Mechanistic insight into the peroxidase catalyzed nitration of tyrosine derivatives by nitrite and hydrogen peroxide.

Peroxidases perform the nitration of tyrosine and tyrosyl residues in proteins, in the presence of nitrite and hydrogen peroxide. The nitrating species is still unknown but it is usually assumed to be nitrogen dioxide. In the present investigation, the nitration of phenolic compounds derived from tyrosine by lactoperoxidase and horseradish peroxidase was studied, with the aim of elucidating the mechanism of the reaction. The results indicate that nitrogen dioxide cannot be the only nitrating species and suggest the presence of two simultaneously operative pathways, one proceeding through enzyme-generated nitrogen dioxide and another through a more reactive species, assumed to be complexed peroxynitrite, which is generated by reaction of hydrogen peroxide with the enzyme-nitrite complex. The importance of the two pathways depends on peroxide and nitrite concentrations. With lactoperoxidase, nitration through the highly reactive intermediate is preferred except at very low nitrite concentration, while with horseradish peroxidase, the nitrogen dioxide driven mechanism is preferred except at very high nitrite concentration. The preferred mechanism for the two enzymes is that operative in the physiological nitrite concentration range.     

Ethyl gallate, a scavenger of hydrogen peroxide that inhibits lysozyme-induced hydrogen peroxide signaling in vitro, reverses hypotension in canine septic shock

Although hydrogen peroxide (H2O2) is a well-described reactive oxygen species that is known for its cytotoxic effects and associated tissue injury, H2O2 has recently been established as an important signaling molecule. We previously demonstrated that lysozyme (Lzm-S), a mediator of sepsis that is released from leukocytes, could produce vasodilation in a phenylephrine-constricted carotid artery preparation by H2O2 signaling. We found that Lzm-S could intrinsically generate H2O2 and that this generation activated H2O2-dependent pathways. In the present study, we used this carotid artery preparation as a bioassay to define those antioxidants that could inhibit Lzm-S's vasodilatory effect. We then determined whether this antioxidant could reverse the hypotension that developed in an Escherichia coli bacteremic model. Of the many antioxidants tested, we found that ethyl gallate (EG), a nonflavonoid phenolic compound, was favorable in inhibiting Lzm-S-induced vasodilation. In our E. coli model, we found that EG reversed the hypotension that developed in this model and attenuated end-organ dysfunction. By fluorometric H2O2 assay and electrochemical probe techniques, we showed that EG could scavenge H2O2 and that it could reduce H2O2 production in model systems. These results show that EG, an antioxidant that was found to scavenge H2O2 in vitro, was able to attenuate cardiovascular dysfunction in a canine in vivo preparation. Antioxidants such as EG may be useful in the treatment of hemodynamic deterioration in septic shock.     

Glycolipid changes in tissues of wheat leaves induced by exogenous hydrogen peroxide

The results of the study of H2O2 treatment of wheat leaf glycolipid composition are presented. The fraction changes depended upon H2O2 concentration, way of treatment and exposition. The most typical reaction was galactolipid (MGDG, DGDG) content increase while lower sulpholipid variations at low doses of the root treatment and at leaf spraying.     

Stobadine inhibits lysosomal enzyme release in vivo and in vitro

This study investigated the ability of stobadine, an effective cardioprotective drug with antiarrhythmic, antihypoxic and oxygen free radical scavenging properties, to protect cells against cyclophosphamide-induced toxic and cytotoxic damage in vivo and in vitro. Cyclophosphamide-induced toxic damage in female ICR mice was accompanied by marked increase in the activity of lysosomal enzymes in the spleen and kidney. Administration of stobadine prior to cyclophosphamide inhibited these biochemical changes. The in vivo protective effect of stobadine was comparable with its in vitro effect established in HeLa cells.     

In vivo and in vitro cytogenetic damage induced by sodium nitrite

The mutagenicity of sodium nitrite was assayed by in vivo and in vitro experiments. The in vivo experiments were carried out in male rats and mice intragastrically treated twice, with an interval of 24 h, with nitrite in doses of 1.72, 5.18, 15.55 and 46.66 mg/kg body weight and in male rabbits treated with the same doses of nitrite administered daily in drinking water for 3 months. Chromosomal aberration analysis was conducted in all 3 species of animals and micronucleus induction was only evaluated in mice. Nitrite induced increases in aberrant metaphases in all 3 species of animals. Likewise, in mice it induced increases of the numbers of micronucleated polychromatic erythrocytes and a light bone marrow depression. Neither in the increases of the numbers of chromosomal aberrations nor in that of micronuclei, were dose-related responses observed. The in vitro experiments were carried out on BSC-1 and HeLa cells grown in cultures with nitrite in doses of 0.265 and 0.530 mg/ml for 24 h. Both doses produced significant increases of the percentage of chromosomal aberrations but also without demonstration of positive dose-effect relationships.     

