Reactive Peroxynitrite Research Articles

Effect of distal His mutation on the peroxynitrite reactivity of Leishmania major peroxidase

The conserved distal histidine in peroxidases has been considered to play a major role as a general acid-base catalyst for heterolytic cleavage of an OO bond in H2O2. However, heme peroxidases react with peroxynitrite to form transient intermediates but the role of the distal histidine in this reaction is still unknown. In order to investigate catalytic roles of the histidine at the distal cavity, two Leishmania major peroxidase (LmP) mutants (H68E, H68V) were prepared. The rate of transition from ferric H68V to Compound ES by H2O2 is decreased by approximately five orders of magnitude relative to wild type, which is consistent with electron donor oxidation data where the H68V is ~1000 fold less active than wild type. In the reaction with peroxynitrite, the formation rate of intermediates in the mutants is not significantly lower than that for the wild type, indicating that the His68 has no major role in homolytic cleavage of an OO bond in peroxynitrite. EPR spectroscopic data suggest that the transient intermediates formed by the reaction of LmP with H2O2 exhibits an intense and stable signal similar to CCP Compound ES whereas in case of the reaction with peroxynitrite, this signal disappears, indicating that the transient intermediate is Compound II. Rapid kinetics data suggest that the distal His68 mutants display higher decay rates of Compound II than wild type. Thus, His68 mutations minimize Compound II formation (inactive species in peroxynitrite scavenging cycles) by increasing decay rates during the steady state and results in higher peroxynitrite degrading activity.

One-electron oxidation and reduction of glycosaminoglycan chloramides: A kinetic study

Hypochlorous acid and its acid–base counterpart, hypochlorite ions, produced under inflammatory conditions, may produce chloramides of glycosaminoglycans, these being significant components of the extracellular matrix (ECM). This may occur through the binding of myeloperoxidase directly to the glycosaminoglycans. The N–Cl group in the chloramides is a potential selective target for both reducing and oxidizing radicals, leading possibly to more efficient and damaging fragmentation of these biopolymers relative to the parent glycosaminoglycans. In this study, the fast reaction techniques of pulse radiolysis and nanosecond laser flash photolysis have been used to generate both oxidizing and reducing radicals to react with the chloramides of hyaluronan (HACl) and heparin (HepCl). The strong reducing formate radicals and hydrated electrons were found to react rapidly with both HACl and HepCl with rate constants of 1–1.7×108 and 0.7–1.2×108M−1s−1 for formate radicals and 2.2×109 and 7.2×108M−1s−1 for hydrated electrons, respectively. The spectral characteristics of the products of these reactions were identical and were consistent with initial attack at the N–Cl groups, followed by elimination of chloride ions to produce nitrogen-centered radicals, which rearrange subsequently and rapidly to produce C-2 radicals on the glucosamine moiety, supporting an earlier EPR study by M.D. Rees et al. (J. Am. Chem. Soc.125: 13719–13733; 2003). The oxidizing hydroxyl radicals also reacted rapidly with HACl and HepCl with rate constants of 2.2×108 and 1.6×108M−1s−1, with no evidence from these data for any degree of selective attack on the N–Cl group relative to the N–H groups and other sites of attack. The carbonate anion radicals were much slower with HACl and HepCl than hydroxyl radicals (1.0×105 and 8.0×104M−1s−1, respectively) but significantly faster than with the parent molecules (3.5×104 and 5.0×104M−1s−1, respectively). These findings suggest that these potential in vivo radicals may react in a site-specific manner with the N–Cl group in the glycosaminoglycan chloramides of the ECM, possibly to produce more efficient fragmentation. This is the first study therefore to conclusively demonstrate that reducing radicals react rapidly with glycosaminoglycan chloramides in a site-specific attack at the N–Cl group, probably to produce a 100% efficient biopolymer fragmentation process. Although less reactive, carbonate radicals, which may be produced in vivo via reactions of peroxynitrite with serum levels of carbon dioxide, also appear to react in a highly site-specific manner at the N–Cl group. It is not yet known if such site-specific attacks by this important in vivo species lead to a more efficient fragmentation of the biopolymers than would be expected for attack by the stronger oxidizing species, the hydroxyl radical. It is clear, however, that the N–Cl group formed under inflammatory conditions in the extracellular matrix does present a more likely target for both reactive oxygen species and reducing species than the N–H groups in the parent glycosaminoglycans.

