Peroxynitrate Formation Research Articles

Impact of temperature-dependent non-PAN peroxynitrate formation, RO2NO2, on nighttime atmospheric chemistry.

The formation of peroxynitrates (RO2NO2) from the reaction of peroxy radicals (RO2) and nitrogen dioxide (NO2) and their subsequent redissociation are typically not included in chemical mechanisms. This is often done to save computational time as the assumption is that the equilibrium is strongly towards the RO2 + NO2 reaction for most conditions. Exceptions are the reactions of the methyl peroxy radical due to its abundance in the atmosphere and of acyl-RO2 radicals due to the long lifetime of peroxyacyl nitrates RO2NO2 (PANs). In this study, the nighttime oxidation of cis-2-butene and trans-2-hexene in the presence of NO2 is investigated in the atmospheric simulation chamber SAPHIR, Forschungszentrum Jülich, Germany, at atmospherically-relevant conditions at different temperatures (≈276 K, ≈293 K, ≈305 K). Measured concentrations of peroxy and hydroperoxy radicals as well as other trace gases (ozone, NO2, volatile organic compounds) are compared to state-of-the-art zero-dimensional box model calculations. Good model-measurement agreement can only be achieved when reversible RO2 + NO2 reactions are included for all RO2 species using literature values available from the latest SAR by [Jenkin et al., Atmos. Chem. Phys., 2019, 19, 7691]. The good agreement observed gives confidence that the SAR, derived originally for aliphatic RO2, can be applied to a large range of substituted RO2 radicals, simplifying generalised implementation in chemical models. RO2NO2 concentrations from non-acyl RO2 radicals of up to 2 × 10 cm-3 are predicted at 276 K, impacting effectively the kinetics of RO2 radicals. Under these conditions, peroxy radicals are slowly regenerated downwind of the pollution source and may be lost in the atmosphere through deposition of RO2NO2. Based on this study, 60% of RO2 radicals would be stored as RO2NO2 at a temperature of 10 °C and in the presence of a few ppbv of NO2. The fraction increases further at colder temperatures and/or higher NO2 mixing ratios. This does not only affect the expected concentrations of RO2 radicals but, as the peroxynitrates can react with OH radicals or photolyse, they could comprise a net sink for RO2 radicals as well as increase the production of NOx (= NO + NO2) in different locations depending on their lifetime. Omitting this chemistry from the kinetic model can lead to misinterpreted product formation and may prevent reconciling observations and model predictions.

High doses of sodium nitrate prior to exhaustive exercise increases plasma peroxynitrite levels in well-trained subjects: randomized, double-blinded, crossover study.

The aim of this study was to investigate the effect of different doses of pre-workout sodium nitrate supplementation on nitric oxide, peroxynitrite levels, and performance parameters. Ten well-trained male subjects participated in a randomized, double-blinded, crossover study. They ingested 8, 16, and 24 mmol sodium nitrate or placebo (NaCl) dissolved in water at 2.5 h before an incremental exercise test. Respiratory gases (oxygen consumption, carbon dioxide production, respiratory exchange ratio) were measured throughout the exercise trials and 3 blood samples (pre-ingestion, 2.5 h post-ingestion and postexercise) were taken to analyze nitrate/nitrite (NOx) and peroxynitrite levels. Data were analyzed using repeated-measures ANOVA at significance level of P < 0.05. NOx levels significantly increased following sodium nitrate ingestion compared with placebo (placebo: 40.86 ± 10.7 μmol/L, 8 mmol: 203.69 ± 25.1 μmol/L, 16 mmol: 289.41 ± 30.1 μmol/L, and 24 mmol: 300.95 ± 42.4 μmol/L, respectively) (P = 0.0001). However, this did not induce any significant change in oxygen consumption (P = 0.351), blood lactate concentration (P = 0.245), and time-to-exhaustion (P = 0.147). Peroxynitrite levels were similar compared with placebo when participants ingested 8 and 16 mmol of inorganic nitrate but a significant increase was observed after exercise at maximal intensity when participants were supplemented with 24 mmol (mean = 14.60 ± 1.3 μmol/L, P = 0.001). Pre-workout ingestion of high dose of sodium nitrate (24 mmol) induced peroxynitrate formation, a marker of oxidative stress. Caution must be taken regarding administration of higher doses before benefits or adverse effects are established in this population.

