damage, neuronal apoptosis, and improved neurological outcomes in the ischemic stroke rat model with delayed t-PA treatment. In conclusion, peroxynitrite-mediated HMGB1/TLR2 signaling contributes to hemorrhagic transformation, and glycyrrhizin could be a potential adjuvant therapy to attenuate hemorrhagic transformation, possibly through inhibiting the ONOO - /HMGB1/TLR2 signaling cascades.

Tea is one of the most popular beverages in the world. Camellia sinensis tea (CST) or green tea is widely regarded as a potent antioxidant. In Thailand, Pluchea indica (L.) Less. tea (PIT) has been commercially available as a health-promoting drink. This study focused on free radical scavenging activities of PIT, and its ability to protect isolated human low-density lipoproteins (LDL) from oxidation by chemical agents. A preliminary study to investigate the antioxidant nature of PIT was undertaken. These included common antioxidant assays involving 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), hypochlorous acid (HOCl), and its potential to scavenge peroxynitrite. In separated experiments, isolated human LDL was challenged with either 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), copper (Cu 2+ ), or 3-Morpholinosydnonimine hydrochloride (SIN-1) to induce LDL oxidation. PIT exhibited antioxidant activity in all test systems and performed significantly better than CST in both DPPH ( P < 0.05; IC 50 PIT = 245.85 ± 15.83 and CST = 315.41 ± 24.18  μ g/ml) and peroxynitrite scavenging assays. PIT at 75  μ g/ml almost fully prevented the peroxynitrite over a 5 h period. Moreover, it displayed similar properties to CST during the antioxidation of isolated human LDL using AAPH, Cu 2+ , SIN-1, and hypochlorous acid scavenging assays. However, it revealed a significantly lower ABTS scavenging activity than CST ( P < 0.05; IC 50 PIT = 30.47 ± 2.20 and CST = 21.59 ± 0.67  μ g/ml). The main constituents of the PIT were identified using LC-MS/MS. It contained 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), and 4,5-O-dicaffeoylquinic acid (4,5-CQ). In conclusion, caffeoyl derivatives in PIT could play an important role in potent antioxidant properties. So, it may be further developed to be antioxidant beverages for preventing atherosclerosis and cardiovascular diseases associated with oxidative stress., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Kittipot Sirichaiwetchakoon et al.)

The cytochromes P450 are heme-dependent enzymes that catalyze many vital reaction processes in the human body related to biodegradation and biosynthesis. They typically act as mono-oxygenases; however, the recently discovered P450 subfamily TxtE utilizes O 2 and NO to nitrate aromatic substrates such as L-tryptophan. A direct and selective aromatic nitration reaction may be useful in biotechnology for the synthesis of drugs or small molecules. Details of the catalytic mechanism are unknown, and it has been suggested that the reaction should proceed through either an iron(III)-superoxo or an iron(II)-nitrosyl intermediate. To resolve this controversy, we used stopped-flow kinetics to provide evidence for a catalytic cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite species that is able to nitrate l-Trp efficiently. We show that the rate of binding of O 2 is faster than that of NO and also leads to l-Trp nitration, while little evidence of product formation is observed from the iron(II)-nitrosyl complex. To support the experimental studies, we performed density functional theory studies on large active site cluster models. The studies suggest a mechanism involving an iron(III)-peroxynitrite that splits homolytically to form an iron(IV)-oxo heme (Compound II) and a free NO 2 radical via a small free energy of activation. The latter activates the substrate on the aromatic ring, while compound II picks up the ipso -hydrogen to form the product. The calculations give small reaction barriers for most steps in the catalytic cycle and, therefore, predict fast product formation from the iron(III)-peroxynitrite complex. These findings provide the first detailed insight into the mechanism of nitration by a member of the TxtE subfamily and highlight how the enzyme facilitates this novel reaction chemistry.