Activation of receptor for advanced glycation end products contributes to aortic remodeling and endothelial dysfunction in sinoaortic denervated rats

ObjectiveThe aim of present study was to test the hypothesis that activation of receptor for advanced glycation end products (RAGE) pathway contributes to aortic remodeling and endothelial dysfunction in sinoaortic denervated (SAD) rats. Methods and resultsExperiment 1: 8 weeks after sinoaortic denervation, aortas were removed for measurement of AGE/RAGE pathway. Sinoaortic denervation in rats resulted in enhanced activity of aldose reductase, reduced activity of glyoxalase 1, accumulation of methylglyoxal and AGE, and upregulated expression of RAGE in aortas. Experiment 2: 5 weeks after sinoaortic denervation, the rats received intraperitoneal injections of 500 μg soluble RAGE (sRAGE) daily for 3 weeks. Treatment of SAD rats with sRAGE attenuated aortic remodeling, marked by reduction in AW/length, wall thickness, proliferation of SMC, and collagen deposition, and improvement of endothelial function. Treatment of SAD rats with sRAGE abated aortic oxidative stress, marked by reduction in formation of malondialdehyde, reactive oxygen species, superoxide, peroxynitrite and 3-nitrotyrosine, and enhancement of ratio of GSH/GSSG. Treatment of SAD rats with sRAGE attenuated aortic mitochondrial dysfunction. Treatment of SAD rats with sRAGE suppressed aortic NFκB nuclear translocation and inflammation. Treatment of SAD rats with sRAGE restored aortic NO formation through upregulating eNOS and dimethylarginine dimethylaminohydrolase-2 and downregulating protein arginine methyltransferase-1. ConclusionActivated RAGE contributed to aortic remodeling and endothelial dysfunction in SAD rats, possibly via induction of oxidative stress and inflammation, impairment of mitochondrial function, and reduction in NO bioavailability.

The role of oxidative and nitrosative bursts caused by azoles and amphotericin B against the fungal pathogen Cryptococcus gattii

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

Cyclic stretch stimulates nitric oxide synthase-1-dependent peroxynitrite formation by neonatal rat pulmonary artery smooth muscle

Peroxynitrite, the reaction product of nitric oxide and superoxide, contributes to the pathogenesis of chronic pulmonary hypertension in immature animals by stimulating proliferation of pulmonary arterial smooth muscle cells (PASMCs). Pulmonary vasoconstriction, secondary to hypoxia and other stimuli, leads to enhanced pulsatile stretch of cells in the vascular wall, particularly in smooth muscle, which we hypothesized would cause increased peroxynitrite generation. Our objectives in this study were to determine whether cyclic mechanical stretch, at supraphysiologic levels, would cause increased production of reactive oxygen species (ROS), nitric oxide, and peroxynitrite in vitro. Early passage neonatal rat PASMCs were seeded and grown to subconfluence on collagen-coated elastomer-bottom plates and subjected to cyclic mechanical stretch (10% (“physiologic”) or 20% (“supraphysiologic”) at 0.5 Hz) for up to 24 h. Compared to nonstretched controls and to cells subjected to 10% stretch, 20% stretch increased H2O2 (stable marker of ROS) and nitrate/nitrite (stable marker of nitric oxide) in conditioned medium. These effects were accompanied by increased peroxynitrite, as evidenced by increased in situ dihydroethidium fluorescence and immunoreactive nitrotyrosine and by increased expression of nitric oxide synthase (NOS)-1 and NADPH oxidase 4 (NOX4), but not NOS-2. Stretch-induced H2O2 release and increased dihydroethidium fluorescence were prevented by pretreatment with a superoxide scavenger, nonspecific inhibitors of NADPH oxidase or NOS, or a peroxynitrite decomposition catalyst. Short-interfering RNA-mediated knockdown of NOS-1 or NOX4 attenuated increased nitric oxide and H2O2 content, respectively, in stretched-cell-conditioned medium. Knockdown of NOS-1 also attenuated increased immunoreactive nitrotyrosine content and stretch-induced proliferation, whereas knockdown of NOS-2 had no effect. We conclude that increased peroxynitrite generation by neonatal rat PASMCs subjected to supraphysiologic levels of cyclic stretch is NOS-1-dependent and that increased ROS production is predominantly mediated by NADPH oxidase, specifically NOX4.