Ceruloplasmin enhances DNA damage induced by hydrogen peroxide in vitro

Ceruloplasmin (Cp) was found to promote the oxidative damage to DNA, as evidenced by the formation of 8-hydroxy-2'-deoxyguanosine and strand breaks, when incubated with H₂O₂ in vitro. The capacity of Cp to enhance oxidative damage to DNA was inhibited by hydroxyl radical scavengers such as sodium azide and mannitol, a metal chelator, diethylenetriaminepenta-acetic acid, and catalase. Although the oxidized protein resulted in an increase in the content of carbonyl groups, the ferroxidase activity and the proteolytic susceptibility were not significantly altered. The release of a portion of Cu from Cp was observed, and conformational alterations were indicated by the changes in fluorescence spectra. Based on these results, we suggest that damage to DNA is mediated in the H₂O₂/Cp system via the generation of ^·OH by released Cu²⁺ and/or loosely bound Cu exposed from oxidatively damaged Cp through the conformational change. The release of Cu from Cp during oxidative stress could enhance the formation of reactive oxygen species and could also potentiate cellular damage.     

Haemonchus contortus utilises catalase in defence against exogenous hydrogen peroxide in vitro

The toxicity of activated oxygen species towards adult Haemonchus contortus nematodes was examined in in vitro assays using ingestion of [³H]inulin to assess nematode viability. Both glucose/glucose oxidase (generation of hydrogen peroxide) and xanthine/xanthine oxidase (generation of superoxide anion) systems showed concentration-dependant toxicity to the nematodes. Both adult and larval Haemonchus contortus enzyme preparations showed significant catalase activities. Adult nematodes exposed to aminotriazole for 24 h showed catalase activities reduced to less than 20% of controls. Aminotriazole-treated nematodes exposed to a glucose/glucose oxidase system were significantly more susceptible to the toxic effects of the oxidant-generating system than controls (no aminotriazole pre-treatment). The concentration of glucose oxidase required to inhibit feeding by 50% was decreased 33-fold in aminotriazole-treated nematodes compared with controls. The effect of aminotriazole pre-treatment implicates hydrogen peroxide as a significant toxic agent in the glucose/glucose oxidase system. It is apparent that inhibition of Haemonchus contortus catalase increases the susceptibility of the parasite to the toxic effects of hydrogen peroxide, demonstrating a protective role for this enzyme. This suggests that catalase has the potential to play a significant role in the defence of this parasite against hydrogen peroxide produced as part of the respiratory burst of activated phagocytes within the host during its response to nematode infection.     

Nitrotyrosine, dityrosine, and nitrotryptophan formation from metmyoglobin, hydrogen peroxide, and nitrite

The biological relevance of tyrosine nitration is a subject of much interest, because extensive evidence supports formation of 3-nitrotyrosine in vivo under a variety of different pathological conditions. Several reagents are likely to be responsible for nitration in vivo, among others peroxynitrite and nitrite in the presence of H(2)O(2)/peroxidases. In this work we show that also metmyoglobin and methemoglobin can nitrate free tyrosine in the presence of nitrite and H(2)O(2). The results of these studies are simulated rather well by using a scheme that comprehends all the possible reactions that can take place in the system. Thus, a good understanding of the factors that determine the yields is achieved. Finally, we demonstrate that the system metMb/H(2)O(2)/NO(2)(-) can also lead to the nitration of tryptophan and produces, in particular, 6-, 4-, and 5-nitrotryptophan.     

Synthesis of peroxynitrite using isoamyl nitrite and hydrogen peroxide in a homogeneous solvent system

A method for the synthesis of peroxynitrite is described. It involves nitrosation of H2O2 at pH> or = 12.5 by isoamyl or butyl nitrite in mixed solvents of isopropyl alcohol (IPA) and water at 25+/-1 degrees C. Maximum yields of peroxynitrite are obtained after 15 min of incubation at IPA concentrations of 30-70% (v/v). The solutions of peroxynitrite are processed for removal of IPA and isoamyl alcohol by solvent extraction. Unreacted H2O2 is removed by catalytic decomposition on granular MnO(2). The post processed solutions of peroxynitrite are useful in several chemical and biochemical investigations where bolus additions are required. The method as reported is amenable for large scale synthesis as it involves sequential mixing of solvents (water and IPA) to alkali followed by the addition of H2O2 and alkyl nitrite.     

