The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins.

Hydrogen peroxide (H2O2) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H2O2 in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H2O2 is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H2O2 between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H2O2 permeation. The permeability coefficient of H2O2 through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10-4 and 5 × 10-3 cm s-1, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H2O2 at 37 °C, on the other hand, was 1.6 × 10-3 cm s-1. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H2O2. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H2O2 as normal human RBCs. Therefore, these results indicate that H2O2 does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.

Diffusion and Transport of Reactive Species Across Cell Membranes.

This chapter includes an overview of the structure of cell membranes and a review of the permeability of membranes to biologically relevant oxygen and nitrogen reactive species, namely oxygen, singlet oxygen, superoxide, hydrogen peroxide, hydroxyl radical, nitric oxide, nitrogen dioxide, peroxynitrite and also hydrogen sulfide. Physical interactions of these species with cellular membranes are discussed extensively, but also their relevance to chemical reactions such as lipid peroxidation. Most of these species are involved in different cellular redox processes ranging from physiological pathways to damaging reactions against biomolecules. Cell membranes separate and compartmentalize different processes, inside or outside cells, and in different organelles within cells. The permeability of these membranes to reactive species varies according to the physicochemical properties of each molecule. Some of them, such as nitric oxide and oxygen, are small and hydrophobic and can traverse cellular membranes virtually unhindered. Nitrogen dioxide and hydrogen sulfide find a slightly higher barrier to permeation, but still their diffusion is largely unimpeded by cellular membranes. In contrast, the permeability of cellular membranes to the more polar hydrogen peroxide, is up to five orders of magnitude lower, allowing the formation of concentration gradients, directionality and effective compartmentalization of its actions which can be further regulated by specific aquaporins that facilitate its diffusion through membranes. The compartmentalizing effect on anionic species such as superoxide and peroxynitrite is even more accentuated because of the large energetic barrier that the hydrophobic interior of membranes presents to ions that may be overcome by protonation or the use of anion channels. The large difference in cell membrane permeability for different reactive species indicates that compartmentalization is possible for some but not all of them.

N-acetylcysteine improves the quality of red blood cells stored for transfusion.

Storage inflicts a series of changes on red blood cells (RBC) that compromise the cell survival and functionality; largely these alterations (storage lesions) are due to oxidative modifications. The possibility of improving the quality of packed RBC stored for transfusion including N-acetylcysteine (NAC) in the preservation solution was explored. Relatively high concentrations of NAC (20-25 mM) were necessary to prevent the progressive leakage of hemoglobin, while lower concentrations (≥2.5 mM) were enough to prevent the loss of reduced glutathione during the first 21 days of storage. Peroxiredoxin-2 was also affected during storage, with a progressive accumulation of disulfide-linked dimers and hetero-protein complexes in the cytosol and also in the membrane of stored RBC. Although the presence of NAC in the storage solution was unable to avoid the formation of thiol-mediated protein complexes, it partially restored the capacity of the cell to metabolize H2O2, indicating the potential use of NAC as an additive in the preservation solution to improve RBC performance after transfusion.

Immunoglobulins against tyrosine-nitrated epitopes in coronary artery disease.

BACKGROUND: Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine-nitrated proteins, a footprint of oxygen- and nitrogen-derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of patients with coronary artery disease (CAD). However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored. METHODS AND RESULTS: Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of patients with CAD. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in patients with CAD (n=374) and subjects without CAD (non-CAD controls, n=313). A 10-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in patients with CAD (3.75±1.8 µg antibody Eq/mL plasma versus 0.36±0.8 µg antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD. CONCLUSIONS: The results of this cross-sectional study suggest that posttranslational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neo-epitopes for the elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD.

Oxidants and Antioxidants in the Redox Biochemistry of Human Red Blood Cells.