NLRP3 Inflammasome is activated in the kidneys of Metabolic Syndrome animals

Inflammasomes are oligomeric protein complexes formed in response to Danger‐associated molecular patterns (DAMPS) such as uric acid crystals and asbestos and have been shown to contribute to the pathogenesis of metabolic syndrome (MetS). Since uric acid is increased in chronic kidney disease, we hypothesized that increase uric acid might induce NLRP3 inflammasomes and exacerbate MetS via disrupting the redox balance and causing structural and functional impairment of the kidneys. Male Lean zucker controls (LZR) and Obese (OZR) underwent acute renal clearance and hemodynamic study. Compared to the OZR rats, the LZR exhibited higher glomerular filtration rate, higher renal blood flow and lower renal vascular resistance. Renal histopathology results revealed glomerulosclerosis, interstitial nephritis and proteinuria, and Real time RT PCR showed an increased gene expression of kidney injury markers and inflammasomes in OZR rats. Free radical production rates measured by electron paramagnetic spectroscopy showed a reduced total reactive oxygen species, superoxide and peroxynitrite levels in LZR animals. The gene and protein expression levels of IL‐1β and IL‐18 were higher in OZR rats. The plasma uric acid levels were higher in OZR rats. Our results indicate that inflammasomes are activated in the kidneys of MetS, thereby contributing to increased oxidative stress and renal damage. Research supported by LSU.

Blueberry treatment improves renal function and reduces oxidative stress in Metabolic Syndrome animals – Role of Toll‐like receptor 4 (TLR4)

Metabolic syndrome (MetS) is characterized by a cluster of health factors that indicate a higher risk for cardiac disease, renal disabilities and diabetes. Although inflammation and oxidative stress in renal dysfunction has been well studied, the mechanism by which natural antioxidants protect against MetS is not clear. Blueberries (BB) are among the richest source of antioxidants known to have anti‐inflammatory and antioxidant properties. We hypothesize that BB protect against MetS via inhibition of TLR4, which is an upstream receptor in several inflammatory pathways. Male zucker rats – Lean controls (LZR) and Obese (OZR) were both fed with a BB‐enriched diet or a control diet for 15 weeks. Compared to controls, the rats fed with BB exhibited higher glucose sensitivity, lower blood pressure and improved renal function. Free radical production rates measured by electron paramagnetic spectroscopy (EPR) showed a decreased total reactive oxygen species, superoxide and peroxynitrite levels in BB treated rats. Reduction in renal pathology and improvement in antioxidant levels in the BB‐fed rat kidneys were consistent with the EPR results. BB treated animals also expressed lower gene and protein levels of TLR4 and proinflammatory cytokines. In conclusion, TLR4 inhibition is a mechanism by which blueberries protect against oxidative stress and renal damage, and prevent the onset of MetS. Research supported by NIH.

Cerium oxide nanoparticles accelerate the decay of peroxynitrite (ONOO−)

Cerium oxide nanoparticles (CeO2 NPs) have been shown to possess a substantial oxygen storage capacity via the interchangeable surface reduction and oxidation of cerium atoms, cycling between the Ce(4+) and Ce(3+) redox states. It has been well established in many studies that depending on their reactivity and surface chemistry, CeO2 NPs can effectively convert both reactive oxygen species (superoxide, O2 (•-), and hydrogen peroxide) into more inert species and scavenge reactive nitrogen species (RNS)(nitric oxide, •NO), both in vitro and in vivo. Since much of damage attributed to •NO and O2 (•-) is actually the result of oxidation or nitration by peroxynitrite or its breakdown products and due to the multiple species that these nanoparticles target in vivo, it was logical to test their interaction with the highly reactive molecule peroxynitrite (ONOO(-)). Here, we report that CeO2 NPs significantly accelerated the decay of ONOO(-) by three independent methods. Additionally, our data suggest the ability of CeO2 NPs to interact with ONOO(-) is independent of the Ce(3+)/Ce(4+) ratio on the surface of the CeO2 NPs. The accelerated decay was not observed when reactions were carried out in an inert gas (argon), suggesting strongly that the decay of peroxynitrite is being accelerated due to a reaction of CeNPs with the carbonate radical anion. These results suggest that one of the protective effects of CeO2 NPs during RNS is likely due to reduction in peroxynitrite or its reactive breakdown products.