Inorganic Nitrate Supplementation Improves Diastolic Function in Cancer Survivors treated with Anthracycline Chemotherapy

Cancer survivors treated with anthracycline‐based chemotherapy have an increased risk of cardiotoxicity, resulting in the development of cardiac complications up to 10 years after chemotherapy cessation. Thus, cardiovascular events and heart failure are the second leading cause of morbidity and mortality in these women. Notably, the increased generation of reactive oxygen species relative to antioxidant defenses in the cardiomyocyte with anthracyclines results in lipid peroxidation and peroxynitrate formation, at the consequence of a decreased nitric oxide (NO) bioavailability, which over time results in ventricular systolic and diastolic dysfunction. Recently, orally ingested inorganic nitrates (NO3−), which increase NO bioavailability via the nitrate‐nitrite‐nitric oxide pathway and high antioxidant capacity, have shown to improve cardiac function in mice given anthracycline chemotherapy. Therefore, we hypothesized that inorganic NO3− administration would improve of left ventricular (LV) systolic and diastolic function, arterial stiffness, and ventricular‐arterial coupling in human cancer survivors with a history of anthracycline chemotherapy. Ten cancer survivors, 9 with breast cancer and 1 with lymphoma, participated in this randomized, double blind, crossover study comparing a week of daily doses NO3− rich beetroot juice with a nitrate‐depleted placebo. Standard and Tissue Doppler echocardiography were used to assess LV and carotid artery function during systole and diastole. NO3− supplementation significantly improved parameters of LV diastolic function, specifically strain rate in early filling (1.07±0.17 versus 0.80±0.29 1/s; P&lt;0.5), early mitral septal wall annular velocity (0.07±0.02 versus 0.06±0.02 m/s; P=0.02, and mitral A‐wave velocity (0.65±0.23 versus 0.52±0.17 cm/s; P=0.009). There were no differences in LV systolic parameters; ejection fraction (P=0.2) ventricular‐arterial coupling (P =0.10), systolic strain rate (P =0.49). Arterial stiffness (P =0.38) was not different between conditions. These findings suggest that decrease in NO bioavailability may have a mechanistic role in determining LV diastolic function in cancer survivors treated with anthracycline‐based chemotherapy. This is critical information that can be used to developed therapeutic strategies targeting NO‐dependent changes in left ventricular function that occur following anthracycline chemotherapy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sodium nitrite potentiates antimicrobial photodynamic inactivation: possible involvement of peroxynitrate.

We have recently shown that a wide range of different inorganic salts can potentiate antimicrobial photodynamic inactivation (aPDI) and TiO2-mediated antimicrobial photocatalysis. Potentiation has been shown with azide, bromide, thiocyanate, selenocyanate, and most strongly, with iodide. Here we show that sodium nitrite can also potentiate broad-spectrum aPDI killing of Gram-positive MRSA and Gram-negative Escherichia coli bacteria. Literature reports have previously shown that two photosensitizers (PS), methylene blue (MB) and riboflavin, when excited by broad-band light in the presence of nitrite could lead to tyrosine nitration. Addition of up to 100 mM nitrite gave 6 logs of extra killing in the case of Rose Bengal excited by green light against E. coli, and 2 logs of extra killing against MRSA (eradication in both cases). Comparable results were obtained for other PS (TPPS4 + blue light and MB + red light). Some bacterial killing was obtained when bacteria were added after light using a functionalized fullerene (LC15) + nitrite + blue light, and tyrosine ester amide was nitrated using both "in" and "after" modes with all four PS. The mechanism could involve formation of peroxynitrate by a reaction between superoxide radicals and nitrogen dioxide radicals; formation of the latter species was demonstrated by spin trapping with nitromethane.