Red blood cells (RBCs) are exposed to both external and internal sources of oxidants that challenge their integrity and compromise their physiological function and supply of oxygen to tissues. Autoxidation of oxyhemoglobin is the main source of endogenous RBC oxidant production, yielding superoxide radical and then hydrogen peroxide. In addition, potent oxidants from other blood cells and the surrounding endothelium can reach the RBCs. Abundant and efficient enzymatic systems and low molecular weight antioxidants prevent most of the damage to the RBCs and also position the RBCs as a sink of vascular oxidants that allow the body to maintain a healthy circulatory system. Among the antioxidant enzymes, the thiol-dependent peroxidase peroxiredoxin 2, highly abundant in RBCs, is essential to keep the redox balance. A great part of the RBC antioxidant activity is supported by an active glucose metabolism that provides reducing power in the form of NADPH via the pentose phosphate pathway. There are several RBC defects and situations that generate oxidative stress conditions where the defense mechanisms are overwhelmed, and these include glucose-6-phosphate dehydrogenase deficiencies (favism), hemoglobinopathies like sickle cell disease and thalassemia, as well as packed RBCs for transfusion that suffer from storage lesions. These oxidative stress-associated pathologies of the RBCs underline the relevance of redox balance in these anucleated cells that lack a mechanism of DNA-inducible antioxidant response and rely on a complex and robust network of antioxidant systems.

Oxidative Stress in Healthy and Pathological Red Blood Cells.

Red cell diseases encompass a group of inherited or acquired erythrocyte disorders that affect the structure, function, or production of red blood cells (RBCs). These disorders can lead to various clinical manifestations, including anemia, hemolysis, inflammation, and impaired oxygen-carrying capacity. Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms, plays a significant role in the pathophysiology of red cell diseases. In this review, we discuss the most relevant oxidant species involved in RBC damage, the enzymatic and low molecular weight antioxidant systems that protect RBCs against oxidative injury, and finally, the role of oxidative stress in different red cell diseases, including sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, and pyruvate kinase deficiency, highlighting the underlying mechanisms leading to pathological RBC phenotypes.

Sphingosine-1-phosphate receptor-3 is a novel biomarker in acute lung injury.

The inflamed lung exhibits oxidative and nitrative modifications of multiple target proteins, potentially reflecting disease severity and progression. We identified sphingosine-1-phosphate receptor-3 (S1PR3), a critical signaling molecule mediating cell proliferation and vascular permeability, as a nitrated plasma protein in mice with acute lung injury (ALI). We explored S1PR3 as a potential biomarker in murine and human ALI. In vivo nitrated and total S1PR3 concentrations were determined by immunoprecipitation and microarray studies in mice, and by ELISA in human plasma. In vitro nitrated S1PR3 concentrations were evaluated in human lung vascular endothelial cells (ECs) or within microparticles shed from ECs after exposure to barrier-disrupting agonists (LPS, low-molecular-weight hyaluronan, and thrombin). The effects of S1PR3-containing microparticles on EC barrier function were assessed by transendothelial electrical resistance (TER). Nitrated S1PR3 was identified in the plasma of murine ALI and in humans with severe sepsis-induced ALI. Elevated total S1PR3 plasma concentrations (> 251 pg/ml) were linked to sepsis and ALI mortality. In vitro EC exposure to barrier-disrupting agents induced S1PR3 nitration and the shedding of S1PR3-containing microparticles, which significantly reduced TER, consistent with increased permeability. These changes were attenuated by reduced S1PR3 expression (small interfering RNAs). These results suggest that microparticles containing nitrated S1PR3 shed into the circulation during inflammatory lung states, and represent a novel ALI biomarker linked to disease severity and outcome.

Discovery of new orally effective analgesic and anti-inflammatory hybrid furoxanyl N-acylhydrazone derivatives.