We recently showed that red blood cells (RBCs) from patients with type 2 diabetes mellitus (T2DM-RBCs) induce endothelial dysfunction through a mechanism involving arginase I and reactive oxygen species. Peroxynitrite is known to activate arginase in endothelial cells. Whether peroxynitrite regulates arginase activity in RBCs, and whether it is involved in the cross-talk between RBCs and the vasculature in T2DM, is unclear and elusive. The present study was designed to test the hypothesis that endothelial dysfunction induced by T2DM-RBCs is driven by peroxynitrite and upregulation of arginase. RBCs were isolated from patients with T2DM and healthy age matched controls. RBCs were co-incubated with aortae isolated from wild type rats for 18 h in the absence and presence of peroxynitrite scavenger FeTTPS. Evaluation of endothelial function in organ chambers by cumulative addition of acetylcholine as well as measurement of RBC and vessel arginase activity was performed. In another set of experiments, RBCs isolated from healthy subjects (Healthy RBCs) were incubated with the peroxynitrite donor SIN-1 with subsequent evaluation of endothelial function and arginase activity. T2DM-RBCs, but not Healthy RBCs, induced impairment in endothelial function, which was fully reversed by scavenging of RBC but not vascular peroxynitrite with FeTPPS. Arginase activity was up-regulated by the peroxynitrite donor SIN-1 in Healthy RBCs, an effect that was inhibited by FeTTPS. Healthy RBCs co-incubated with aortae in the presence of SIN-1 caused impairment of endothelial function, which was inhibited by FeTTPS or the arginase inhibitor ABH. T2DM-RBCs induced up-regulation of vascular arginase, an effect that was fully inhibited by FeTTPS. Collectively, our data indicate that RBCs impair endothelial function in T2DM via an effect that is driven by a peroxynitrite-mediated increase in arginase activity. This mechanism may be targeted in patients with T2DM for improvement in endothelial function.

A series of near-infrared and photostable Si-oxazine fluorescent dyes was synthesized using a simple three-step procedure, and one of their reduced products, i.e. hydro-Si-oxazine HSiO3, has been utilized to sensitively detect hypochlorous acid and peroxynitrite generation by phagocytes in inflamed and pulmonary fibrosis diseased mice with emission wavelength beyond 750 nm.

Deep tissue imaging in the second near-infrared (NIR-II) window holds great promise for widespread fundamental research. However, inhomogeneous signal attenuation due to tissue absorption and scattering hampers its application for accurate in vivo biosensing. Here, lifetime-based in situ hepatocellular carcinoma (HCC) detection in NIR-II region is presented using a tumor-microenvironment (peroxynitrite, ONOO - )-responsive lanthanide-cyanine Förster resonance energy transfer (FRET) nanosensor. A specially designed ONOO - -responsive NIR-II dye, MY-1057, is synthesized as the FRET acceptor. Robust lifetime sensing is demonstrated to be independent of tissue penetration depth. Tumor lesions are accurately distinguished from normal tissue due to the recovery lifetime. Magnetic resonance imaging and liver dissection results illustrate the reliability of lifetime-based detection in single and multiple HCC models. Moreover, the ONOO - amount can be calculated according to the standard curve., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study aimed to further understand the toxicity of high concentrations of nitrogen dioxide (NO 2 ) to plants, especially to plant photosynthesis. Tobacco plants in the six-leaf stage were exposed to 16.0 μL L -1 NO 2 to determine the activities of photosystem II (PSII) and photosystem I (PSI) reaction centers, the blocking site of PSII electron transport, the degree of membrane peroxidation and the relative expression of PsbA, PsbO and PsaA genes in the third fully expanded leaves by using gas exchange and chlorophyll fluorescence techniques, biochemical and RT-PCR analysis. The results showed that 16.0 μL L -1 NO 2 caused necrotic lesions to form on leaves and significantly increased the generation rate of superoxide anions (O 2 - ) and the content of peroxynitrite (ONOO - ) in leaves of tobacco seedling, leading to damage to cell membrane, chlorophyll content and net photosynthetic rate reduction, and photosynthetic apparatus destruction. Fumigation with 16.0 μL L -1 NO 2 decreased the activity of PSII reaction center and oxygen evolution complex, and the relative expression of PabA in leaves of tobacco seedlings to inhibit the electron transport from the donor side to the receptor side of PSII, especially blocking the electron transport from Q A to Q B on the receptor side. The activity of the PSI reaction center and the relative expression of PsaA decreased, weakening the ability to accept electrons and inhibiting the electron transfer from PSII to PSI, which further increased the damage of PSII of tobacco seedling leaves caused by 16.0 μL L -1 NO 2 . Therefore, 16.0 μL L -1 NO 2 leaded to the accumulation of O 2 - and ONOO - , which damaged the cell membrane and thylakoid membrane, inhibit the electron transport, and destroyed the photosynthetic apparatus in leaves of tobacco seedlings. The results from this study emphasized the importance of reducing the NO 2 concentration in the atmosphere., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Main Conclusion: Nitro/oxidative modifications of proteins and RNA nitration resulted from altered peroxynitrite generation are elements of the indirect mode of action of canavanine and meta-tyrosine in plants Environmental conditions and stresses, including supplementation with toxic compounds, are known to impair reactive oxygen (ROS) and reactive nitrogen species (RNS) homeostasis, leading to modification in production of oxidized and nitrated derivatives. The role of nitrated and/or oxidized biotargets differs depending on the stress factors and developmental stage of plants. Canavanine (CAN) and meta-tyrosine (m-Tyr) are non-proteinogenic amino acids (NPAAs). CAN, the structural analog of arginine, is found mostly in seeds of Fabaceae species, as a storage form of nitrogen. In mammalian cells, CAN is used as an anticancer agent due to its inhibitory action on nitric oxide synthesis. m-Tyr is a structural analogue of phenylalanine and an allelochemical found in root exudates of fescues. In animals, m-Tyr is recognized as a marker of oxidative stress. Supplementation of plants with CAN or m-Tyr modify ROS and RNS metabolism. Over the last few years of our research, we have collected the complex data on ROS and RNS metabolism in tomato (Solanum lycopersicum L.) plants exposed to CAN or m-Tyr. In addition, we have shown the level of nitrated RNA (8-Nitro-guanine) in roots of seedlings, stressed by the tested NPAAs. In this review, we describe the model of CAN and m-Tyr mode of action in plants based on modifications of signaling pathways induced by ROS/RNS with a special focus on peroxynitrite induced RNA and protein modifications.

