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Amyotrophic lateral sclerosis (ALS) is a fatal disease that causes degeneration of motor neurons (MNs) and paralysis. ALS can be caused by mutations in the gene that encodes copper/zinc superoxide dismutase (SOD1). SOD1 is known mostly as a cytosolic antioxidant protein, but SOD1 is also in the nucleus of non-transgenic (tg) and human SOD1 (hSOD1) tg mouse MNs. SOD1's nuclear presence in different cell types and subnuclear compartmentations are unknown, as are the nuclear functions of SOD1. We examined hSOD1 nuclear localization and DNA damage in tg mice expressing mutated and wildtype variants of hSOD1 (hSOD1-G93A and hSOD1-wildtype). We also studied ALS patient-derived induced pluripotent stem (iPS) cells to determine the nuclear presence of SOD1 in undifferentiated and differentiated MNs. In hSOD1-G93A and hSOD1-wildtype tg mice, choline acetyltransferase (ChAT)-positive MNs had nuclear hSOD1, but while hSOD1-wildtype mouse MNs also had nuclear ChAT, hSOD1-G93A mouse MNs showed symptom-related loss of nuclear ChAT. The interneurons had preserved parvalbumin nuclear positivity in hSOD1-G93A mice. hSOD1-G93A was seen less commonly in spinal cord astrocytes and, notably, oligodendrocytes, but as the disease emerged, the oligodendrocytes had increased mutant hSOD1 nuclear presence. Brain and spinal cord subcellular fractionation identified mutant hSOD1 in soluble nuclear extracts of the brain and spinal cord, but mutant hSOD1 was concentrated in the chromatin nuclear extract only in the spinal cord. Nuclear extracts from mutant hSOD1 tg mouse spinal cords had altered protein nitration, footprinting peroxynitrite presence, and the intact nuclear extracts had strongly increased superoxide production as well as the active NADPH oxidase marker, p47phox. The comet assay showed that MNs from hSOD1-G93A mice progressively (6–14 weeks of age) accumulated DNA single-strand breaks. Ablation of the NCF1 gene, encoding p47phox, and pharmacological inhibition of NADPH oxidase with systemic treatment of apocynin (10 mg/kg, ip) extended the mean lifespan of hSOD1-G93A mice by about 25% and mitigated genomic DNA damage progression. In human postmortem CNS, SOD1 was found in the nucleus of neurons and glia; nuclear SOD1 was increased in degenerating neurons in ALS cases and formed inclusions. Human iPS cells had nuclear SOD1 during directed differentiation to MNs, but mutant SOD1-expressing cells failed to establish wildtype MN nuclear SOD1 levels. We conclude that SOD1 has a prominent nuclear presence in the central nervous system, perhaps adopting aberrant contexts to participate in ALS pathobiology. [ABSTRACT FROM AUTHOR]

Food-grade titanium dioxide (E171) and zinc oxide nanoparticles (ZnO NPs) are common food additives for human consumption. We examined multi-organ toxicity of both compounds on Wistar rats orally exposed for 90 days. Rats were divided into three groups: (1) control (saline solution), (2) E171-exposed, and (3) ZnO NPs-exposed. Histological examination was performed with hematoxylin–eosin (HE) staining and transmission electron microscopy (TEM). Ceramide (Cer), 3-nitrotyrosine (NT), and lysosome-associated membrane protein 2 (LAMP-2) were detected by immunofluorescence. Relevant histological changes were observed: disorganization, inflammatory cell infiltration, and mitochondrial damage. Increased levels of Cer, NT, and LAMP-2 were observed in the liver, kidney, and brain of E171- and ZnO NPs-exposed rats, and in rat hearts exposed to ZnO NPs. E171 up-regulated Cer and NT levels in the aorta and heart, while ZnO NPs up-regulated them in the aorta. Both NPs increased LAMP-2 expression in the intestine. In conclusion, chronic oral exposure to metallic NPs causes multi-organ injury, reflecting how these food additives pose a threat to human health. Our results suggest how complex interplay between ROS, Cer, LAMP-2, and NT may modulate organ function during NP damage. [ABSTRACT FROM AUTHOR]

Visual loss in acute optic neuritis is typically attributed to axonal conduction block due to inflammatory demyelination, but the mechanisms remain unclear. Recent research has highlighted tissue hypoxia as an important cause of neurological deficits and tissue damage in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) and, here, we examine whether the optic nerves are hypoxic in experimental optic neuritis induced in Dark Agouti rats. At both the first and second peaks of disease expression, inflamed optic nerves labelled significantly for tissue hypoxia (namely, positive for hypoxia inducible factor-1α (HIF1α) and intravenously administered pimonidazole). Acutely inflamed nerves were also labelled significantly for innate markers of oxidative and nitrative stress and damage, including superoxide, nitric oxide and 3-nitrotyrosine. The density and diameter of capillaries were also increased. We conclude that in acute optic neuritis, the optic nerves are hypoxic and come under oxidative and nitrative stress and damage. Tissue hypoxia can cause mitochondrial failure and thus explains visual loss due to axonal conduction block. Tissue hypoxia can also induce a damaging oxidative and nitrative environment. The findings indicate that treatment to prevent tissue hypoxia in acute optic neuritis may help to restore vision and protect from damaging reactive oxygen and nitrogen species. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal disease that causes degeneration of motor neurons (MNs) and paralysis. ALS can be caused by mutations in the gene that encodes copper/zinc superoxide dismutase (SOD1). SOD1 is known mostly as a cytosolic antioxidant protein, but SOD1 is also in the nucleus of non-transgenic (tg) and human SOD1 (hSOD1) tg mouse MNs. SOD1’s nuclear presence in different cell types and subnuclear compartmentations are unknown, as are the nuclear functions of SOD1. We examined hSOD1 nuclear localization and DNA damage in tg mice expressing mutated and wildtype variants of hSOD1 (hSOD1-G93A and hSOD1-wildtype). We also studied ALS patient-derived induced pluripotent stem (iPS) cells to determine the nuclear presence of SOD1 in undifferentiated and differentiated MNs. In hSOD1-G93A and hSOD1-wildtype tg mice, choline acetyltransferase (ChAT)-positive MNs had nuclear hSOD1, but while hSOD1-wildtype mouse MNs also had nuclear ChAT, hSOD1-G93A mouse MNs showed symptom-related loss of nuclear ChAT. The interneurons had preserved parvalbumin nuclear positivity in hSOD1-G93A mice. hSOD1-G93A was seen less commonly in spinal cord astrocytes and, notably, oligodendrocytes, but as the disease emerged, the oligodendrocytes had increased mutant hSOD1 nuclear presence. Brain and spinal cord subcellular fractionation identified mutant hSOD1 in soluble nuclear extracts of the brain and spinal cord, but mutant hSOD1 was concentrated in the chromatin nuclear extract only in the spinal cord. Nuclear extracts from mutant hSOD1 tg mouse spinal cords had altered protein nitration, footprinting peroxynitrite presence, and the intact nuclear extracts had strongly increased superoxide production as well as the active NADPH oxidase marker, p47phox. The comet assay showed that MNs from hSOD1-G93A mice progressively (6–14 weeks of age) accumulated DNA single-strand breaks. Ablation of the NCF1 gene, encoding p47phox, and pharmacological inhibition of NADPH oxidase with systemic treatment of apocynin (10 mg/kg, ip) extended the mean lifespan of hSOD1-G93A mice by about 25% and mitigated genomic DNA damage progression. In human postmortem CNS, SOD1 was found in the nucleus of neurons and glia; nuclear SOD1 was increased in degenerating neurons in ALS cases and formed inclusions. Human iPS cells had nuclear SOD1 during directed differentiation to MNs, but mutant SOD1-expressing cells failed to establish wildtype MN nuclear SOD1 levels. We conclude that SOD1 has a prominent nuclear presence in the central nervous system, perhaps adopting aberrant contexts to participate in ALS pathobiology.