Modeling of the chemistry in oxidation flow reactors with high initial NO

Abstract. Oxidation flow reactors (OFRs) are increasingly employed in atmospheric chemistry research because of their high efficiency of OH radical production from low-pressure Hg lamp emissions at both 185 and 254 nm (OFR185) or 254 nm only (OFR254). OFRs have been thought to be limited to studying low-NO chemistry (in which peroxy radicals (RO2) react preferentially with HO2) because NO is very rapidly oxidized by the high concentrations of O3, HO2, and OH in OFRs. However, many groups are performing experiments by aging combustion exhaust with high NO levels or adding NO in the hopes of simulating high-NO chemistry (in which RO2 + NO dominates). This work systematically explores the chemistry in OFRs with high initial NO. Using box modeling, we investigate the interconversion of N-containing species and the uncertainties due to kinetic parameters. Simple initial injection of NO in OFR185 can result in more RO2 reacted with NO than with HO2 and minor non-tropospheric photolysis, but only under a very narrow set of conditions (high water mixing ratio, low UV intensity, low external OH reactivity (OHRext), and initial NO concentration (NOin) of tens to hundreds of ppb) that account for a very small fraction of the input parameter space. These conditions are generally far away from experimental conditions of published OFR studies with high initial NO. In particular, studies of aerosol formation from vehicle emissions in OFRs often used OHRext and NOin several orders of magnitude higher. Due to extremely high OHRext and NOin, some studies may have resulted in substantial non-tropospheric photolysis, strong delay to RO2 chemistry due to peroxynitrate formation, VOC reactions with NO3 dominating over those with OH, and faster reactions of OH–aromatic adducts with NO2 than those with O2, all of which are irrelevant to ambient VOC photooxidation chemistry. Some of the negative effects are the worst for alkene and aromatic precursors. To avoid undesired chemistry, vehicle emissions generally need to be diluted by a factor of &gt; 100 before being injected into an OFR. However, sufficiently diluted vehicle emissions generally do not lead to high-NO chemistry in OFRs but are rather dominated by the low-NO RO2 + HO2 pathway. To ensure high-NO conditions without substantial atmospherically irrelevant chemistry in a more controlled fashion, new techniques are needed.

Hyperhomocysteinemia, Suppressed Immunity, and Altered Oxidative Metabolism Caused by Pathogenic Microbes in Atherosclerosis and Dementia.

Many pathogenic microorganisms have been demonstrated in atherosclerotic plaques and in cerebral plaques in dementia. Hyperhomocysteinemia, which is a risk factor for atherosclerosis and dementia, is caused by dysregulation of methionine metabolism secondary to deficiency of the allosteric regulator, adenosyl methionine. Deficiency of adenosyl methionine results from increased polyamine biosynthesis by infected host cells, causing increased activity of ornithine decarboxylase, decreased nitric oxide and peroxynitrate formation and impaired immune reactions. The down-regulation of oxidative phosphorylation that is observed in aging and dementia is attributed to deficiency of thioretinaco ozonide oxygen complexed with nicotinamide adenine dinucleotide and phosphate, which catalyzes oxidative phosphorylation. Adenosyl methionine biosynthesis is dependent upon thioretinaco ozonide and adenosine triphosphate (ATP), and the deficiency of adenosyl methionine and impaired immune function in aging are attributed to depletion of thioretinaco ozonide from mitochondrial membranes. Allyl sulfides and furanonaphthoquinones protect against oxidative stress and apoptosis by increasing the endogenous production of hydrogen sulfide and by inhibiting electron transfer to the active site of oxidative phosphorylation. Diallyl trisulfide and napabucasin inhibit the signaling by the signal transducer and activator of transcription 3 (Stat3), potentially enhancing immune function by effects on T helper lymphocytes and promotion of apoptosis. Homocysteine promotes endothelial dysfunction and apoptosis by the unfolded protein response and endoplasmic reticulum stress through activation of the N-methyl D-aspartate (NMDA) receptor, causing oxidative stress, calcium influx, apoptosis and endothelial dysfunction. The prevention of atherosclerosis and dementia may be accomplished by a proposed nutritional metabolic homocysteine-lowering protocol which enhances immunity and corrects the altered oxidative metabolism in atherosclerosis and dementia.