To investigate the association between systemic nitrotyrosine (NT) levels and primary angle-closure glaucoma (PACG) and primary open-angle glaucoma (POAG) and the mechanism involved. A case control study was conducted in the Department of Ophthalmology & Visual Science, Eye & ENT Hospital, Fudan University from April 2017 to December 2017. A total of 400 participants were consecutively recruited into this study (100 PACG, 100 POAG and 200 controls). Multivariable logistic regression analysis was performed to identify the association between serum NT level and PACG or POAG. Clinical results were validated in cell and animal models. Among 200 glaucoma patients, 101 (50.5%) were women; the age was 57.07 ± 14.51 years. 106 (53%) control participants were women and age was 58.34 ± 14.04 years. Serum levels of NT in PACG and POAG patients are significantly higher than controls (1808.53 ± 417.76 nmol/L vs. 1270.62 ± 454.60 nmol/L, p < 0.001; 1718.63 ± 437.29 nmol/L vs. 1258.38 ± 460.72 nmol/L, p < 0.001). Further, elevated serum NT level increases the risk of developing PACG (OR = 1.003, 95% CI: 1.002 to 1.004, p < 0.001) and POAG (OR = 1.002, 95% CI: 1.002 to 1.003, p < 0.001). Consistent with the clinical data, serum and aqueous humour NT levels are significantly higher in caveolin 1 knockout (Cav1 KO) mice, an animal model of glaucoma. More importantly, peroxynitrite (PN) scavenger MnTMPyP and its transduction molecule PARP inhibitor significantly reduce intraocular pressure in Cav1 KO mice. Our data show for the first time that NT is a systemic risk factor and local treatment target of glaucoma., Competing Interests: Declaration of competing interest No conflicting relationship exists for any author., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Microcystin-LR (MC-LR) is a widely known hepatotoxin which could induce the occurrence and metastasis of hepatocellular carcinoma. In recent years, with the frequent outbreak of cyanobacteria, the harm of MC-LR has gradually attracted more attention. Hence, this study focused on the effect of MC-LR on DNA damage in HepG2 cells, identifying the types and sources of free radicals that make an important function on this issue. Our data suggested that MC-LR induced concentration- and time-dependent increasement of DNA double-strand breaks (DSBs). After exposure to 1 μM MC-LR for 3 days, the protein expression and immunofluorescence staining of γ-H2AX was significantly increased. Using a scavenger of mitochondrial O 2 .- (4-hydroxy-tempo), a inhibitor of mitochondrial NOS (7-nitroindazole), and a scavenger of ONOO - (uric acid), it was revealed that ONOO - originated from mitochondria made a significant contribution to the genotoxicity of MC-LR. Moreover, a significant decreasement of mitochondrial membrane potential (MMP) was observed. These findings suggested that peroxynitrite targeting mitochondria plays a vital role in the MC-LR-induced genotoxic response in mammalian cells., Competing Interests: Declaration of competing interest There are no conflicts to declare. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and death worldwide. Peroxynitrite, formed from nitric oxide, which is derived from inducible nitric oxide synthase, and superoxide, has been implicated in the development of emphysema, but the source of the superoxide was hitherto not characterized. Here, we identify the non-phagocytic NADPH oxidase organizer 1 (NOXO1) as the superoxide source and an essential driver of smoke-induced emphysema and pulmonary hypertension development in mice. NOXO1 is consistently upregulated in two models of lung emphysema, Cybb (also known as NADPH oxidase 2, Nox2)-knockout mice and wild-type mice with tobacco-smoke-induced emphysema, and in human COPD. Noxo1-knockout mice are protected against tobacco-smoke-induced pulmonary hypertension and emphysema. Quantification of superoxide, nitrotyrosine and multiple NOXO1-dependent signalling pathways confirm that peroxynitrite formation from nitric oxide and superoxide is a driver of lung emphysema. Our results suggest that NOXO1 may have potential as a therapeutic target in emphysema.