We report the design, the synthesis and the biological evaluation of the analgesic and anti-inflammatory activities of furoxanyl N-acylhydrazones (furoxanyl-NAH) by applying molecular hybridization approach. Hybrid compounds with IL-8-release inhibition capabilities were identified. Among them, furoxanyl-NAH, 17, and benzofuroxanyl-derivative, 24, together with furoxanyl-NAH derivative, 31, without IL-8 inhibition displayed both orally analgesic and anti-inflammatory activities. These hybrid derivatives do not have additional LOX- or COX-inhibition activities. For instance, LOX-inhibition by furoxanyl-NAH derivative, 42, emerged as a structural lead to develop new inhibitors. The lack of mutagenicity of the active derivatives 17, 31, and 42, allow us to propose them as candidates for further clinical studies. These results confirmed the success in the exploitation of hybridization strategy for identification of novel N-acylhydrazones (NAH) with optimized activities.

Oxidative Modification of Proteins: From Damage to Catalysis, Signaling, and Beyond.

Significance: The systematic investigation of oxidative modification of proteins by reactive oxygen species started in 1980. Later, it was shown that reactive nitrogen species could also modify proteins. Some protein oxidative modifications promote loss of protein function, cleavage or aggregation, and some result in proteo-toxicity and cellular homeostasis disruption. Recent Advances: Previously, protein oxidation was associated exclusively to damage. However, not all oxidative modifications are necessarily associated with damage, as with Met and Cys protein residue oxidation. In these cases, redox state changes can alter protein structure, catalytic function, and signaling processes in response to metabolic and/or environmental alterations. This review aims to integrate the present knowledge on redox modifications of proteins with their fate and role in redox signaling and human pathological conditions. Critical Issues: It is hypothesized that protein oxidation participates in the development and progression of many pathological conditions. However, no quantitative data have been correlated with specific oxidized proteins or the progression or severity of pathological conditions. Hence, the comprehension of the mechanisms underlying these modifications, their importance in human pathologies, and the fate of the modified proteins is of clinical relevance. Future Directions: We discuss new tools to cope with protein oxidation and suggest new approaches for integrating knowledge about protein oxidation and redox processes with human pathophysiological conditions. Antioxid. Redox Signal. 35, 1016-1080.

Identification of chalcones as in vivo liver monofunctional phase II enzymes inducers.

Cancer preventive agents (CPA) are drugs able to suppress the carcinogen metabolic activation or block the formation of ultimate carcinogens. CPA could act through various molecular mechanisms, for example by interfering with the action of procarcinogen. This could be attained by increasing the phase II enzymes levels of quinone reductase (QR) and glutathione S-transferase (GST). New flavonoids, especially chalcones, have been identified as in vivo monofunctional phase II enzymes inducers. Oral administration of chalcone, 4, and both p-methoxy-substituted chalcones, 6 and 14, increased hepatic QR activity with concomitant decrease in CYP1A1 activity, a member of the most important group of phase I enzymes cytochrome P450. Among them, 4 also increased GST activity. While p-bromo-substituted chalcone 8 was the best inducer of QR it decreased hepatic GST expression and cytochrome P450, being the most effective decreasing cytochrome P450-expression. Thienyl-chalcone 20 being the bioisostere of chalcone 4 did not display the same in vivo profile in the phase I level modification. As chalcone 4 its bioisostere, chalcone 20, displayed low DNA strand breakage and absence of mutagenicity. Also, in our preliminary in vivo tumourigenesis/chemopreventive and acute-toxicity studies, chalcones 4, 6 and 8 showed the best behaviours as CPA justifying additional studies that are ongoing.

Cannabis sativa extracts protect LDL from Cu2+-mediated oxidation.