Metformin associated with photodynamic therapy – A novel oncological direction

The aim of our study was to assess the effect of the combined treatment of Metformin (Metf) and 5, 10, 15, 20-tetra-sulfophenyl-porphyrin (TSPP)–mediated photodynamic therapy (PDT) on an in vivo tumour model. Wistar male rats were divided in 6 groups: group 1, treated with TSPP; groups 2 and 4 treated with TSPP and Metf, respectively, and irradiated 24h thereafter; group 3 was treated with Metf and the last two groups received the combined treatment, Metf administered prior (group 5) or after (group 6) irradiation. 72h from the start of the treatment, tumour tissue was sampled for the investigation of oxidative and nitrosative stress. The apoptotic rate, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions and matrix metalloproteinases activities were also quantified. Malondialdehyde and glutathione levels were significantly elevated in the groups treated with combined therapy (p<0.05). Metf associated with TSPP–PDT reduced iNOS and COX-2 expressions and enhanced nitrotyrosine levels in both therapeutic regimens. Peroxynitrate formation and its cytotoxic effect on tumour cells were related to an elevated index of apoptosis and necrosis. Moreover, MMP-2 activity reached a minimum in the groups which received combined therapy. Our results confirmed that the association of Metf with PDT might prove a new and promising oncological approach.

Direct evidence of singlet molecular oxygen generation from peroxynitrate, a decomposition product of peroxynitrite

The decomposition of peroxynitrite to nitrite and dioxygen at neutral pH follows complex kinetics, compared to its isomerization to nitrate at low pH. Decomposition may involve radicals or proceed by way of the classical peracid decomposition mechanism. Peroxynitrite (ONOOH/ONOO(-)) decomposition has been proposed to involve formation of peroxynitrate (O(2)NOOH/O(2)NOO(-)) at neutral pH (D. Gupta, B. Harish, R. Kissner and W. H. Koppenol, Dalton Trans., 2009, DOI: 10.1039/b905535e, see accompanying paper in this issue). Peroxynitrate is unstable and decomposes to nitrite and dioxygen. This study aimed to investigate whether O(2)NOO(-) formed upon ONOOH/ONOO(-) decomposition generates singlet molecular oxygen [O(2) ((1)Delta(g))]. As unequivocally revealed by the measurement of monomol light emission in the near infrared region at 1270 nm and by chemical trapping experiments, the decomposition of ONOO(-) or O(2)NOOH at neutral to alkaline pH generates O(2) ((1)Delta(g)) at a yield of ca. 1% and 2-10%, respectively. Characteristic light emission, corresponding to O(2) ((1)Delta(g)) monomolecular decay was observed for ONOO(-) and for O(2)NOOH prepared by reaction of H(2)O(2) with NO(2)BF(4) and of H(2)O(2) with NO(2)(-) in HClO(4). The generation of O(2) ((1)Delta(g)) from ONOO(-) increased in a concentration-dependent manner in the range of 0.1-2.5 mM and was dependent on pH, giving a sigmoid profile with an apparent pK(a) around pD 8.1 (pH 7.7). Taken together, our results clearly identify the generation of O(2) ((1)Delta(g)) from peroxynitrate [O(2)NOO(-) --> NO(2)(-) + O(2) ((1)Delta(g))] generated from peroxynitrite and also from the reactions of H(2)O(2) with either NO(2)BF(4) or NO(2)(-) in acidic media.

Cytoplasmic CXCR4 expression in breast cancer: induction by nitric oxide and correlation with lymph node metastasis and poor prognosis

BackgroundLymph nodes constitute the first site of metastasis for most malignancies, and the extent of lymph node involvement is a major criterion for evaluating patient prognosis. The CXC chemokine receptor 4 (CXCR4) has been shown to play an important role in lymph node metastasis. Nitric oxide (NO) may also contribute to induction of metastatic ability in human cancers.MethodsCXCR4 expression was analyzed in primary human breast carcinoma with long-term follow-up. The relationship between nitrotyrosine levels (a biomarker for peroxynitrate formation from NO in vivo) and lymph node status, CXCR4 immunoreactivity, and other established clinico-pathological parameters, as well as prognosis, was analyzed. Nitrite/nitrate levels and CXCR4 expressions were assessed in MDA-MB-231 and SK-BR-3 breast cancer cell lines after induction and/or inhibition of NO synthesis.ResultsCXCR4 staining was predominantly cytoplasmic; this was observed in 50%(56/113) of the tumors. Cytoplasmic CXCR4 expression was significantly correlated with nitrotyrosine levels and lymph node metastasis. Kaplan-Meier survival curves showed that cytoplasmic CXCR4 expression was associated with reduced disease-free and overall survival. In multivariate analysis, cytoplasmic CXCR4 expression emerged as a significant independent predictor for overall and disease-free survival. Cytoplasmic expression of functional CXCR4 in MDA-MB-231 and SK-BR-3 cells was increased by treatment with the NO donor DETA NONOate. This increase was abolished by L-NAME, an inhibitor of NOS.ConclusionOur data showed a role for NO in stimulating cytoplasmic CXCR4 expression in vitro. Formation of the biomarker nitrotyrosine was also correlated with CXCR4 expression and lymph node metastasis in vivo. In addition, cytoplasmic CXCR4 expression may serve as a significant prognostic factor for long-term survival in breast cancer.