Antioxidant Function and Health Implications of Vitamin E

Superoxide, a key precursor of important reactive oxygen/nitrogen species (ROS/RNS), may release iron from its protein complex. By mediating the generation and/level of superoxide, vitamin E, the most important fat-soluble anti-oxidant and free radical scavenger, may exert its antioxidant function by limiting the formation of reactive hydroxyl radi-cals and peroxynitrite. The antioxidant function of vitamin E is augmented by GSH peroxidase and related metabolic sys-tems which respond adaptively and compensatively to oxidative stress. By mediating the levels of ROS/RNS, vitamin E may also modulate the activation and/or expression of redox-sensitive biological response modifiers, and thereby attenu-ate the cellular events leading to the onset and development of aging and other degenerative disorders. Earlier epidemiol-ogical data and retrospective studies show an association between increased intake of vitamin E and reduced risk of car-diovascular disease, cancer, and other disorders. However, subsequent prospective randomized placebo-controlled studies and interventional trials have provided inconsistent findings. Data available from recent large-scale interventional trials suggest that vitamin E supplements at high doses are not beneficial.

Non-hydrolyzed in digestive tract and blood natural L-carnosine peptide (“bioactivated Jewish penicillin”) as a panacea of tomorrow for various flu ailments: signaling activity attenuating nitric oxide (NO) production, cytostasis, and NO-dependent inhibition of influenza virus replication in macrophages in the human body infected with the virulent swine influenza A (H1N1) virus

Influenza (flu) is caused by a highly contagious virus that is spread by coughs and sneezes. Flu symptoms include high fever, chills and sweating, sore throat, weakness, headache, muscle and joint pains, and cough. Older people and those with an underlying medical condition are more likely to develop serious complications, including secondary bacterial pneumonia, primary influenza pneumonia, and inflammation of the brain or heart. There are three types of flu virus: A, B, and C. The flu virus has a unique ability to change its surface structure. This allows it to escape recognition by the body's immune system and cause widespread illness (epidemics and pandemics). Most cases of influenza occur within a 6- to 8-week period during winter and spring. Epidemics occur when there are minor changes in the nature of the virus so that more people within a community are susceptible. Influenza A is more likely to cause epidemics. Pandemics (worldwide epidemics) occur when there are major changes in the virus so that the disease affects a large proportion of people in a geographic region or on more than one continent. The findings presented in this article have many important implications for understanding the influenza A (H1N1) viral pathogenesis, prevention, and treatment. Direct viral cytotoxicity (referred cytopathic effect) is only a fraction of several types of events induced by virus infection. Nitric oxide and oxygen free radicals such as superoxide anion (O2-·) are generated markedly in influenza A (including H1N1) virus-infected host boosts, and these molecular species are identified as the potent pathogenic agents. The mutual interaction of nitric oxide (NO) with O2-· resulting in the formation of peroxynitrite is operative in the pathogenic mechanism of influenza virus pneumonia. Influenza virus infection involves pathological events in which oxygen free radicals play an important role in the pathogenesis. The toxicity and reactivity of oxygen radicals generated in excessive amounts mediate the overreaction of the host's immune response against the organs or tissues in which viruses are replicating, and this may explain the mechanism of tissue injuries observed in influenza virus infection of various types. In this article, the types of protection of carnosine in its bioavailable non-hydrolyzed forms in formulations are considered against reactive oxygen radical species-dependent injury, peroxynitrite damage, and other types of viral injuries in which impaired immune responses to viral pathogens are usually involved. Carnosine (β-alanyl-L-histidine) shows the pharmacological intracellular correction of NO release, which might be one of the important factors of natural immunity in controlling the initial stages of influenza A virus infection (inhibition of virus replication) and virus-induced regulation of cytokine gene expression. The protective effects of orally applied non-hydrolyzed formulated species of carnosine include at least the direct interaction with NO, inhibition of cytotoxic NO-induced proinflammatory condition, and attenuation of the effects of cytokines and chemokines that can exert profound effects on inflammatory cells. These data are consistent with the hypothesis that natural products, such as chicken soup and chicken breast extracts rich in carnosine and its derivative anserine (β-alanyl-1-methyl-L-histidine), could contribute to the pathogenesis and prevention of influenza virus infections and cold but have a limitation due to the susceptibility to enzymatic hydrolysis of dipeptides with serum carnosinase and urine excretion after oral ingestion of a commercial chicken extract. The formulations of non-hydrolyzed in digestive tract and blood natural carnosine peptide and isopeptide (γ-glutamyl-carnosine) products, manufactured at the cGMP-certified facility and patented by the authors, have promise in the control and prevention of influenza A (H1N1) virus infection, cough, and cold.