BACKGROUND: Multiple therapeutic properties have been attributed to Cannabis sativa. However, further research is required to unveil the medicinal potential of Cannabis and the relationship between biological activity and chemical profile. OBJECTIVES: The primary objective of this study was to characterize the chemical profile and antioxidant properties of three varieties of Cannabis sativa available in Uruguay during progressive stages of maturation. METHODS: Fresh samples of female inflorescences from three stable Cannabis sativa phenotypes, collected at different time points during the end of the flowering period were analyzed. Chemical characterization of chloroform extracts was performed by 1H-NMR. The antioxidant properties of the cannabis sativa extracts, and pure cannabinoids, were measured in a Cu2+-induced LDL oxidation assay. RESULTS: The main cannabinoids in the youngest inflorescences were tetrahydrocannabinolic acid (THC-A, 242 ± 62 mg/g) and tetrahydrocannabinol (THC, 7.3 ± 6.5 mg/g). Cannabinoid levels increased more than twice in two of the mature samples. A third sample showed a lower and constant concentration of THC-A and THC (177 ± 25 and 1 ± 1, respectively). The THC-A/THC rich cannabis extracts increased the latency phase of LDL oxidation by a factor of 1.2-3.5 per µg, and slowed down the propagation phase of lipoperoxidation (IC50 1.7-4.6 µg/mL). Hemp, a cannabidiol (CBD, 198 mg/g) and cannabidiolic acid (CBD-A, 92 mg/g) rich variety, also prevented the formation of conjugated dienes during LDL oxidation. In fact, 1 µg of extract was able to stretch the latency phase 3.7 times and also to significantly reduce the steepness of the propagation phase (IC50 of 8 µg/mL). Synthetic THC lengthened the duration of the lag phase by a factor of 21 per µg, while for the propagation phase showed an IC50 ≤ 1 µg/mL. Conversely, THC-A was unable to improve any parameter. Meanwhile, the presence of 1 µg of pure CBD and CBD-A increased the initial latency phase 4.8 and 9.4 times, respectively, but did not have an effect on the propagation phase. CONCLUSION: Cannabis whole extracts acted on both phases of lipid oxidation in copper challenged LDL. Those effects were just partially related with the content of cannabinoids and partially recapitulated by isolated pure cannabinoids. Our results support the potentially beneficial effects of cannabis sativa whole extracts on the initial phase of atherosclerosis.

Increased protein nitration burden in the atherosclerotic lesions and plasma of apolipoprotein A-I deficient mice.

Apolipoprotein A-I (apoA-I), the major protein constituent within high-density lipoprotein (HDL), has been associated with antiatherogenic protection by mechanisms that include reverse cholesterol transport and antiinflammatory functions. To evaluate the proposed protective function of apoA-I, proteins modified by nitrating oxidants were evaluated in the aortic tissue and plasma of mice lacking the low-density lipoprotein receptor and apobec (LA) and LA mice with genetic deletion of apoA-I (LA-apoA-I(-/-)). The levels of nitrated proteins in aortic tissue quantified by liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) were 6-fold higher in the LA-apoA-I(-/-) as compared with the LA mice. The quantitative analyses were corroborated by immunohistochemical and high-resolution immunoelectron microscopic evaluation of the lesions, which revealed abundant staining for nitrated proteins in the aortic root lesions of LA-apoA-I(-/-) as compared with the LA mice. Proteomic approaches based on affinity enrichment and site-specific adduct mapping identified unique specific protein targets for nitration in the plasma of LA-apoA-I(-/-) that were not present in the plasma of LA mice. In particular the nitration of fibrinogen was shown to accelerate fibrin clot formation. Another consequence of the augmented levels of nitrated proteins was the induction of humoral responses documented by the increased circulating immunoglobulins that recognize nitrotyrosine in LA-apoA-I(-/-) as compared with the LA mice. These data collectively support a protective function of apoA-I diminishing the burden of nitrative oxidants in these mice models of atherosclerosis.

Kinetics of peroxiredoxins and their role in the decomposition of peroxynitrite.