CXCR4 expression in papillary thyroid carcinoma: induction by nitric oxide and correlation with lymph node metastasis

BackgroundMetastasis to regional lymph nodes is a common step in the progression of cancer. Recent evidence suggests that tumor production of CXCR4 promotes lymph node metastasis. Nitric oxide (NO) may also increase metastatic ability in human cancers.MethodsNitrite/nitrate levels and functional CXCR4 expression were assessed in K1 and B-CPAP papillary thyroid carcinoma (PTC) cells after induction and/or inhibition of NO synthesis. CXCR4 expression was also analyzed in primary human PTC. The relationship between nitrotyrosine levels, which are a biomarker for peroxynitrate formation from NO in vivo, CXCR4 expression, and lymph node status was also analyzed.ResultsProduction of nitrite/nitrate and functional CXCR4 expression in both cell lines was increased by treatment with the NO donor DETA NONOate. The NOS inhibitor L-NAME eliminated this increase. Positive CXCR4 immunostaining was observed in 60.7% (34/56) of PTCs. CXCR4 expression was significantly correlated with nitrotyrosine levels and lymph node metastasis in human PTC.ConclusionOur data indicate that NO stimulates CXCR4 expression in vitro. Formation of the NO biomarker nitrotyrosine was also correlated with CXCR4 expression and lymph node metastasis in human PTC. NO may induce lymph node metastasis via CXCR4 induction in papillary thyroid carcinoma.

Nitric oxide in breast cancer: induction of vascular endothelial growth factor-C and correlation with metastasis and poor prognosis.

Metastasis to regional lymph nodes through the lymphatic vessels is a common step in the progression of cancer. Recent evidence suggests that tumor production of vascular endothelial growth factor-C (VEGF-C) promotes lymphagiogenesis, which in turn promotes lymphatic metastasis. Nitric oxide (NO) may also increase metastatic ability in human cancers. Nitrite/nitrate levels and VEGF-C production were assessed in MDA-MB-231 breast cancer cells after induction and/or inhibition of NO synthesis. Formation of nitrotyrosine, a biomarker for peroxynitrate formation from NO in vivo, was analyzed in primary human breast carcinoma with long-term follow-up. The relationship between nitrotyrosine levels and lymph node status, VEGF-C immunoreactivity, and other established clinicopathologic variables, as well as prognosis, was analyzed. Production of nitrite/nitrate and VEGF-C in MDA-MB-231 cells was increased by treatment with the NO donor DETA NONOate. The NO synthase inhibitor N(G)-nitro-l-arginine methyl ester eliminated this increase. High-grade nitrotyrosine staining was observed in 57.5% (65 of 113) of the invasive breast carcinomas. Nitrotyrosine levels were significantly correlated with VEGF-C immunoreactivity and lymph node metastasis. Survival curves determined by the Kaplan-Meier method showed that high nitrotyrosine levels were associated with reduced disease-free and overall survival. In multivariate analysis, high nitrotyrosine levels emerged as a significant independent predictor for overall survival. Our data showed a role for NO in stimulating VEGF-C expression in vitro. Formation of its biomarker nitrotyrosine was also correlated with VEGF-C expression and lymph node metastasis. Furthermore, high nitrotyrosine levels may serve as a significant prognostic factor for long-term survival in breast cancer.