Reactive oxygen species and reactive nitrogen species (RNS) are damaging for many biomolecules. Peroxynitrite (ONOO−) is the most toxic molecular species among RNS. Betalains are known to possess ONOO− scavenging ability. Betanin, a betalain isolated from red beet, possesses antioxidant, anti-inflammatory, and antitumor activities; however, detailed studies of this isolated pigment have not been conducted, owing to its instability under physiological conditions. This study aimed to isolate highly purified betanin from red beetroots using an improved purification method involving deproteinization and citric acid co-precipitation and evaluated its antioxidant activities. The purified betanin thus obtained had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin examined in the 2,2-diphenyl-1-picrylhydrazyl assay, the direct ONOO− reaction, ONOO−-dependent DNA damage, and lipid peroxidation reactions revealed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H2O2 and ONOO− cytotoxicity, respectively, in cultured mouse fibroblasts. To the best of our knowledge, this is the first study to demonstrate the cytoprotective effects of betanin against ONOO− toxicity. The highly purified betanin obtained in this study will aid in further exploring its physiological functions. [ABSTRACT FROM AUTHOR]

Food-grade titanium dioxide (E171) and zinc oxide nanoparticles (ZnO NPs) are common food additives for human consumption. We examined multi-organ toxicity of both compounds on Wistar rats orally exposed for 90 days. Rats were divided into three groups: (1) control (saline solution), (2) E171-exposed, and (3) ZnO NPs-exposed. Histological examination was performed with hematoxylin–eosin (HE) staining and transmission electron microscopy (TEM). Ceramide (Cer), 3-nitrotyrosine (NT), and lysosome-associated membrane protein 2 (LAMP-2) were detected by immunofluorescence. Relevant histological changes were observed: disorganization, inflammatory cell infiltration, and mitochondrial damage. Increased levels of Cer, NT, and LAMP-2 were observed in the liver, kidney, and brain of E171- and ZnO NPs-exposed rats, and in rat hearts exposed to ZnO NPs. E171 up-regulated Cer and NT levels in the aorta and heart, while ZnO NPs up-regulated them in the aorta. Both NPs increased LAMP-2 expression in the intestine. In conclusion, chronic oral exposure to metallic NPs causes multi-organ injury, reflecting how these food additives pose a threat to human health. Our results suggest how complex interplay between ROS, Cer, LAMP-2, and NT may modulate organ function during NP damage.

Visual loss in acute optic neuritis is typically attributed to axonal conduction block due to inflammatory demyelination, but the mechanisms remain unclear. Recent research has highlighted tissue hypoxia as an important cause of neurological deficits and tissue damage in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) and, here, we examine whether the optic nerves are hypoxic in experimental optic neuritis induced in Dark Agouti rats. At both the first and second peaks of disease expression, inflamed optic nerves labelled significantly for tissue hypoxia (namely, positive for hypoxia inducible factor-1α (HIF1α) and intravenously administered pimonidazole). Acutely inflamed nerves were also labelled significantly for innate markers of oxidative and nitrative stress and damage, including superoxide, nitric oxide and 3-nitrotyrosine. The density and diameter of capillaries were also increased. We conclude that in acute optic neuritis, the optic nerves are hypoxic and come under oxidative and nitrative stress and damage. Tissue hypoxia can cause mitochondrial failure and thus explains visual loss due to axonal conduction block. Tissue hypoxia can also induce a damaging oxidative and nitrative environment. The findings indicate that treatment to prevent tissue hypoxia in acute optic neuritis may help to restore vision and protect from damaging reactive oxygen and nitrogen species.