Methodologies and results of studies on the kinetics of peroxiredoxins (Prx) are reviewed. Peroxiredoxins are broad-spectrum peroxidases that catalyze the reduction of H2O2, organic hydroperoxides and peroxynitrite by thiols. Their catalytic cycle starts with the oxidation of a particularly reactive cysteine residue (C(P)) to a sulfenic acid derivative by the peroxide substrate, the sulfenic acid then reacts with a thiol to form a disulfide, and the cycle is completed by thiol/disulfide exchange reactions that regenerate the ground-state enzyme. Depending on the subtype of peroxiredoxin, the thiol reacting with the primary oxidation product (E-SOH) may be a cysteine residue of a second subunit (typical 2-Cys Prx), a cysteine residue of the same subunit (atypical 2-Cys Prx) or reducing substrate (1-Cys Prx and at least one example of an atypical 2-Cys Prx). In a typical 2-Cys Prx the intra-subunit disulfide formation with the second "resolving" cysteine (C(R)) is mandatory for the reduction by the specific substrate, which is a protein characterized by a CXXC motif such as thioredoxin, tryparedoxin or AhpF. These consecutive redox reactions define the catalysis as an enzyme substitution mechanism, which is corroborated by a ping-pong pattern that is commonly observed in steady-state analyses, chemical identification of catalytic intermediates and stopped-flow analyses of partial reactions. More complex kinetic patterns are discussed in terms of cooperativity between the subunits of the oligomeric enzymes, generation of different oxidized intermediates or partial over-oxidation of C(P) to a sulfinic acid. Saturation kinetics is often not observed indicating that a typical complex between reduced enzyme and hydroperoxide is not formed and that, in these cases, formation of the complex between the oxidized enzyme and its reducing substrate is slower than the reaction within this complex. Working with sulphur catalysis, Prxs are usually less efficient than the heme- or selenium-containing peroxidases, but in some cases the k(+1) values (bimolecular rate constant for oxidation of reduced E by ROOH) are comparable, the overall range being 2 x 10(3)-4 x 10(7) M(-1)s(-1) depending on the hydroperoxide and the individual Prx. For the reduction of peroxynitrite k(+1) values of 1 x 10(6) up to 7 x 10(7) M(-1)s(-1) have been measured. The net forward rate constants k'(+2) for the reductive part of the cycle range between 2 x 10(4)-1 x 10(7) M(-1)s(-1). These kinetic characteristics qualify the peroxiredoxins as moderately efficient devices to detoxify hydroperoxides, which is pivotal to organisms devoid of more efficient peroxidases, and as most relevant to the detoxification of peroxynitrite. In higher organisms, their specific role is seen in the regulation of signalling cascades that are modulated by H2O2, lipid hydroperoxides or peroxynitrite.

Kinetic and stoichiometric constraints determine the pathway of H2O2 consumption by red blood cells.

Red blood cells (RBC) are considered as a circulating sink of H2O2, but a significant debate remains over the role of the different intraerythocyte peroxidases. Herein we examined the kinetic of decomposition of exogenous H2O2 by human RBC at different cell densities, using fluorescent and oxymetric methods, contrasting the results against a mathematical model. Fluorescent measurements as well as oxygen production experiments showed that catalase was responsible for most of the decomposition of H2O2 at cell densities suitable for both experimental settings (0.1-10â€¯× 1010 cell L-1), since sodium azide but not N-ethylmaleimide (NEM) inhibited H2O2 consumption. Oxygen production decreased at high cell densities until none was detected above 1.1 × 1012 cell L-1, being recovered after inhibition of the thiol dependent systems by NEM. This result underlined that the consumption of H2O2 by catalase prevail at RBC densities regularly used for research, while the thiol dependent systems predominate when the cell density increases, approaching the normal number in blood (5â€¯× 1012 cell L-1). The mathematical model successfully reproduced experimental results and at low cell number it showed a time sequence involving Prx as the first line of defense, followed by catalase, with a minor role by Gpx. The turning points were given by the total consumption of reduced Prx in first place and reduced GSH after that. However, Prx alone was able to account for the added H2O2 (50 µM) at physiological RBC density, calling attention to the importance of cell density in defining the pathway of H2O2 consumption and offering an explanation to current apparently conflicting results in the literature.