We selected five substances among the organochlorine pesticides (OCPs), chlordane, heptachlor, p,p'-DDT, β-HCH, and hexachlorobenzene, and investigated whether low-concentration exposure to the OCP compounds affected glucose metabolism. The exposure of L6 myotubes to the OCP compounds (1 or 5 μM) for 24 and 48 h significantly inhibited glucose uptake with an excessive production of intracellular reactive oxygen species (ROS) and peroxynitrite anions (ONOO - ) compared to control cells. In contrast, the production of nitric oxide was highly reduced by exposure to the OCP compounds. The protein expression of glucose transporter 4 (GLUT4) in the L6 myotubes was significantly suppressed by exposure to the OCP compounds. In addition, exposure to the OCP compounds for 1 h in RIN-m5F pancreatic beta cells remarkably suppressed insulin secretion but the ability to secrete insulin recovered to control levels after 24 h exposure to the OCP compounds. The abundant ROS generated by 1 h exposure to OCP compounds was inversely related to insulin secretion in RIN-m5F pancreatic beta cells. Therefore, these results suggest that low-concenration exposure of skeletal muscle and pancreatic beta cells to OCP compounds may affect insulin secretion and insulin-dependent glucose uptake through extreme oxidative stress and inactivation of the glucose transport protein., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020. Published by Elsevier Ltd.)

Parkinson's disease (PD) is a chronic low-grade inflammatory process in which activated microglia generate cytotoxic factors-most prominently peroxynitrite-which induce the death and dysfunction of neighboring dopaminergic neurons. Dying neurons then release damage-associated molecular pattern proteins such as high mobility group box 1 which act on microglia via a range of receptors to amplify microglial activation. Since peroxynitrite is a key mediator in this process, it is proposed that nutraceutical measures which either suppress microglial production of peroxynitrite, or which promote the scavenging of peroxynitrite-derived oxidants, should have value for the prevention and control of PD. Peroxynitrite production can be quelled by suppressing activation of microglial NADPH oxidase-the source of its precursor superoxide-or by down-regulating the signaling pathways that promote microglial expression of inducible nitric oxide synthase (iNOS). Phycocyanobilin of spirulina, ferulic acid, long-chain omega-3 fatty acids, good vitamin D status, promotion of hydrogen sulfide production with taurine and N-acetylcysteine, caffeine, epigallocatechin-gallate, butyrogenic dietary fiber, and probiotics may have potential for blunting microglial iNOS induction. Scavenging of peroxynitrite-derived radicals may be amplified with supplemental zinc or inosine. Astaxanthin has potential for protecting the mitochondrial respiratory chain from peroxynitrite and environmental mitochondrial toxins. Healthful programs of nutraceutical supplementation may prove to be useful and feasible in the primary prevention or slow progression of pre-existing PD. Since damage to the mitochondria in dopaminergic neurons by environmental toxins is suspected to play a role in triggering the self-sustaining inflammation that drives PD pathogenesis, there is also reason to suspect that plant-based diets of modest protein content, and possibly a corn-rich diet high in spermidine, might provide protection from PD by boosting protective mitophagy and thereby aiding efficient mitochondrial function. Low-protein diets can also promote a more even response to levodopa therapy.