Peroxynitrite (ONOO−) is a crucial reactive oxygen species that plays a vital role in cellular signal transduction and homeostatic regulation. Determining and visualizing peroxynitrite accurately in biological systems is important for understanding its roles in physiological and pathological activity. Among the various detection methods, fluorescent probe-based spectroscopic detection offers real-time and minimally invasive detection, high sensitivity and selectivity, and easy structural and property modification. This review categorizes fluorescent probes by their fluorophore structures, highlighting their chemical structures, recognition mechanisms, and response behaviors in detail. We hope that this review could help trigger novel ideas for potential medical diagnostic applications of peroxynitrite-related molecular diseases. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) and nitric oxide (NO) are two relevant signal molecules that can affect protein function throughout post-translational modifications (PTMs) such as persulfidation, S-nitrosation, metal-nitrosylation, and nitration. Lipoxygenases (LOXs) are a group of non-heme iron enzymes involved in a wide range of plant physiological functions including seed germination, plant growth and development, and fruit ripening and senescence. Likewise, LOXs are also involved in the mechanisms of response to diverse environmental stresses. Using purified soybean (Glycine max L.) lipoxygenase type 1 (LOX 1) and nitrosocysteine (CysNO) and sodium hydrosulfide (NaHS) as NO and H2S donors, respectively, the present study reveals that both compounds negatively affect LOX activity, suggesting that S-nitrosation and persulfidation are involved. Mass spectrometric analysis of nitrated soybean LOX 1 using a peroxynitrite (ONOO−) donor enabled us to identify that, among the thirty-five tyrosine residues present in this enzyme, only Y214 was exclusively nitrated by ONOO−. The nitration of Y214 seems to affect its interaction with W500, a residue involved in the substrate binding site. The analysis of the structure 3PZW demonstrates the existence of several tunnels that directly communicate the surface of the protein with different internal cysteines, thus making feasible their potential persulfidation, especially C429 and C127. On the other hand, the CysNO molecule, which is hydrophilic and bulkier than H2S, can somehow be accommodated throughout the tunnel until it reaches C127, thus facilitating its nitrosation. Overall, a large number of potential persulfidation targets and the ease by which H2S can reach them through the diffuse tunneling network could be behind their efficient inhibition. [ABSTRACT FROM AUTHOR]

Pathological similarities between sarcoidosis (SA) and tuberculosis (TB) suggest the role of mycobacterial antigens in the etiopathogenesis of SA. The Dubaniewicz group revealed that not whole mycobacteria, but Mtb-HSP70, Mtb-HSP 65, and Mtb-HSP16 were detected in the lymph nodes, sera, and precipitated immune complexes in patients with SA and TB. In SA, the Mtb-HSP16 concentration was higher than that of Mtb-HSP70 and that of Mtb-HSP65, whereas in TB, the Mtb-HSP16 level was increased vs. Mtb-HSP70. A high Mtb-HSP16 level, induced by low dose-dependent nitrate/nitrite (NOx), may develop a mycobacterial or propionibacterial genetic dormancy program in SA. In contrast to TB, increased peroxynitrite concentration in supernatants of peripheral blood mononuclear cell cultures treated with Mtb-HSP may explain the low level of NOx detected in SA. In contrast to TB, monocytes in SA were resistant to Mtb-HSP-induced apoptosis, and CD4+T cell apoptosis was increased. Mtb-HSP-induced apoptosis of CD8+T cells was reduced in all tested groups. In Mtb-HSP-stimulated T cells, lower CD8+γδ+IL-4+T cell frequency with increased TNF-α,IL-6,IL-10 and decreased INF-γ,IL-2,IL-4 production were present in SA, as opposed to an increased presence of CD4+γδ+TCR cells with increased TNF-α,IL-6 levels in TB, vs. controls. Mtb-HSP modulating the level of co-stimulatory molecules, regulatory cells, apoptosis, clonal deletion, epitope spread, polyclonal activation and molecular mimicry between human and microbial HSPs may also participate in the induction of autoimmunity, considered in SA. In conclusion, in different genetically predisposed hosts, the same antigens, e.g., Mtb-HSP, may induce the development of TB or SA, including an autoimmune response in sarcoidosis. [ABSTRACT FROM AUTHOR]

The binding of nitric oxide (NO) to heme in the β1 subunit of soluble guanylyl cyclase (sGC) activates both the heterodimeric α1β1 and α2β1 isoforms of the enzyme, leading to the increased production of cGMP from GTP. In cultured human mast cells, exogenous NO is able to inhibit mast cell degranulation via NO-cGMP signaling. However, under inflammatory oxidative or nitrosative stress, sGC becomes insensitive to NO. The occurrence of mast cells in healthy and inflamed human tissues and the in vivo expression of the α1 and β1 subunits of sGC in human mast cells during inflammation remain largely unresolved and were investigated here. Using peroxidase and double immunohistochemical incubations, no mast cells were found in healthy dental pulp, whereas the inflammation of dental pulp initiated the occurrence of several mast cells expressing the α1 and β1 subunits of sGC. Since inflammation-induced oxidative and nitrosative stress oxidizes Fe2+ to Fe3+ in the β1 subunit of sGC, leading to the desensitization of sGC to NO, we hypothesize that the NO- and heme-independent pharmacological activation of sGC in mast cells may be considered as a regulatory strategy for mast cell functions in inflamed human dental pulp. [ABSTRACT FROM AUTHOR]

Reactive oxygen species and reactive nitrogen species (RNS) are damaging for many biomolecules. Peroxynitrite (ONOO−) is the most toxic molecular species among RNS. Betalains are known to possess ONOO− scavenging ability. Betanin, a betalain isolated from red beet, possesses antioxidant, anti-inflammatory, and antitumor activities; however, detailed studies of this isolated pigment have not been conducted, owing to its instability under physiological conditions. This study aimed to isolate highly purified betanin from red beetroots using an improved purification method involving deproteinization and citric acid co-precipitation and evaluated its antioxidant activities. The purified betanin thus obtained had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin examined in the 2,2-diphenyl-1-picrylhydrazyl assay, the direct ONOO− reaction, ONOO−-dependent DNA damage, and lipid peroxidation reactions revealed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H2O2 and ONOO− cytotoxicity, respectively, in cultured mouse fibroblasts. To the best of our knowledge, this is the first study to demonstrate the cytoprotective effects of betanin against ONOO− toxicity. The highly purified betanin obtained in this study will aid in further exploring its physiological functions.

Peroxynitrite (ONOO−) is a crucial reactive oxygen species that plays a vital role in cellular signal transduction and homeostatic regulation. Determining and visualizing peroxynitrite accurately in biological systems is important for understanding its roles in physiological and pathological activity. Among the various detection methods, fluorescent probe-based spectroscopic detection offers real-time and minimally invasive detection, high sensitivity and selectivity, and easy structural and property modification. This review categorizes fluorescent probes by their fluorophore structures, highlighting their chemical structures, recognition mechanisms, and response behaviors in detail. We hope that this review could help trigger novel ideas for potential medical diagnostic applications of peroxynitrite-related molecular diseases.