Myocarditis

Myocarditis is clinically and pathologically defined as “inflammation of the myocardium.” Despite its rather clear-cut definition, the classification, diagnosis, and treatment of myocarditis continue to prompt considerable debate. The more routine use of endomyocardial biopsy has helped to better define the natural history of human myocarditis and to clarify clinicopathological correlations. Clinical presentations of the disease range from nonspecific systemic symptoms (fever, myalgias, palpitations, or exertional dyspnea) to fulminant hemodynamic collapse and sudden death. The extreme diversity of clinical manifestations has made the true incidence of myocarditis difficult to determine. Recent prospective postmortem data have implicated myocarditis in sudden cardiac death of young adults at rates of 8.6% to 12%.1,2 Furthermore, it has been identified as a cause of dilated cardiomyopathy in 9% of cases in a large prospective series.3 Recent molecular techniques have facilitated new insights into inflammatory autoimmune processes that affect the myocardium and ultimately result in acute or chronic dilated cardiomyopathy. Despite the well-established morbidity and mortality associated with myocarditis,4–7 clinical practice guidelines with regard to its evaluation and treatment are lacking.8 The wide variety of etiologies implicated in myocarditis and its heterogeneous clinical presentations5,7,9 have impeded patient identification and consensus on the most appropriate diagnostic criteria. The Dallas pathological criteria, published in 1986, served as the first attempt to develop standardized diagnostic guidelines for the histopathological classification of myocarditis.10 Active myocarditis is characterized by an inflammatory cellular infiltrate with evidence of myocyte necrosis (Figure 1), whereas borderline myocarditis demonstrates an inflammatory cellular infiltrate without evidence of myocyte injury (Figure 2). The inflammatory infiltrate should be further described as lymphocytic, eosinophilic, or granulomatous (Figure 3). The amount of inflammation may be mild, moderate, or severe, and its distribution may be focal, confluent, or diffuse, respectively. A retrospective study of 112 consecutive …

Nitric Oxide in Papillary Thyroid Carcinoma: Induction of Vascular Endothelial Growth Factor D and Correlation with Lymph Node Metastasis

Vascular endothelial growth factor-D (VEGF-D) plays an important role in lymph node metastasis via lymphangiogenesis in papillary thyroid carcinoma (PTC). Although PTC metastasizes to regional lymph nodes at a high frequency, the regulation of VEGF-D expression is largely unknown. Nitrite/nitrate levels and VEGF-D production were assessed in K1 papillary thyroid carcinoma cells after induction and/or inhibition of nitric oxide (NO) synthesis. Formation of nitrotyrosine, a biomarker for peroxynitrate formation from NO in vivo, was analyzed in primary human PTC. The production of nitrite/nitrate and VEGF-D in K1 cells was increased by treatment with the NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate). The NO synthase inhibitor N(G)-nitro-l-arginine methyl ester inhibited the increase in nitrate/nitrite and eliminated the increase in VEGF-D. High-grade nitrotyrosine staining was observed in 51.8% (29 of 56) of PTCs. Nitrotyrosine levels were significantly correlated with VEGF-D immunoreactivity and lymph node metastasis. Our data showed a role for NO in stimulating VEGF-D expression in vitro. The formation of its biomarker, nitrotyrosine, was also correlated with VEGF-D expression in human PTC. NO may induce lymph node metastasis via VEGF-D stimulation in PTC.

Investigation of the reaction of peroxynitrite with propofol at acid pH: Predominant production of oxidized, nitrated, and halogenated derivatives

We reported here the reaction, in acidic conditions, of peroxynitrite (ONOO −) with the anaesthetic agent propofol (2,6-diisopropylphenol, PPF). The most interesting finding is that peroxynitrite is able to nitrate and to oxidize propofol leading to 4-nitropropofol, quinone, and diphenylquinone as the major products. More surprisingly, we also found that peroxynitrite is capable of halogenating propofol in the presence of halide ions, suggesting the formation of nitrosyl chloride (NOCl) or nitryl chloride (NO 2Cl) from the reaction of peroxynitrite with chloride ions. A significant enhancement of the halogenation yield is observed with a simultaneous decrease of the yields of the other products in the presence of methanol or hydrogen peroxide. Increased halogenation of PPF probably results from the formation of peroxynitrate (O 2NOO −), that further oxidizes chloride ions in hypochlorous acid (HOCl) or molecular chlorine (Cl 2). Spontaneous decay of peroxynitrate is relatively slow in acidic medium, thus explaining the decrease of the yields of the other products. By direct EPR techniques, we also observed that this reaction occurs via a radical pathway.

The pathobiochemistry of nitrogen dioxide.