Nitric oxide and coral bleaching: is peroxynitrite generation required for symbiosis collapse?

The temperature-induced collapse ('bleaching') of the coral-dinoflagellate symbiosis is hypothesised to result from symbiont oxidative stress and a subsequent host innate immune-like response. This includes the production of nitric oxide (NO), which is involved in numerous microbial symbioses. Much of NO's cytotoxicity has been attributed to its conversion, in the presence of superoxide (O2(-)), to highly reactive peroxynitrite (ONOO(-)). However, ONOO(-) generation has yet to be observed in either a lower invertebrate or an intracellular mutualism. Using confocal laser scanning microscopy with the fluorescent ONOO(-) indicator aminophenyl fluorescein (APF), we observed strong evidence that ONOO(-) is generated in symbiotic Aiptasia pulchella under conditions known to induce thermal bleaching. However, a role for ONOO(-) in bleaching remains unclear as treatment with a peroxynitrite scavenger had no significant effect on thermal bleaching. Therefore, while ONOO(-) may have a potential for cytotoxicity, in vivo levels of the compound may be insufficient to affect bleaching.

Photodynamic inhibition of Trichophyton rubrum: in vitro activity and the role of oxidative and nitrosative bursts in fungal death

Antimicrobial photodynamic inhibition (aPI) is based on the use of a light source and a photosensitizer to kill pathogens. Little is known about aPI of dermatophytic fungi and its mechanism of action. We aimed to evaluate aPI of Trichophyton rubrum. We performed tests using toluidine blue (TBO) as a photosensitizer and a 630 nm light-emitting diode (LED) as a source of light to target 12 T. rubrum isolates. Susceptibility testing with cyclopiroxolamine, time-kill curves and quantification of reactive oxygen species (ROS), peroxynitrite (ONOO·) and nitric oxide (NO·) were performed. The optimal conditions for in vitro aPI were 10 mg/L for TBO and 48 J/cm(2) for LED; these conditions were fungicidal or inhibited >98% of fungal growth depending on the strain tested. LED or TBO treatment alone did not inhibit growth. The MICs of cyclopiroxolamine were 2.0 mg/L for 90% of the strains. Analysis of time-kill curves revealed that pathogen death occurred 24 h post-treatment. Quantification of ROS, ONOO· and NO· revealed improvement after aPI. Photodynamic inhibition was more efficient in promoting cell death than the antifungal cyclopiroxolamine against T. rubrum. ROS, ONOO· and NO· were important in the fungicidal activity of aPI. A suggested mechanism for this activity is that TBO is excited by LED light (630 nm), reacts with biomolecules and increases the availability of transition electrons and substrates for nitric oxide synthase, thereby increasing the oxidative and nitrosative bursts in the fungal cell.

Protective Effect of SnCl2 on K2Cr2O7-Induced Toxicity in LLC-PK1 Cells

The exposure to hexavalent chromium is often known to cause acute renal failure. It has been found that nonenzymatic antioxidants and the induction of heme oxygenase 1 have protective effects against nephrotoxicity induced by potassium dichromate in vivo. In this work, the effect of stannous chloride, an inducer of heme oxygenase 1, on potassium dichromate-induced toxicity in proximal tubular epithelial cells was studied. Hexavalent chromium levels, peroxynitrite content, reduced thiol content, heme oxygenase activity, reactive oxygen species production, and stannous chloride scavenging capacity were measured. It was found that stannous chloride protects proximal tubular epithelial cells from potassium dichromate-induced cell death. The decrease in extracellular and intracellular hexavalent chromium concentration, the induction of heme oxygenase 1, and the ability to scavenge reactive oxygen species and peroxynitrite are involved in the mechanism by which stannous chloride protects proximal tubular epithelial cells from potassium dichromate-induced toxicity.