NCX4040, the non-steroidal anti-inflammatory-NO donor, is cytotoxic to several human tumors, including ovarian tumor cells. We have found that NCX4040 is also cytotoxic against both OVCAR-8 and its adriamycin resistant (NCI/ADR-RES) tumor cell lines. Here, we have examined mechanism(s) for the cytotoxicity of NCX4040 in OVCAR-8 and NCI/ADR-RES cell lines. We found that NCX4040 induced significant apoptosis in both cell lines. Furthermore, NCX4040 treatment caused significant depletion of cellular glutathione, causing oxidative stress due to the formation of reactive oxygen/nitrogen species (ROS/RNS). Significantly more ROS/RNS were detected in OVCAR-8 cells than in NCI/ADR-RES cells which may have resulted from increased activities of SOD, glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. NCX4040 treatment resulted in the formation of double-strand DNA breaks in both cells; however, more of these DNA breaks were detected in OVCAR-8 cells. RT-PCR studies indicated that NCX4040-induced DNA damage was not repaired as efficiently in NCI/ADR-RES cells as in OVCAR-8 cells which may lead to a differential cell death. Pretreatment of OVCAR-8 cells with N-acetylcysteine (NAC) significantly decreased cytotoxicity of NCX4040 in OVCAR-8 cells; however, NAC had no effects on NCX4040 cytotoxicity in NCI/ADR-RES cells. In contrast, FeTPPS, a peroxynitrite scavenger, completely blocked NCX4040-induced cell death in both cells, suggesting that NCX4040-induced cell death could be mediated by peroxynitrite formed from NCX4040 following cellular metabolism. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) and nitric oxide (NO) are two relevant signal molecules that can affect protein function throughout post-translational modifications (PTMs) such as persulfidation, S-nitrosation, metal-nitrosylation, and nitration. Lipoxygenases (LOXs) are a group of non-heme iron enzymes involved in a wide range of plant physiological functions including seed germination, plant growth and development, and fruit ripening and senescence. Likewise, LOXs are also involved in the mechanisms of response to diverse environmental stresses. Using purified soybean (Glycine max L.) lipoxygenase type 1 (LOX 1) and nitrosocysteine (CysNO) and sodium hydrosulfide (NaHS) as NO and H2S donors, respectively, the present study reveals that both compounds negatively affect LOX activity, suggesting that S-nitrosation and persulfidation are involved. Mass spectrometric analysis of nitrated soybean LOX 1 using a peroxynitrite (ONOO−) donor enabled us to identify that, among the thirty-five tyrosine residues present in this enzyme, only Y214 was exclusively nitrated by ONOO−. The nitration of Y214 seems to affect its interaction with W500, a residue involved in the substrate binding site. The analysis of the structure 3PZW demonstrates the existence of several tunnels that directly communicate the surface of the protein with different internal cysteines, thus making feasible their potential persulfidation, especially C429 and C127. On the other hand, the CysNO molecule, which is hydrophilic and bulkier than H2S, can somehow be accommodated throughout the tunnel until it reaches C127, thus facilitating its nitrosation. Overall, a large number of potential persulfidation targets and the ease by which H2S can reach them through the diffuse tunneling network could be behind their efficient inhibition.

Pathological similarities between sarcoidosis (SA) and tuberculosis (TB) suggest the role of mycobacterial antigens in the etiopathogenesis of SA. The Dubaniewicz group revealed that not whole mycobacteria, but Mtb-HSP70, Mtb-HSP 65, and Mtb-HSP16 were detected in the lymph nodes, sera, and precipitated immune complexes in patients with SA and TB. In SA, the Mtb-HSP16 concentration was higher than that of Mtb-HSP70 and that of Mtb-HSP65, whereas in TB, the Mtb-HSP16 level was increased vs. Mtb-HSP70. A high Mtb-HSP16 level, induced by low dose-dependent nitrate/nitrite (NOx), may develop a mycobacterial or propionibacterial genetic dormancy program in SA. In contrast to TB, increased peroxynitrite concentration in supernatants of peripheral blood mononuclear cell cultures treated with Mtb-HSP may explain the low level of NOx detected in SA. In contrast to TB, monocytes in SA were resistant to Mtb-HSP-induced apoptosis, and CD4+T cell apoptosis was increased. Mtb-HSP-induced apoptosis of CD8+T cells was reduced in all tested groups. In Mtb-HSP-stimulated T cells, lower CD8+γδ+IL-4+T cell frequency with increased TNF-α,IL-6,IL-10 and decreased INF-γ,IL-2,IL-4 production were present in SA, as opposed to an increased presence of CD4+γδ+TCR cells with increased TNF-α,IL-6 levels in TB, vs. controls. Mtb-HSP modulating the level of co-stimulatory molecules, regulatory cells, apoptosis, clonal deletion, epitope spread, polyclonal activation and molecular mimicry between human and microbial HSPs may also participate in the induction of autoimmunity, considered in SA. In conclusion, in different genetically predisposed hosts, the same antigens, e.g., Mtb-HSP, may induce the development of TB or SA, including an autoimmune response in sarcoidosis.