Because of the low tissue penetration depth and poor photostability of organic cyanine dye, in addition to environmental interference, it is a great challenge to monitor the degree of drug-induced hepatotoxicity by the in vivo detection of peroxynitrite (ONOO-). Herein, we fabricated heptamethine cyanine dye (P-cy7)-coordinated upconversion nanoparticles (UCNPs), namely UCY7, as a fluorescent nanoprobe for evaluating drug-induced hepatotoxicity. Due to the luminescence resonance energy transfer (LRET) between UCNPs and the cyanine dye (P-cy7), the irradiation changed from visible light at 660 nm to near infrared (NIR) light at 980 nm; therefore, the issues of poor photostability and severe photobleaching of cyanine dye can be effectively solved. After injecting via the tail vein, the nanoprobes are rapidly concentrated in the liver. Since the level of ONOO- is up-regulated during the drug-induced liver injury, the LRET between UCNPs and P-cy7 is disrupted to release the upconversion luminescence at 656 nm, while the upconversion luminescence at 800 nm remains constant, thus achieving the ratio-fluorescent imaging (RFLI) of ONOO- in the liver to calibrate the influence of the environment. In addition, the reduction in the absorption of nanoprobes in the presence of ONOO- allows for sensitive photoacoustic imaging (PAI). Based on the RFLI and PAI of the liver, the real-time monitoring and accurate diagnosis of different degrees of hepatotoxicity using the model of Acetaminophen (APAP) induction was achieved successfully, providing a new approach for the clinical evaluation of drug-induced hepatotoxicity.

Nitric oxide (NO) and superoxide anions (O 2 •- ) are two noteworthy reactive species implicated in various physiological and pathological processes, such as ROS-induced lysosomal cell death. The interaction ("crosstalk") between them may form a new mediator peroxynitrite (ONOO - ) which has implications for cancer, diabetes, Alzheimer's disease, and liver-damage. It is therefore essential to investigate lysosomal NO/O 2 •- crosstalk in vivo through ONOO - -responsive molecular tools in order to fully comprehend the physiological and pathological mechanisms involved. In this study, a lysosome-targeting iridium(III) complex, Ir-NIR , has been investigated as a near-infrared (NIR) phosphorescent probe for visualizing NO/O 2 •- crosstalk by the phosphorescent detection of endogenous ONOO - levels in vivo. Ir-NIR exhibits a rapid (within 200 s), highly sensitive, and approximately 100-fold enhanced response to ONOO - in phosphorescence intensity. Thus, these characteristics, coupled with good cell permeability and low cytotoxicity, enable the probe to be used to detect intracellular ONOO - living organisms both in vitro and in vivo.

Background: Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca 2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca 2+ signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca 2+ influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels., Methods: We used endothelium-specific knockout mice and high-fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals., Results: We show that Ca 2+ influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients., Conclusions: These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