Inhibition of LDL oxidation and inflammasome assembly by nitroaliphatic derivatives. Potential use as anti-inflammatory and anti-atherogenic agents.

We have previously shown the antioxidant and anti-inflammatory properties of several para-substituted arylnitroalkenes. Since oxidative stress and inflammation are key processes that drive the initiation and progression of atherosclerosis, in the present work the antioxidant, anti-inflammatory and anti-atherogenic properties of an extended library of aryl-nitroaliphatic derivatives, including several newly designed nitroalkanes, was explored. The antioxidant capacity of the nitroaliphatic compounds, measured using the oxygen radical absorbance capacity assay (ORAC) showed that the p-methylthiophenyl-derivatives were about three times more effective than Trolox to prevent fluorescein oxidation, independently of the presence or the absence of the double bond next to the nitro group. The peroxyl radical scavenger capacity of the p-dimethylaminophenyl-derivatives was even higher, being the reduced form of these compounds even more active. In fact, while the antioxidant capacity of 1-dimethylamino-4-(2-nitro-1Z-ethenyl)benzene and 1-dimethylamino-4-(2-nitro-1Z-propenyl)benzene was 4.2 ±â€¯0.1 and 5.4 ±â€¯0.1 Trolox Eq/mol, respectively; ORAC values obtained with the ethyl and the propyl derivatives were 10 ±â€¯1 and 13 ±â€¯2 Trolox Eq/mol, respectively. The p-dimethylamino-derivatives, especially the nitroalkanes, were also able to prevent LDL oxidation mediated by peroxyl radicals. Oxygen consumption due to the oxidation of fatty acids was delayed in the presence of the dimethylamino substituted compounds, only the alkanes interrupted the chain of lipid oxidations decreasing the rate of oxygen consumption. Although the formation of foam cells in the presence of oxidized-LDL (oxLDL) remained unaffected, the molecules containing the dimethylamino moiety were able to decrease the expression of IL-1ß in LPS/INF-γ challenged macrophages.

Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease.

In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO(2)Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO(2)Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO(2)Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO(2)Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.

Subcellular localization of tyrosine-nitrated proteins is dictated by reactive oxygen species generating enzymes and by proximity to nitric oxide synthase.

Using high-resolution immuno-electron microscopy the steady-state subcellular distribution of tyrosine-nitrated proteins in different cells and tissues was evaluated. In quiescent eosinophils and neutrophils in the bone marrow intracellular nitrated proteins were mainly restricted to the peroxidase-containing secretory granules. The inducible nitric oxide synthase (iNOS) was expressed in the same granules. Proteins nitrated on tyrosine residues were also abundant in the cytosol of circulating erythrocytes. In the vasculature, nitrated proteins were mainly located in mitochondria and endoplasmic reticulum of the endothelial cells, fibroblasts, and smooth muscle cells. Endogenous nitrated proteins were also found in chondrocytes in cartilage, where it was typically associated with the cytoplasmic interface of the endoplasmic reticulum membrane. Nitrated proteins were also prominent in the peroxisomes of liver hepatocytes and of secretory cells in the lacrimal gland. Challenge of mouse dendritic cells with lipopolysaccharide induced iNOS protein expression in cytosol and peroxisomes and was associated with an increased 3-nitrotyrosine formation in cytosol, mitochondria, and peroxisomes. These data indicate that nitric oxide-dependent protein tyrosine nitration is a physiologically relevant process localized within specific subcellular compartments in close proximity to iNOS and to enzymes capable of peroxidative chemistry and reactive oxygen species production.

3-nitrotyrosine modified proteins in atherosclerosis.