DNA damage is closely related to cancer and many aging-related diseases. Peroxynitrite is a strong oxidant, thus a typical DNA damage agent, and is a major mediator of the inflammation-associated pathogenesis. For the first time, we directly visualized the process of DNA damage by peroxynitrite and DNA protection by ectoine via atomic force microscopy in liquid. We found that the persistence length of DNA decreases significantly by adding a small amount of peroxynitrite, but the observed DNA chains are still intact. Specifically, the persistence length of linear DNA in a low concentration of peroxynitrite (0 µM to 200 µM) solution decreases from about 47 nm to 4 nm. For circular plasmid DNA, we observed the enhanced superhelices of plasmid DNA due to the chain soften. When the concentration of peroxynitrite was above 300 µM, we observed the fragments of DNA. Interestingly, we also identified single-stranded DNAs during the damage process, which is also confirmed by ultraviolet spectroscopy. However, if we added 500 mM ectoine to the high concentration PN solution, almost no DNA fragments due to double strand breaks were observed because of the protection of ectoine. This protection is consistent with the similar effect for DNA damage caused by ionizing radiation and oxygenation. We ascribe DNA protection to the preferential hydration of ectoine. [ABSTRACT FROM AUTHOR]

NCX4040, the non-steroidal anti-inflammatory-NO donor, is cytotoxic to several human tumors, including ovarian tumor cells. We have found that NCX4040 is also cytotoxic against both OVCAR-8 and its adriamycin resistant (NCI/ADR-RES) tumor cell lines. Here, we have examined mechanism(s) for the cytotoxicity of NCX4040 in OVCAR-8 and NCI/ADR-RES cell lines. We found that NCX4040 induced significant apoptosis in both cell lines. Furthermore, NCX4040 treatment caused significant depletion of cellular glutathione, causing oxidative stress due to the formation of reactive oxygen/nitrogen species (ROS/RNS). Significantly more ROS/RNS were detected in OVCAR-8 cells than in NCI/ADR-RES cells which may have resulted from increased activities of SOD, glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. NCX4040 treatment resulted in the formation of double-strand DNA breaks in both cells; however, more of these DNA breaks were detected in OVCAR-8 cells. RT-PCR studies indicated that NCX4040-induced DNA damage was not repaired as efficiently in NCI/ADR-RES cells as in OVCAR-8 cells which may lead to a differential cell death. Pretreatment of OVCAR-8 cells with N-acetylcysteine (NAC) significantly decreased cytotoxicity of NCX4040 in OVCAR-8 cells; however, NAC had no effects on NCX4040 cytotoxicity in NCI/ADR-RES cells. In contrast, FeTPPS, a peroxynitrite scavenger, completely blocked NCX4040-induced cell death in both cells, suggesting that NCX4040-induced cell death could be mediated by peroxynitrite formed from NCX4040 following cellular metabolism.

DNA damage is closely related to cancer and many aging-related diseases. Peroxynitrite is a strong oxidant, thus a typical DNA damage agent, and is a major mediator of the inflammation-associated pathogenesis. For the first time, we directly visualized the process of DNA damage by peroxynitrite and DNA protection by ectoine via atomic force microscopy in liquid. We found that the persistence length of DNA decreases significantly by adding a small amount of peroxynitrite, but the observed DNA chains are still intact. Specifically, the persistence length of linear DNA in a low concentration of peroxynitrite (0 µM to 200 µM) solution decreases from about 47 nm to 4 nm. For circular plasmid DNA, we observed the enhanced superhelices of plasmid DNA due to the chain soften. When the concentration of peroxynitrite was above 300 µM, we observed the fragments of DNA. Interestingly, we also identified single-stranded DNAs during the damage process, which is also confirmed by ultraviolet spectroscopy. However, if we added 500 mM ectoine to the high concentration PN solution, almost no DNA fragments due to double strand breaks were observed because of the protection of ectoine. This protection is consistent with the similar effect for DNA damage caused by ionizing radiation and oxygenation. We ascribe DNA protection to the preferential hydration of ectoine.

Dinitrosyl iron complexes (DNICs) are a physiological form of nitric oxide (•NO) in an organism. They are able not only to deposit and transport •NO, but are also to act as antioxidant and antiradical agents. However, the mechanics of hemoglobin-bound DNICs (Hb-DNICs) protecting Hb against peroxynitrite-caused, mediated oxidative modification have not yet been scrutinized. Through EPR spectroscopy we show that Hb-DNICs are destroyed under the peroxynitrite action in a dose-dependent manner. At the same time, DNICs inhibit the oxidation of tryptophan and tyrosine residues and formation of carbonyl derivatives. They also prevent the formation of covalent crosslinks between Hb subunits and degradation of a heme group. These effects can arise from the oxoferryl heme form being reduced, and they can be connected with the ability of DNICs to directly intercept peroxynitrite and free radicals, which emerge due to its homolysis. These data show that DNICs may ensure protection from myocardial ischemia.

Azanone (HNO) is an elusive electrophilic reactive nitrogen species of growing pharmacological and biological significance. Here, we present a comparative kinetic study of HNO reactivity toward selected cyclic C-nucleophiles under aqueous conditions at pH 7.4. We applied the competition kinetics method, which is based on the use of a fluorescein-derived boronate probe FlBA and two parallel HNO reactions: with the studied scavenger or with O2 (k = 1.8 × 104 M−1s−1). We determined the second-order rate constants of HNO reactions with 13 structurally diverse C-nucleophiles (k = 33–20,000 M−1s−1). The results show that the reactivity of HNO toward C-nucleophiles depends strongly on the structure of the scavenger. The data are supported with quantum mechanical calculations. A comprehensive discussion of the HNO reaction with C-nucleophiles is provided.

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

Excessive levels of reactive nitrogen species (RNS) produce nitrosative stress. Among RNS is peroxynitrite, a highly reactive free radical generated when nitric oxide reacts with superoxide anion. Peroxynitrite effects have been mainly studied in somatic cells, and in spermatozoa the majority of studies are focused in humans. The aim of this study is to investigate the in vitro peroxynitrite effect on boar spermatozoa functions and the molecular mechanisms involved. Spermatozoa were exposed to the donor 3-morpholinosydnonimine (SIN-1) in non-capacitating or capacitating medium, motility was evaluated by CASA, functional parameters by flow cytometry and sperm protein phosphorylation by Western blotting. SIN-1 treatment, that significantly increases peroxynitrite levels in boar spermatozoa, potentiates the capacitating-stimulated phosphorylation of cAMP-dependent protein kinase 1 (PKA) substrates and GSK-3α. SIN-1 induced peroxynitrite does not decrease sperm viability, but significantly reduces sperm motility, progressive motility, velocities and motility coefficients. Concomitantly, peroxynitrite does not affect mitochondrial membrane potential, plasma membrane fluidity, or A23187-induced acrosome reaction. However, peroxynitrite significantly increases sperm lipid peroxidation in both media. In conclusion, peroxynitrite compromises boar sperm motility without affecting mitochondrial activity. Although peroxynitrite potentiates the phosphorylation of pathways leading to sperm motility, it also causes oxidative stress that might explain, at least partially, the motility impairment.