Cardiolipin-Mediated Facilitation of Cytochrome C Tyrosine Nitration by Peroxynitrite

Cytochrome c, which functions as carrier in the mitochondrial respiratory chain and as a pro-apoptotic mediator can also act as a peroxidase when exposed to high doses of hydrogen peroxide, the phospholipid cardiolipin (CL) or after its modification by tyrosine (Y) nitration. Since mitochondria are key places of intracellular-generated peroxynitrite, cyt c nitration becomes a biologically-relevant event. In this work, we studied the effect of CL on horse-heart cyt c tyrosine nitration and free radical formation when exposed to peroxynitrite, and the relevance of each of the four Y residues in the process. The effect of CL in cytochrome c exposed to peroxynitrite was studied by EPR analysis, affinity studies, MS and immunochemical detection. The relevance of each Y in the reaction of peroxynitrite with cyt c in presence of CL was assessed with Y>F mutants at each of the Y. Notably, the increase of nitration, changes of binding constant and distribution of nitrated species observed using WT cyt c were not observed when using the Y67F mutant, suggesting a key role of Y67 in those processes. The data supports that a structural change induced by cardiolipin in cytochrome c increases tyrosine nitration yields, particularly in the heme-adjacent Y67, suggesting an iron-catalyzed event.

Reactivity of Resorufin-Based Fluorogenic Probe PC1 toward ROS/RNS and its use in the Studies of Peroxynitrite Reactivity

Reactivity of Resorufin-Based Fluorogenic Probe PC1 toward ROS/RNS and its use in the Studies of Peroxynitrite Reactivity

Reaction of Human Cytochrome P450 3A4 with Peroxynitrite: Nitrotyrosine Formation on the Proximal Side Impairs Its Interaction with NADPH-Cytochrome P450 Reductase

The reaction of peroxynitrite (PN) with purified human cytochrome P450 3A4 (CYP3A4) resulted in the loss of the reduced-CO difference spectrum, but the absolute absorption spectrum of the heme was not significantly altered. The loss of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) O-debenzylation activity of CYP3A4 was concentration-dependent with respect to PN, and the loss of BFC activity supported by NADPH-cytochrome P450 reductase (CPR) was much greater than that supported by tert-butyl hydroperoxide. Moreover, the PN-treated CYP3A4 exhibited a reduced-CO spectrum when reduced by CPR that was much smaller than when it was reduced by dithionite. These results suggest that modification of CYP3A4 by PN may impair its interaction with CPR, leading to the loss of catalytic activity. Tyrosine nitration, as measured by an increase in mass of 45 Da due to the addition of a nitro group, was used as a biomarker for protein modification by PN. PN-treated CYP3A4 was digested by trypsin and endoproteinase Glu C, and nitrotyrosine formation was then determined by using electrospray ionization-liquid chromatography-tandem mass spectrometry. Tyr residues 99, 307, 347, 430, and 432 were found to be nitrated. Using the GRAMM-X docking program, the structure for the CYP3A4-CPR complex shows that Tyr99, Tyr347, and Tyr430 are on the proximal side of CYP3A4 and are in close contact with three acidic residues in the FMN domain of CPR, suggesting that modification of one or more of these tyrosine residues by PN may influence CPR binding or the transfer of electrons to CYP3A4. Mutagenesis of Tyr430 to Phe or Val revealed that both the aromatic and the hydroxyl groups of Tyr are required for CPR-dependent catalytic activity and thus support the idea that the proximal side Tyr participates in the 3A4-CPR interaction. In conclusion, modification of tyrosine residues by PN and their subsequent identification can be used to enhance our knowledge of the structure/function relationships of the P450s with respect to the electron transfer steps, which are critical for P450 activity.