Molecular hydrogen is thought to have an inhibitory effect on oxidative stress, thereby attenuating the onset and progression of various diseases including cardiovascular disease; however, few reports have assessed the preventive effect of constitutive inhalation of hydrogen gas on of vascular remodeling. Here, we investigated the effect of constitutive inhalation of hydrogen gas on vascular neointima formation using a cuff-induced vascular injury mouse model. After constitutive inhalation of compressed hydrogen gas (O2 21%, N2 77.7%, hydrogen 1.3%) or compressed air only (O2 21%, N2 79%) by C57BL/6 mice for 2 weeks from 8 weeks of age in a closed chamber, inflammatory cuff injury was induced by polyethylene cuff placement around the femoral artery under anesthesia, and hydrogen gas administration was continued until sampling of the femoral artery. Neointima formation, accompanied by an increase in cell proliferation, was significantly attenuated in the hydrogen group compared with the control group. NADPH oxidase NOX1 downregulation in response to cuff injury was shown in the hydrogen group, but the expression levels of NADPH oxidase subunits, p40phox and p47phox, did not differ significantly between the hydrogen and control groups. Although the increase in superoxide anion production did not significantly differ between the hydrogen and control groups, DNA damage was decreased as a result of reduction of reactive oxygen species such as hydroxyl radical (⋅OH) and peroxynitrite (ONOO-) in the hydrogen group. These results demonstrate that constitutive inhalation of hydrogen gas attenuates vascular remodeling partly via reduction of oxidative stress, suggesting that constitutive inhalation of hydrogen gas at a safe concentration in the living environment could be an effective strategy for prevention of vascular diseases such as atherosclerosis., Competing Interests: The authors have declared that no competing interests exist.

Drug-induced liver injury (DILI) is a widespread clinical problem. The pathophysiological mechanisms of DILI are complicated, and the traditional diagnostic methods for DILI have their limitations. Owing to its convenient operation, high sensitivity, and high specificity, luminescent sensing and imaging as an indispensable tool in biological research and clinical trials may provide an important means for DILI study. Herein, we report the rational design and preparation of a near-infrared dual-phosphorescent polymeric probe ( P-ONOO ) for exploring the DILI via specific imaging of peroxynitrite (ONOO - ) elevation in vivo, which was one of early markers of DILI and very difficult to be detected due to its short half-life and high reactive activity. With the utilization of P-ONOO , the raised ONOO - was visualized successfully in the drug-treated hepatocytes with a high signal-to-noise ratio via ratiometric and time-resolved photoluminescence imaging. Importantly, the ONOO - boost in the acetaminophen-induced liver injury in real time was verified, and the direct observation of the elevated ONOO - production in ketoconazole-induced liver injury was achieved for the first time. Our findings may contribute to understanding the exact mechanism of ketoconazole-induced hepatotoxicity that is still ambiguous. Notably, this luminescent approach for revealing the liver injury works fast and conveniently.

Peroxynitrite (ONOO - ) is a primary kind of reactive oxygen species. Excessive ONOO - can induce oxidative damage to biomolecules and further results in various diseases. So, quantitative monitoring ONOO - with excellent selectivity and sensitivity is imperative for elucidating its role in biological processes. In this study, a novel pyridinium fluorescent ONOO - probe (CPC) has been constructed base on ICT-modulated by combining coumarin fluorophore and diphenylphosphinate recognition group. The fluorescence response of CPC for ONOO - is realized via the removal of diphenylphosphinate group. The probe CPC shows prominent features for detection of ONOO - including fast response rate (within 3 min), excellent selectivity and sensitivity, distinct colorimetric (red to green), and a large emission wavelength shift (105 nm). The emission intensity ration (I 538 /I 643 ) exhibits 153-fold enhancement along with the increasing ONOO - and the detection limit is as low as 1.60 × 10 - 8  M. These good response properties make CPC possible to quantitative detection of ONOO - concentration. By using the strategy, the ratiometric CPC has been employed to detection of mitochondrial ONOO - in live cell successfully., Competing Interests: Declaration of competing interest The authors declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Peroxynitrite (ONOO - ) is involved in neurodegenerative, inflammatory, cardiovascular disorders, cancers, and other pathological progress. However, current imaging methods for sensing ONOO - usually suffer from high background/autofluorescence for fluorescent probes and poor selectivity/short emission wavelength for chemiluminescent probes. Herein, we present a novel chemiluminescent molecule (oxygen-embedded quinoidal pentacene) responsive to ONOO - for the first time, on the basis of which we rationally construct a near-infrared nanoprobe for detecting ONOO - via chemiluminescence resonance energy transfer (CRET) mechanism. Notably, our nanoprobe exhibits good selectivity, ultrahigh sensitivity (nanomole level), low background noise, fast response, and high water solubility. Moreover, the near-infrared emission from CRET offers higher tissue penetration of the chemiluminescent signal. Finally, our nanoprobe is further successfully applied to detecting endogenous ONOO - in mice with abdominal inflammation, drug-induced hepatotoxicity, or tumor models in vivo. In summary, the self-luminescing nanoprobes can act as an alternative visualizable tool for illuminating the mechanism of ONOO - involved in the specific pathological process.