Sepsis is associated with an increase in reactive oxygen species (ROS), however, the precise role of ROS in the septic process remains unknown. We hypothesized that treatment with EUK-134 (manganese-3-methoxy N,N'-bis(salicyclidene)ethylene-diamine chloride), a compound with superoxide dismutase and catalase activity, attenuates the vascular manifestations of sepsis in vivo. Pigs were instrumented to measure cardiac output and blood flow in renal, superior mesenteric and femoral arteries, and portal vein. Animals were treated with saline (control), lipopolysaccharide (LPS; 10 µg·kg−1·h−1), EUK-134, or EUK-134 plus LPS. Results show that an LPS-induced increase in pulmonary artery pressure (PAP) as well as a trend towards lower blood pressure (BP) were both attenuated by EUK-134. Renal blood flow decreased with LPS whereas superior mesenteric, portal and femoral flows did not change. Importantly, EUK-134 decreased the LPS-induced fall in renal blood flow and this was associated with a corresponding decrease in LPS-induced protein nitrotyrosinylation in the kidney. PO2, pH, base excess and systemic vascular resistance fell with LPS and were unaltered by EUK-134. EUK-134 also had no effect on LPS-associated increase in CO. Interestingly, EUK-134 alone resulted in higher CO, BP, PAP, mean circulatory filling pressure, and portal flow than controls. Taken together, these data support a protective role for EUK-134 in the renal circulation in sepsis.

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa.

Exposure of U937 cells to peroxynitrite promotes mitochondrial superoxide formation via a mechanism dependent on both inhibition of complex III and increased mitochondrial Ca2+ accumulation. Otherwise inactive concentrations of the oxidant produced the same maximal effects in the presence of either complex III inhibitors or agents mobilizing Ca2+ from the ryanodine receptor and enforcing its mitochondrial accumulation. L-Ascorbic acid (AA) produced similar enhancing effects in terms of superoxide formation, DNA strand scission and cytotoxicity. However, AA failed to enhance the intra-mitochondrial concentration of Ca2+ and the effects observed in cells supplemented with peroxinitrite, while insensitive to manipulations preventing the mobilization of Ca2+, or the mitochondrial accumulation of the cation, were also detected in human monocytes and macrophages, which do not express the ryanodine receptor. In all these cell types, mitochondrial permeability transition-dependent toxicity was detected in cells exposed to AA/peroxynitrite and, based on the above criteria, these responses also appeared Ca2+-independent. The enhancing effects of AA are therefore similar to those mediated by bona fide complex III inhibitors, although the vitamin failed to directly inhibit complex III, and in fact enhanced its sensitivity to the inhibitory effects of peroxynitrite. [ABSTRACT FROM AUTHOR]

The NO/ONOO-cycle is a primarily local, biochemical vicious cycle mechanism, centered on elevated peroxynitrite and oxidative stress, but also involving 10 additional elements: NF-κB, inflammatory cytokines, iNOS, nitric oxide (NO), superoxide, mitochondrial dysfunction (lowered energy charge, ATP), NMDA activity, intracellular Ca2+, TRP receptors and tetrahydrobiopterin depletion. All 12 of these elements have causal roles in heart failure (HF) and each is linked through a total of 87 studies to specific correlates of HF. Two apparent causal factors of HF, RhoA and endothelin-1, each act as tissue-limited cycle elements. Nineteen stressors that initiate cases of HF, each act to raise multiple cycle elements, potentially initiating the cycle in this way. Different types of HF, left vs. right ventricular HF, with or without arrhythmia, etc., may differ from one another in the regions of the myocardium most impacted by the cycle. None of the elements of the cycle or the mechanisms linking them are original, but they collectively produce the robust nature of the NO/ONOO-cycle which creates a major challenge for treatment of HF or other proposed NO/ONOO-cycle diseases. Elevated peroxynitrite/NO ratio and consequent oxidative stress are essential to both HF and the NO/ONOO-cycle.

The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 μM) with an EC50 value of 5 μM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 μM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 μM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.

Congenital heart defects with increased pulmonary blood flow (PBF) result in pulmonary endothelial dysfunction that is dependent, at least in part, on decreases in nitric oxide (NO) signaling. Utilizing a lamb model with left-to-right shunting of blood and increased PBF that mimics the human disease, we have recently shown that a disruption in carnitine homeostasis, due to a decreased carnitine acetyl transferase (CrAT) activity, correlates with decreased bioavailable NO. Thus, we undertook this study to test the hypothesis that the CrAT enzyme plays a major role in regulating NO signaling through its effect on mitochondrial function. We utilized the siRNA gene knockdown approach to mimic the effect of decreased CrAT activity in pulmonary arterial endothelial cells (PAEC). Our data indicate that silencing the CrAT gene disrupted cellular carnitine homeostasis, reduced the expression of mitochondrial superoxide dismutase-and resulted in an increase in oxidative stress within the mitochondrion. CrAT gene silencing also disrupted mitochondrial bioenergetics resulting in reduced ATP generation and decreased NO signaling secondary to a reduction in eNOS/Hsp90 interactions. Thus, this study links the disruption of carnitine homeostasis to the loss of NO signaling observed in children with CHD. Preserving carnitine homeostasis may have important clinical implications that warrant further investigation.