Cardiovascular disease is the leading cause of premature death worldwide, and atherosclerosis is the main contributor. Lipid-laden macrophages, known as foam cells, accumulate in the subendothelial space of the lesion area and contribute to consolidate a chronic inflammatory environment where oxygen and nitrogen derived oxidants are released. Oxidatively modified lipids and proteins are present both in plasma as well as atherosclerotic lesions. A relevant oxidative posttranslational protein modification is the addition of a nitro group to the hydroxyphenyl ring of tyrosine residues, mediated by nitric oxide derived oxidants. Nitrotyrosine modified proteins were found in the lesion and also in plasma from atherosclerotic patients. Despite the fact of the low yield of nitration, immunogenic, proatherogenic, and prothrombotic properties acquired by 3-nitrotyrosine modified proteins are in agreement with epidemiological studies showing a significant correlation between the level of nitration found in plasma proteins and the prevalence of cardiovascular disease, supporting the usefulness of this biomarker to predict the outcome and to take appropriate therapeutic decisions in atherosclerotic disease.

Analgesic and Anti-Inflammatory Properties of Arylnitroalkenes.

In a recent work, we described the design and synthesis of arylnitroalkenes, able to scavenge macrophagederived oxidants, in particular peroxynitrite and peroxynitrite derived radicals. Four compounds emerged as potential leads, 1,1-dimethylamino-4-(2-nitro-1Z-ethenyl)benzene (1), 1,1-dimethylamino-4-(2-nitro-1Z-propenyl)benzene (2), 5- (2-nitro-1Z-ethenyl)benzo[d][1,3]dioxol (3), and 5-(2-nitro-1Z-ethenyl)benzo[d][1,3]dioxol (4). In the present work, the possibility of the preclinical validation of these molecules as anti-inflammatory and analgesic was explored in appropriate in vivo mouse models. Compounds 1, 2 and 4, administered orally as a single dose (30 µmol kg-(1)) to the mice showed anti-inflammatory and analgesic properties similar to classic nonsteroidal anti-inflammatory agents. The pharmacological effects were consistent with the inhibitory effect observed on prostaglandin endoperoxide H synthase (PGHS). In fact, both PGHS-1 and PGHS-2 were inhibited by the compounds, with compound 2 being more specific as PGHS-2 inhibitor with a specificity index superior to 70%. Conversely to classical nonsteroidal anti-inflammatory drugs, compound 2 inhibited peroxidase half reaction of the enzyme (IC50 2.3 µM) while the cyclooxygenase activity of hrPGHS-2 remained unchanged. In vitro experiments were reinforced by docking and molecular dynamics simulations showing arylnitroalkene moiety located in the region of the peroxidase active site, competing with the peroxide intermediate. The absence of toxicity and mutagenicity of the compounds was also demonstrated.

Arylnitroalkenes as scavengers of macrophage-generated oxidants.

Oxygen and nitrogen derived molecules mediated oxidation and nitration have been involved in several pathological conditions. Conversely, nitric oxide and hydrogen peroxide are important signalization intermediates, whose concentrations are tightly regulated by specialized enzyme repertoires and should remain undisturbed by the addition of exogenous antioxidant molecules, as already demonstrated by intervention studies with antioxidant vitamins. Our goal was to develop specific antioxidants able to scavenge peroxynitrite anion, as well the radicals derived from the homolytic decomposition of its conjugated acid, nitrogen dioxide and hydroxyl radical. Fourteen substituted nitroalkenes, seven 4-substituted 1-(2-nitro-1Z-ethenyl)benzene, and seven 4-substituted (2-nitro-1Z-propenyl)benzene, with different stereochemical and electronic characteristics were synthesized and tested. Compounds with the electron donor group N,N-dimethylamino showed the highest reaction rates against peroxynitrite, and also reacted with its homolytic decomposition products, OH and NO2. While 1,1-dimethylamino-4-(2-nitro-1Z-ethenyl)benzene came up as a lead for future developments without the risk of interfering with signalization pathways, since it was highly specific for peroxynitrite and peroxynitrite derived radicals, its methylated analogous 1,1-dimethylamino-4-(2-nitro-1Z-propenyl)benzene was less specific and also reacted with NO and O2(-), the biological precursor of H2O2.