Parkinson's disease (PD) is a neurodegenerative disease that devastatingly affects people's lives. Numerous research studies have shown that peroxynitrite (ONOO - ) plays a pivotal role in the pathogenesis of PD. However, a suitable tool that could quickly and sensitively detect ONOO - in various PD models is still lacking. To this end, we designed and synthesized a series of near-infrared probes that could detect ONOO - within seconds by near-infrared fluorescent imaging in an ultrafast and highly selective manner. It is noteworthy that one of those developed probes, NIR-PN1 , showed excellent sensing performance and blood-brain barrier penetrating ability. NIR-PN1 was successfully applied for imaging of ONOO - fluxes in multiple PD models including PC12 cell, Drosophila, C. elegans , and mouse brain, indicating its great potential application not only for understanding the biological roles that ONOO - played in PD but also for early PD diagnosis and treatment.

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment., Competing Interests: The authors declare no conflict of interest.

Peroxynitrite is an ion acting as a powerful oxidant and nucleophile, which plays a key role in the inflammation and aging process by nitrating tyrosine or tryptophan residues of the proteins. Nitration of a target protein is considered to be a proper method to study the behavioral change of the proteins being nitrated. The commonly used methods for peroxynitrite preparation in vitro usually contain high concentration of sodium hydroxide, which easily induces hydrolysis of target proteins. Accordingly, the method for peroxynitrite preparation was optimized in vitro by changing the sequence of hydrochloric acid and sodium hydroxide added. After different amount of hydrochloric acid added to the system following sodium nitrite, peroxynitrite can be yielded in a concentration up to 60 mM with sodium hydroxide as low as 17 mM. More importantly, biological activity of the target protein was well maintained after protein nitration since low sodium hydroxide was used.

Human diving is known to induce endothelial dysfunction. The aim of this study was to decipher the mechanism of ROS production during diving through the measure of mitochondrial calcium concentration, peroxynitrite, NO°, and superoxide towards better understanding of dive-induced endothelial dysfunction. Air diving simulation using bovine arterial endothelial cells (compression rate 101 kPa/min to 808 kPa, time at depth 45 min) was performed in a system allowing real-time fluorescent measurement. During compression, the cells showed increased mitochondrial superoxide, peroxynitrite, and mitochondrial calcium, and decreased NO° concentration. MnTBAP (peroxynitrite scavenger) suppressed superoxide, recovered NO° production and promoted stronger calcium influx. Superoxide and peroxynitrite were inhibited by L-NIO (eNOS inhibitor), but were further increased by spermine-NONOate (NO° donor). L-NIO induced stronger calcium influx than spermine-NONOate or simple diving. The superoxide and peroxynitrite were also inhibited by ruthenium red (blocker of mitochondrial Ca 2+ uniporter), but were increased by CGP (an inhibitor of mitochondrial Na + -Ca 2+ exchange). Reactive oxygen and nitrogen species changes are associated, together with calcium mitochondrial storage, with endothelial cell dysfunction during simulated diving. Peroxynitrite is involved in NO° loss, possibly through the attenuation of eNOS and by increasing superoxide which combines with NO° and forms more peroxynitrite. In the field of diving physiology, this study is the first to unveil a part of the cellular mechanisms of ROS production during diving and confirms that diving-induced loss of NO° is linked to superoxide and peroxynitrite.