Monocytes and macrophages are part of the first-line defense against bacterial, fungal, and viral infections during host immune responses; they express high levels of proinflammatory cytokines and cytotoxic molecules, including nitric oxide, reactive oxygen species, and their reaction product peroxynitrite. Peroxynitrite is a short-lived oxidant and a potent inducer of cell death. Honey, in addition to its well-known sweetening properties, is a natural antioxidant that has been used since ancient times in traditional medicine. We examined the ability of Gelam honey, derived from the Gelam tree (Melaleuca spp.), to scavenge peroxynitrite during immune responses mounted in the murine macrophage cell line RAW 264.7 when stimulated with lipopolysaccharide/interferon-γ (LPS/IFN-γ) and in LPS-treated rats. Gelam honey significantly improved the viability of LPS/IFN-γ-treated RAW 264.7 cells and inhibited nitric oxide production—similar to the effects observed with an inhibitor of inducible nitric oxide synthase (1400W). Furthermore, honey, but not 1400W, inhibited peroxynitrite production from the synthetic substrate 3-morpholinosydnonimine (SIN-1) and prevented the peroxynitrite-mediated conversion of dihydrorhodamine 123 to its fluorescent oxidation product rhodamine 123. Honey inhibited peroxynitrite synthesis in LPS-treated rats. Thus, honey may attenuate inflammatory responses that lead to cell damage and death, suggesting its therapeutic uses for several inflammatory disorders.

Thirteen polyphenolics were isolated from fresh pods of Caesalpinia pulcherrima using various methods of column chromatography. The structures of these polyphenolics were elucidated as gallic acid (1), methyl gallate (2), 6-O-galloyl-D-glucoside (3), methyl 6-O-galloyl-β-D-glucoside (4), methyl 3,6-di-O-galloyl-α-D-glucopyranoside (5), gentisic acid 5-O-α-D-(6'-O-galloyl)glucopyranoside (6), guaiacylglycerol 4-O-β-D-(6'-O-galloyl)glucopyranoside (7), 3-methoxy-4-hydroxyphenol 1-O-β-D-(6'-O-galloyl)glucopyranoside (8), (+)-gallocatechin (9), (+)-catechin (10), (+)-gallocatechin 3-O-gallate (11), myricetin 3-rhamnoside (12), and ampelopsin (13). All isolated compounds were tested for their antioxidant activities in the 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and peroxynitrite radicals scavenging assays. Among those compounds, 11, 12, and 2 exhibited the best DPPH-, hydroxyl-, and peroxynitrite radical-scavenging activities, respectively. Compound 7 is a new compound, and possesses better scavenging activities towards DPPH but has equivalent hydroxyl radical scavenging activity when compared to BHT. The paper is the first report on free radical scavenging properties of components of the fresh pods of Caesalpinia pulcherrima. The results obtained from the current study indicate that the free radical scavenging property of fresh pods of Caesalpinia pulcherrima may be one of the mechanisms by which this herbal medicine is effective in several free radical mediated diseases.

Nowadays, there is considerable interest in finding out about antioxidants thatare consumed in the habitual diet. It is known that polyphenols are involved in reducing therisk of diseases associated with oxidative stress. The in vitro antioxidant activity of theprincipal wine polyphenolic compounds (catechins, procyanidins, anthocyanins andpyranoanthocyanins) was studied in this work. Four distinct methods were used to assessthe antioxidant capacity of the tested compounds: inhibition of peroxynitrite mediatedtyrosine nitration, TEAC (Trolox equivalent antioxidant capacity assay), FRAP (Ferricreducing/antioxidant power assay) and TBARS (thiobarbituric acid reactive substances)methods. In general, it could be concluded that procyanidins were, among the in vitrotested groups, the ones which showed more antioxidant capacity using the four differentmethods, followed by anthocyanins and pyranoanthocyanins. On the basis of the simpleregression testing, there was a statistically significant relationship between these differentmethods used in aqueous phase (r > 0.92). However, no correlation was found between theresults obtained in lipid media with the TBARS method and those obtained in the aqueousmedia (peroxynitrite scavenging activity, TEAC and FRAP methods).

Exposure of U937 cells to peroxynitrite promotes mitochondrial superoxide formation via a mechanism dependent on both inhibition of complex III and increased mitochondrial Ca2+ accumulation. Otherwise inactive concentrations of the oxidant produced the same maximal effects in the presence of either complex III inhibitors or agents mobilizing Ca2+ from the ryanodine receptor and enforcing its mitochondrial accumulation. l-Ascorbic acid (AA) produced similar enhancing effects in terms of superoxide formation, DNA strand scission and cytotoxicity. However, AA failed to enhance the intra-mitochondrial concentration of Ca2+ and the effects observed in cells supplemented with peroxinitrite, while insensitive to manipulations preventing the mobilization of Ca2+, or the mitochondrial accumulation of the cation, were also detected in human monocytes and macrophages, which do not express the ryanodine receptor. In all these cell types, mitochondrial permeability transition-dependent toxicity was detected in cells exposed to AA/peroxynitrite and, based on the above criteria, these responses also appeared Ca2+-independent. The enhancing effects of AA are therefore similar to those mediated by bona fide complex III inhibitors, although the vitamin failed to directly inhibit complex III, and in fact enhanced its sensitivity to the inhibitory effects of peroxynitrite.

Sepsis is associated with an increase in reactive oxygen species (ROS), however, the precise role of ROS in the septic process remains unknown. We hypothesized that treatment with EUK-134 (manganese-3-methoxy N,N'-bis(salicyclidene)ethylene-diamine chloride), a compound with superoxide dismutase and catalase activity, attenuates the vascular manifestations of sepsis in vivo. Pigs were instrumented to measure cardiac output and blood flow in renal, superior mesenteric and femoral arteries, and portal vein. Animals were treated with saline (control), lipopolysaccharide (LPS; 10 µg·kg-1·h-1), EUK-134, or EUK-134 plus LPS. Results show that an LPS-induced increase in pulmonary artery pressure (PAP) as well as a trend towards lower blood pressure (BP) were both attenuated by EUK-134. Renal blood flow decreased with LPS whereas superior mesenteric, portal and femoral flows did not change. Importantly, EUK-134 decreased the LPS-induced fall in renal blood flow and this was associated with a corresponding decrease in LPS-induced protein nitrotyrosinylation in the kidney. PO2, pH, base excess and systemic vascular resistance fell with LPS and were unaltered by EUK-134. EUK-134 also had no effect on LPS-associated increase in CO. Interestingly, EUK-134 alone resulted in higher CO, BP, PAP, mean circulatory filling pressure, and portal flow than controls. Taken together, these data support a protective role for EUK-134 in the renal circulation in sepsis. [ABSTRACT FROM AUTHOR]