Interrelationship between hydrogen peroxide,ammonia, glutamine,hydroxylamine and nitrite metabolism in the cyanobacterium Phormidium uncinatum

On transition from nitrogen starvation to ammonia or ammonia/glutamine sufficiency Phormidium uncinatum produces high amounts of H₂O₂, which is consumed by several oxidative reactions catalyzed by thylakoid membrane bound enzymes. These include: oxidation of glutamine to free hydroxylamine, of ammonia to nitrite, of bound hydroxylamine to nitrite, and dismutation of free hydroxylamine to ammonia and nitrite. A possible role of these transformations for detoxification is discussed.Non-standard abbreviations FCCP p-trifluormethoxy carbonylcyanide phenylhydrazone - DCMU dichloromethyl urea     

Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade

The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key post-receptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H(2)O(2) in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H(2)O(2) as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and high M(r) insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases.     

8-prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.     

CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12 h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.     

Embellistatin, a microtubule polymerization inhibitor, inhibits angiogenesis both in vitro and in vivo

The efficient inhibition of angiogenesis is considered as a promising strategy for the treatment of angiogenesis-related diseases including cancer. Herein, we report that embellistatin, a bicyclic ketone compound known as a microtubule polymerization inhibitor, exhibits anti-angiogenic activity. Embellistatin inhibited in vitro angiogenesis of bovine aortic endothelial cells (BAECs) such as bFGF-induced invasion and tube formation as well as bFGF-induced mouse corneal angiogenesis in vivo. Notably, embellistatin exhibited stronger inhibition activity for the growth of BAECs than that of normal and cancer cell lines. Cell cycle analysis revealed that the compound arrests cell cycle at G2/M phase, which is associated with the increased expression of p21(WAF1) and p53 partly. These results demonstrate that embellistatin may serve the basis for the development of new anti-angiogenic agents.     

Gamma-tocopherol supplementation inhibits protein nitration and ascorbate oxidation in rats with inflammation

Gamma-tocopherol (gammaT) complements alpha-tocopherol (alphaT) by trapping reactive nitrogen oxides to form a stable adduct, 5-nitro-gammaT [Christen et al., PNAS 94:3217-3222; 1997]. This observation led to the current investigation in which we studied the effects of gammaT supplementation on plasma and tissue vitamin C, vitamin E, and protein nitration before and after zymosan-induced acute peritonitis. Male Fischer 344 rats were fed for 4 weeks with either a normal chow diet with basal 32 mg alphaT/kg, or the same diet supplemented with approximately 90 mg d-gammaT/kg. Supplementation resulted in significantly higher levels of gammaT in plasma, liver, and kidney of control animals without affecting alphaT, total alphaT+gammaT or vitamin C. Intraperitoneal injection of zymosan caused a marked increase in 3-nitrotyrosine and a profound decline in vitamin C in all tissues examined. Supplementation with gammaT significantly inhibited protein nitration and ascorbate oxidation in the kidney, as indicated by the 29% and 56% reduction of kidney 3-nitrotyrosine and dehydroascorbate, respectively. Supplementation significantly attenuated inflammation-induced loss of vitamin C in the plasma (38%) and kidney (20%). Zymosan-treated animals had significantly higher plasma and tissue gammaT than nontreated pair-fed controls, and the elevation of gammaT was strongly accentuated by the supplementation. In contrast, alphaT did not significantly change in response to zymosan treatment. In untreated control animals, gammaT supplementation lowered basal levels of 3-nitrotyrosine in the kidney and buffered the starvation-induced changes in vitamin C in all tissues examined. Our study provides the first in vivo evidence that in rats with high basal amounts of alphaT, a moderate gammaT supplementation attenuates inflammation-mediated damage, and spares vitamin C during starvation-induced stress without affecting alphaT.     