The kidneys are important organs that are susceptible to aging. Hyperhomocysteinemia (HHcy) is a risk factor for nephropathy and is associated with chronic nephritis, purpuric nephritis, and nephrotic syndrome. Numerous studies have shown that elevated serum homocysteine levels can damage the kidneys; however, the underlying mechanism of HHcy on kidney damage remains unclear. In this study, we make use of a diet-induced HHcy rat model and in vitro cell culture to explore the role of autophagy in HHcy-induced renal aging and further explored the underlying mechanism. We demonstrated that HHcy led to the development of renal aging. Promoted kidney aging and autophagic insufficiency were involved in HHcy-induced renal aging. HHcy decreased the expression of transcription factor EB (TFEB), the key transcription factor of autophagy-related genes in renal tissue. Further experiments showed that nitrative stress levels were increased in the kidney of HHcy rats. Interestingly, pretreatment with the peroxynitrite (ONOO - ) scavenger FeTMPyP not only reduced the Hcy-induced nitrative stress in vitro but also partially attenuated the decrease in TFEB in both protein and mRNA levels. Moreover, our results indicated that HHcy reduced TFEB expression and inhibited TFEB-mediated autophagy activation by elevating nitrative stress. In conclusion, this study showed an important role of autophagic insufficiency in HHcy-induced renal aging, in which downregulation of TFEB plays a major role. Furthermore, downexpression of TFEB was associated with increased nitrative stress in HHcy. This study provides a novel insight into the mechanism and therapeutic strategy for renal aging., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Shangyue Zhang et al.)

Molecular hydrogen (H 2 ) is a colorless, tasteless, odorless, and minimal molecule with high flammability. Although H 2 has been thought to be an inert gas in living bodies for many years, an animal study reported that inhalation of H 2 gas decreased oxidative stress and suppressed brain injury caused by ischemia and reperfusion injury due to its antioxidant action. Since then, the antioxidant action of H 2 has attracted considerable attention and many studies have reported on its benefits. Most studies have reported the effects of H 2 on diseases such as cancer, diabetes, cerebral infarction, and Alzheimer's disease. However, little is known regarding its effects on healthy subjects and exercise. Thus far, including our study, only 6 studies have explored the effect of H 2 on exercise. H 2 is the smallest molecule and therefore can easily penetrate the cellular membrane and rapidly diffuse into organelles. H 2 is thought to be able to selectively reduce hydroxyl radicals and peroxynitrite and does not affect physiologically reactive species. H 2 can be supplied to the body through multiple routes of administration, such as oral intake of H 2 water and H 2 bathing. Therefore, H 2 may be a potential alternative strategy for conventional exogenous antioxidant interventions in sports science. The purpose of this review is to provide evidence regarding the effects of H 2 intake on changes in physiological and biochemical parameters, centering on exercise-induced oxidative stress, for each intake method. Furthermore, this review highlights possible future directions in this area of research., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Takuji Kawamura et al.)

Lactoferrin (LF) is a multifunctional protein that plays important physiological roles as one of the most concentrated proteins in many human and other mammalian fluids and tissues. In particular, LF provides antibacterial properties to human milk, saliva, and tear fluid. LF also protects against stress-induced lipid peroxidation at inflammation sites through its iron-binding ability. Previous studies have shown that LF can be efficiently nitrated via biologically relevant mediators such as peroxynitrite (ONOO - ), which are also present at high intracellular concentrations during inflammation and nitrosative stress. Here, we examine changes in antibacterial properties and structure of LF following ONOO - treatment. The reaction induces nitration of tyrosine and tryptophan residues, which are commonly used as biomarker molecules for several diseases. Treatment with ONOO - at a 10/1 M ratio of ONOO - to tyrosine inhibited all antibacterial activity exhibited by native LF. Secondary structural changes in LF were assessed using circular dichroism spectroscopy. Nitration products with and without the addition of Fe 3+ show significant reduction in alpha-helical properties, suggesting partial protein unfolding. Iron-binding capacity of LF was also reduced after treatment with ONOO - , suggesting a decreased ability of LF to protect against cellular damage. LC-MS/MS spectrometry was used to identify LF peptide fragments nitrated by ONOO - , including tyrosine residue Y92 located in the iron-binding region. These results suggest that posttranslational modification of LF by ONOO - could be an important pathway to exacerbate infection, for example, in inflamed tissues and to reduce the ability of LF to act as an immune responder and decrease oxidative damage., (© 2019 Wiley Periodicals, Inc.)