The NO/ONOO-cycle is a primarily local, biochemical vicious cycle mechanism, centered on elevated peroxynitrite and oxidative stress, but also involving 10 additional elements: NF-?B, inflammatory cytokines, iNOS, nitric oxide (NO), superoxide, mitochondrial dysfunction (lowered energy charge, ATP), NMDA activity, intracellular Ca2+, TRP receptors and tetrahydrobiopterin depletion. All 12 of these elements have causal roles in heart failure (HF) and each is linked through a total of 87 studies to specific correlates of HF. Two apparent causal factors of HF, RhoA and endothelin-1, each act as tissue-limited cycle elements. Nineteen stressors that initiate cases of HF, each act to raise multiple cycle elements, potentially initiating the cycle in this way. Different types of HF, left vs. right ventricular HF, with or without arrhythmia, etc., may differ from one another in the regions of the myocardium most impacted by the cycle. None of the elements of the cycle or the mechanisms linking them are original, but they collectively produce the robust nature of the NO/ONOO-cycle which creates a major challenge for treatment of HF or other proposed NO/ONOO-cycle diseases. Elevated peroxynitrite/NO ratio and consequent oxidative stress are essential to both HF and the NO/ONOO-cycle. [ABSTRACT FROM AUTHOR]

The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 µM) with an EC50 value of 5 µM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 µM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 µM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite. [ABSTRACT FROM AUTHOR]

Congenital heart defects with increased pulmonary blood flow (PBF) result in pulmonary endothelial dysfunction that is dependent, at least in part, on decreases in nitric oxide (NO) signaling. Utilizing a lamb model with left-to-right shunting of blood and increased PBF that mimics the human disease, we have recently shown that a disruption in carnitine homeostasis, due to a decreased carnitine acetyl transferase (CrAT) activity, correlates with decreased bioavailable NO. Thus, we undertook this study to test the hypothesis that the CrAT enzyme plays a major role in regulating NO signaling through its effect on mitochondrial function. We utilized the siRNA gene knockdown approach to mimic the effect of decreased CrAT activity in pulmonary arterial endothelial cells (PAEC). Our data indicate that silencing the CrAT gene disrupted cellular carnitine homeostasis, reduced the expression of mitochondrial superoxide dismutase-and resulted in an increase in oxidative stress within the mitochondrion. CrAT gene silencing also disrupted mitochondrial bioenergetics resulting in reduced ATP generation and decreased NO signaling secondary to a reduction in eNOS/Hsp90 interactions. Thus, this study links the disruption of carnitine homeostasis to the loss of NO signaling observed in children with CHD. Preserving carnitine homeostasis may have important clinical implications that warrant further investigation. [ABSTRACT FROM AUTHOR]

Monocytes and macrophages are part of the first-line defense against bacterial, fungal, and viral infections during host immune responses; they express high levels of proinflammatory cytokines and cytotoxic molecules, including nitric oxide, reactive oxygen species, and their reaction product peroxynitrite. Peroxynitrite is a short-lived oxidant and a potent inducer of cell death. Honey, in addition to its well-known sweetening properties, is a natural antioxidant that has been used since ancient times in traditional medicine. We examined the ability of Gelam honey, derived from the Gelam tree (Melaleuca spp.), to scavenge peroxynitrite during immune responses mounted in the murine macrophage cell line RAW 264.7 when stimulated with lipopolysaccharide/ interferon-γ (LPS/IFN-γ) and in LPS-treated rats. Gelam honey significantly improved the viability of LPS/IFN-γ-treated RAW 264.7 cells and inhibited nitric oxide production--similar to the effects observed with an inhibitor of inducible nitric oxide synthase (1400W). Furthermore, honey, but not 1400W, inhibited peroxynitrite production from the synthetic substrate 3-morpholinosydnonimine (SIN-1) and prevented the peroxynitrite-mediated conversion of dihydrorhodamine 123 to its fluorescent oxidation product rhodamine 123. Honey inhibited peroxynitrite synthesis in LPS-treated rats. Thus, honey may attenuate inflammatory responses that lead to cell damage and death, suggesting its therapeutic uses for several inflammatory disorders. [ABSTRACT FROM AUTHOR]

Dinitrosyl iron complexes (DNICs) are a physiological form of nitric oxide (•NO) in an organism. They are able not only to deposit and transport •NO, but are also to act as antioxidant and antiradical agents. However, the mechanics of hemoglobin-bound DNICs (Hb-DNICs) protecting Hb against peroxynitrite-caused, mediated oxidative modification have not yet been scrutinized. Through EPR spectroscopy we show that Hb-DNICs are destroyed under the peroxynitrite action in a dose-dependent manner. At the same time, DNICs inhibit the oxidation of tryptophan and tyrosine residues and formation of carbonyl derivatives. They also prevent the formation of covalent crosslinks between Hb subunits and degradation of a heme group. These effects can arise from the oxoferryl heme form being reduced, and they can be connected with the ability of DNICs to directly intercept peroxynitrite and free radicals, which emerge due to its homolysis. These data show that DNICs may ensure protection from myocardial ischemia. [ABSTRACT FROM AUTHOR]

Azanone (HNO) is an elusive electrophilic reactive nitrogen species of growing pharmacological and biological significance. Here, we present a comparative kinetic study of HNO reactivity toward selected cyclic C-nucleophiles under aqueous conditions at pH 7.4. We applied the competition kinetics method, which is based on the use of a fluorescein-derived boronate probe FlBA and two parallel HNO reactions: with the studied scavenger or with O2 (k = 1.8 × 104 M−1s−1). We determined the second-order rate constants of HNO reactions with 13 structurally diverse C-nucleophiles (k = 33–20,000 M−1s−1). The results show that the reactivity of HNO toward C-nucleophiles depends strongly on the structure of the scavenger. The data are supported with quantum mechanical calculations. A comprehensive discussion of the HNO reaction with C-nucleophiles is provided. [ABSTRACT FROM AUTHOR]