Nitrogen dioxide (*NO2) is an oxidizing free radical which can initiate a variety of destructive pathways in living systems, and several diseases are suspected to be connected with both exogenously and endogenously formed *NO2. Peroxynitrite (ONOO-/ONOOH) is believed to be an important endogenous source of *NO2 radicals, but other sources, among them enzymatically ones, have been identified recently. It also became clear during the last few years that in vivo formation of 3-nitrotyrosine strictly depends on the availability of *NO2 radicals. Since nitrogen dioxide is a very toxic compound an arsenal of antioxidants (e.g. vitamin C, glutathione, vitamin E, and beta-carotene) must eliminate this harmful radical in vivo. Here the recently identified superoxide (O2*-)-dependent formation of peroxynitrate (O2NOO-) and the central role of vitamin C are of special importance.

Glia-related pathomechanisms in Alzheimer's disease: a therapeutic target?

Reactive glial cell properties could contribute to pathomechanisms underlying Alzheimer's disease by favoring oxidative neuronal damage and β-amyloid toxicity. A critical step is apparently reached when pathological glia activation is no longer restricted to micoglia and includes astrocytes. By giving up their differentiated state, astrocytes may lose their physiological negative feed-back control on microglial NO production and even contribute to neurotoxic peroxynitrate formation. Another consequence is the impairment of the astrocyte-maintained extracellular ion homeostasis favoring excitotoxic damage. By the production of apolipoprotein-E, triggered by the microglial cytokine interleukine-1β, reactive astrocytes could promote the transformation of β-amyloid into the toxic form. A pharmacologically reinforced cAMP signaling in rat glial cell cultures depressed oxygen radical formation in microglia and their release of TNF-α and interleukine-1β, feed-forward signals which mediate oxidative damage and secondary astrocyte activation. Cyclic AMP also favored differentiation and expression of a mature ion channel pattern in astrocytes improving their glutamate buffering. A deficient cholinergic signaling that increases the risk of pathological APP processing was compensated by an adenosine-mediated reinforcement of the second messenger calcium. A combination therapy with acetylcholine-esterase inhibitors together with adenosine raising pharmaca, therefore, may be used to treat cholinergic deficiency in Alzheimer's disease.

Inhibition of peroxynitrite-induced nitration of tyrosine by glutathione in the presence of carbon dioxide through both radical repair and peroxynitrate formation.

Peroxynitrite (ONOO-/ONOOH) is assumed to react preferentially with carbon dioxide in vivo to produce nitrogen dioxide (NO2*) and trioxocarbonate(1-) (CO3*-) radicals. We have studied the mechanism by which glutathione (GSH) inhibits the NO2*/CO3*--mediated formation of 3-nitrotyrosine. We found that even low concentrations of GSH strongly inhibit peroxynitrite-dependent tyrosine consumption (IC50 = 660 microM) as well as 3-nitrotyrosine formation (IC50) = 265 microM). From the determination of the level of oxygen produced or consumed under various initial conditions, it is inferred that GSH inhibits peroxynitrite-induced tyrosine consumption by re-reducing (repairing) the intermediate tyrosyl radicals. An additional protective pathway is mediated by the glutathiyl radical (GS*) through reduction of dioxygen to superoxide (O2*-) and reaction with NO2* to form peroxynitrate (O2NOOH/O2NOO-), which is largely unreactive towards tyrosine. Thus, GSH is highly effective in protecting tyrosine against an attack by peroxynitrite in the presence of CO2. Consequently, formation of 3-nitrotyrosine by freely diffusing NO2* radicals is highly unlikely at physiological levels of GSH.

Peroxynitrite decay in the presence of hydrogen peroxide, mannitol and ethanol: A reappraisal

We have reported previously that the apparent rate of peroxynitrite (ONOO-) decay, as followed from its absorbance at 302 nm, decreases in the presence of hydrogen peroxide, mannitol and ethanol (Alvarez et al., 1995, Chem. Res. Toxicol. 8:859-864; Alvarez et al., 1998, Free Radic. Biol. Med. 24:1331–1337). Recently, two papers confirmed the observation and proposed that this slowing effect was due to the formation of absorbing peroxynitrate (O2NOO-) as intermediate (Goldstein and Czapski, 1998, J. Am. Chem. Soc. 120:3458–3463; Hodges and Ingold, 1999, J. Am. Chem. Soc. 121:10695–10701). Peroxynitrate would be formed from the reaction of peroxynitrite-derived nitrogen dioxide with superoxide. Superoxide, in turn, would arise from the one-electron oxidation of hydrogen peroxide, or from the reaction of reductive radicals derived from mannitol and ethanol with dioxygen. In agreement with this concept, we show herein that under the conditions of our previous work, the slowing effect is prevented by superoxide dismutase and, in the case of mannitol and ethanol, by reducing the dioxygen concentration of the reaction solutions. Thus, superoxide formation is necessary for the decrease in the rate of absorbance decay. In addition, by simulations using known rate constants and absorption coefficients, we show that the slowing effect can be quantitatively accounted for by the formation of peroxynitrate.