Importance of guanine nitration and hydroxylation in DNA in vitro and in vivo

Guanine (Gua) modification by nitrating and hydroxylating systems was investigated in DNA. In isolated calf thymus DNA, 8-NO(2)-Gua and 8-oxo-Gua were dose-dependently formed with peroxynitrite, and 8-NO(2)-Gua was released in substantial amounts. Myeloperoxidase (MPO) with H(2)O(2) and NO(2)(-) reacted with calf thymus DNA to form 8-NO(2)-Gua dose dependently without release of 8-NO(2)-Gua. The frequency of strand breaks was higher than the sum of 8-NO(2)-Gua and 8-oxo-Gua, particularly in the MPO-treated DNA, indicating the importance of other types of damage. The activation of human neutrophils and lymphocytes with phorbol ester did not induce 8-NO(2)-Gua and 8-oxo-Gua in their nuclear DNA. However, 8-NO(2)-Gua was found in calf thymus DNA co-incubated with activated neutrophils in the presence of NO(2)(-). No significant formation of 8-NO(2)-Gua was found in liver DNA from mice treated with Escherichia coli lipopolysaccharide. The incubation of peroxynitrite or MPO-H(2)O(2)-NO(2)(-)-treated DNA with formamidopyrimidine glycosylase (Fpg) released 8-oxo-Gua, but not 8-NO(2)-Gua, indicating that 8-NO(2)-Gua is not a substrate for Fpg. Although 8-NO(2)-Gua was generated in isolated DNA by different nitrating systems, other types of damage were formed in abundance, and the lesion could not be found reliably in nuclear DNA, suggesting that the biological importance is limited.     

Proteomics of the oxidative stress response induced by hydrogen peroxide and paraquat reveals a novel AhpC-like protein in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous pathogen most typically associated with wound infections, but also the main cause of mortality in patients suffering from cystic fibrosis (CF). The ability to adapt to oxidative stress associated with host immune defense may be one mechanism by which P. aeruginosa establishes infection in the cystic fibrosis lung and eventually out-competes other pathogenic bacteria to persist into chronic infection. We utilized a proteomics approach to identify the proteins associated with the oxidative stress response of P. aeruginosa PAO1 to hydrogen peroxide and superoxide-inducing paraquat. 2-DE and MS allowed for the identification of 59 and 58 protein spots that were statistically significantly altered following H(2) O(2) and paraquat treatment, respectively. We observed a unique mass and pI pattern for alkylhydroperoxide reductase C (AhpC) that was replicated by hypothetical protein PA3529 following treatment with 10?mM H(2) O(2) . AhpC belongs to the 2-Cys peroxiredoxin family and is a redox enzyme responsible for removing peroxides in bacterial cells. MS analysis showed that PA3529 was altered by the formation of a dimer via a disulfide bond in a manner analogous to that known for AhpC, and by cysteine overoxidation to Cys-sulfonic acid (SO(3) H) postoxidative stress. PA3529 is therefore a functional AhpC paralog expressed under H(2) O(2) stress. Following paraquat-induced oxidative stress, we also observed the overabundance and likely oxidative modification of a second hypothetical antioxidant protein (PA3450) that shares sequence similarity with 1-Cys peroxiredoxins. Other induced proteins included known oxidative stress proteins (superoxide dismutase and catalase), as well as those involved in iron acquisition (siderophore biosynthesis and receptor proteins FpvA and FptA) and hypothetical proteins, including others predicted to be antioxidants (PA0848). These data suggest that P. aeruginosa contains a plethora of novel antioxidant proteins that contribute to its increased resistance against oxidative stress.     

Hydrogen peroxide inhibits activation, not activity, of cellular caspase-3 in vivo

Oxidants such as H(2)O(2) can induce a low level of apoptosis at low concentrations but at higher concentrations cause necrosis. Higher concentrations of H(2)O(2) also inhibit the induction of apoptosis by chemotherapy drugs. One theory is that, at higher concentrations, H(2)O(2) causes direct oxidative inactivation of caspase-3 activity, thus preventing the apoptotic pathway from being used. We find that treatment of recombinant caspase-3 with H(2)O(2) can partially reduce its enzymatic activity: However, the following findings show that this does not occur in the cell. (1) The inhibition by H(2)O(2) of VP-16-induced apoptosis and cellular caspase-3 activity can be overcome by adding inhibitors of poly(ADP-ribose) polymerase (PARP) at sub-stoichiometric concentrations. (2) Delayed addition of H(2)O(2) to VP-16-treated cells prevents additional caspase induction but does not inhibit the caspase activity that has already been generated. (3) H(2)O(2) is a poor inhibitor of caspase-3 activity in cell lysates. (4) Addition of H(2)O(2) to cells inhibits activation of caspase-9, which is required for activation of caspase-3. We conclude that inhibition of caspase-3 activity in the cell occurs indirectly at a step located upstream of caspase-3 activation. H(2)O(2) acts in part by inducing DNA strand breaks and activating PARP, thus depleting the cells of ATP. When this pathway is blocked, even high concentrations of H(2)O(2) can induce caspase-9 and -3 activation and cause apoptosis.