Enhanced N-nitrosamine formation in pool water by UV irradiation of chlorinated secondary amines in the presence of monochloramine

N-Nitrosamines, in particular N-nitrosodimethylamine (NDMA), are carcinogens, which occur as chlorine disinfection by-products (DBPs) in swimming pools and hot tubs. UV treatment is a commonly used technique in swimming pools for disinfection and DBP attenuation. UV irradiation is known to efficiently degrade N-nitrosamines. However, UV irradiation (at λ = 254 nm) of chlorinated dimethylamine (CDMA) and monochloramine, two NDMA precursors present in swimming pool water, resulted in a substantial UV-induced NDMA formation (∼1–2% molar yield based on initial CDMA concentration) simultaneously to NDMA photolysis. Maximum NDMA concentrations were found at UV doses in the range used for advanced oxidation (350–850 mJ cm−2). Very similar behaviour was found for other chlorinated secondary amines, namely diethylamine and morpholine. Effectiveness of UV irradiation for N-nitrosamine abatement depends on initial N-nitrosamine and precursor concentrations and the applied UV dose. N-Nitrosamine formation is hypothesized to occur via the reaction of nitric oxide or peroxynitrite with the secondary aminyl radical, which are products from the photolysis of monochloramine and chlorinated secondary amines, respectively. Experiments with pool water showed that similar trends were observed under pool water conditions. UV treatment (UV dose: ∼360 mJ cm−2) slightly increased NDMA concentration in pool water instead of the anticipated 50% abatement in the absence of NDMA precursors.

Nitric Oxide- and Heme-Independent Activation of Soluble Guanylate Cyclase Attenuates Peroxynitrite-Induced Endothelial Dysfunction in Rat Aorta

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

Reactions of Amine and Peroxynitrite: Evidence for Hydroxylation as Predominant Reaction and New Insight into the Modulation of CO2

Peroxynitrite is related to numerous diseases including cardiovascular diseases, inflammation, and cancer. In order to expand the understanding for the toxicology of peroxynitrite in biological system, the reactions of amine (morpholine as a probe) with peroxynitrite and the modulation of CO2 were investigated by using DFT methods. The results strongly indicate that the hydroxylation of amine by peroxynitrous acid ONOOH, which was previously overlooked by most studies, is predominant relative to the widely reported nitration and nitrosation in the absence of CO2. The product N-hydroxylamine is proposed to be mainly generated via nonradical pathway (two-electron oxidation). The modulation of CO2 exhibits two main functions: (1) inhibition of hydroxylation due to the promoted consumption of peroxynitrite via fast reaction of CO2 with ONOO¯ to form ONOOCO2¯; (2) dual effect (catalysis and inhibition) of CO2 toward nitration and nitrosation. As a new insight, amine does react with CO2 and produce inert amine carbamate R2NCOO¯. This reaction has the potential to compete with the reaction of CO2 and ONOO¯, which leads to inhibition of nitration and nitrosation. The concentration of CO2 could be a critical factor determining the final effect, catalysis or inhibition. As a new finding, HCO3¯ is probably an effective catalyst for the reaction of amine and CO2. Moreover, further studies on how the different types of the amine might affect the outcome of the reactions would be an interesting topic.

Transcription factor Nrf2 (NF-E2 Related Factor 2) enhances myeloid-derived suppressor cell (MDSC) accumulation and tumor progression (46.13)

Abstract Previous studies have shown that MDSC block adaptive anti-tumor immunity by producing high levels of oxidizing agents such as reactive oxygen species (ROS), nitric oxide (NO), and peroxynitrite. Despite high levels of the toxic oxidizing agents, MDSC survive and suppress anti-tumor immunity. We hypothesize that MDSC survival is mediated by the antioxidant-regulating transcription factor Nrf2. To test this hypothesis, BALB/c wild type and Nrf2-/- mice were injected with 4T1 mammary carcinoma cells and assayed weekly for percentage of MDSC in the blood and for MDSC levels of ROS and glutathione. Mice were also followed for survival. Nrf2-/- MDSC had more ROS and less glutathione than wild type MDSC, indicating that Nrf2-/- MDSC were more oxidatively stressed. Increased oxidative stress is likely to lead to increased apoptosis of MDSC and reduced immune suppression. Tumor-bearing Nrf2-/- and wild type mice accumulated MDSC in the blood with increasing tumor-burden. However, tumor-bearing Nrf2-/- mice had less MDSC than wild type mice for a given sized primary tumor. Therefore, MDSC either accumulate more slowly or they apoptose more quickly in Nrf2-/- vs. wild type mice. Tumor-bearing Nrf2-/- mice lived longer than wild type mice demonstrating that Nrf2 contributes to tumor progression; consistent with our hypothesis that Nrf2 regulates MDSC survival, resulting in fewer MDSC in Nrf2-/- mice, thereby increasing anti-tumor immunity against metastatic disease.