Ascorbate Is a Potent Antioxidant against Peroxynitrite-induced Oxidation Reactions: EVIDENCE THAT ASCORBATE ACTS BY RE-REDUCING SUBSTRATE RADICALS PRODUCED BY PEROXYNITRITE

Peroxynitrite (ONOO(((-)))/ONOOH) is expected in vivo to react predominantly with CO(2), thereby yielding NO(2)(.) and CO(3) radicals. We studied the inhibitory effects of ascorbate on both NADH and dihydrorhodamine 123 (DHR) oxidation by peroxynitrite generated in situ from 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). SIN-1 (150 micrometer)-mediated oxidation of NADH (200 micrometer) was half-maximally inhibited by low ascorbate concentrations (61-75 micrometer), both in the absence and presence of CO(2). Control experiments performed with thiols indicated both the very high antioxidative efficiency of ascorbate and that in the presence of CO(2) in situ-generated peroxynitrite exclusively oxidized NADH via the CO(3) radical. This fact is attributed to the formation of peroxynitrate (O(2)NOO(-)/O(2)NOOH) from reaction of NO(2)(.) with O(2), which is formed from reaction of CO(3) with NADH. SIN-1 (25 micrometer)-derived oxidation of DHR was half-maximally inhibited by surprisingly low ascorbate concentrations (6-7 micrometer), irrespective of the presence of CO(2). Control experiments performed with authentic peroxynitrite revealed that ascorbate was in regard to both thiols and selenocompounds much more effective to protect DHR. The present results demonstrate that ascorbate is highly effective to counteract the oxidizing properties of peroxynitrite in the absence and presence of CO(2) by both terminating CO(3)/HO( small middle dot) reactions and by its repair function. Ascorbate is therefore expected to act intracellulary as a major peroxynitrite antagonist. In addition, a novel, ascorbate-independent protection pathway exists: scavenging of NO(2)(.) by O(2) to yield O(2)NOO(-), which further decomposes into NO(2)(-) and O(2).

Cascading glia reactions: a common pathomechanism and its differentiated control by cyclic nucleotide signaling.

A pathological glia activation, stimulated by inflammatory proteins, beta-amyloid, or brain ischemia, is discussed as a common pathogenic factor for progressive nerve cell damage in vascular and Alzheimer dementia. A critical point seems to be reached, if the cytokine-controlled microglial upregulation causes a secondary activation of astrocytes which loose the negative feedback control, are forced to give up their physiological buffering function, and may add to neuronal damage by the release of nitric oxide (NO) and by promoting toxic beta-amyloid formation. A strengthening of the cyclic adenosine-5',3'-monophosphate (cAMP) signaling exerted a differential inhibition of the stimulatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) released from cultured rat microglia, but maintained the negative feedback signal IL-6; cAMP inhibited also the release of free oxygen radicals (OR) but not of NO. Reinforcement of the NO-induced cyclic guanosine monophosphate (cGMP) increase by blockade of the phosphodiesterase (PDE) subtype-5 with propentofylline counterbalanced the toxic NO action that causes with OR neuronal damage by peroxynitrate formation. In rat cultured astrocytes, a prolonged cAMP elevation favored cell differentiation, the expression of a mature ion channel patter, and an improvement of the extracellular glutamate uptake. Cyclic AMP signaling could be strengthened by PDE blockade and by raising extracellular adenosine, which stimulates A2 receptor-mediated cAMP synthesis. Via an A1 receptor-mediated effect, elevated adenosine was found to overcome a deficient intracellular calcium mobilization resulting from an impaired muscarinic signaling at pathologically decreased acetylcholine concentrations. We suggest that pharmaca, which elevate extracellular adenosine and/or block the degradation of cyclic nucleotides, may be used to counteract glia-related neuronal damage in dementing processes.