The protective role of Wnt3a in peroxynitrite-induced damage of cochlear hair cells in vitro

Abstract Objective To investigate the effect of peroxynitrite on the cultured cochlear hair cells of C57BL/6 P3 mice in vitro as well as the role of Wnt3a, as an activator of the canonical Wnt signaling pathway, underlying the action of such an oxidative stress. Methods The in vitro primary cultured cochlear hair cells were subjected to l00 μM peroxynitrite and l00 μM peroxynitrite +25 ng/mL Wnt3a for 24 h, the cell survival and morphological changes were examined by immunofluorescence and transmission electron microscopy. Results The number of surviving hair cells was significantly reduced in the 100 μM peroxynitrite group, while it was significantly higher in the Wnt3a + peroxynitrite treated group compared with the peroxynitrite treated group. The transmission electron microscopy showed that exposure to peroxynitrite induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, while Wnt3a clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. Conclusion These results indicated that peroxynitrite could cause oxidative damage to the cochlear hair cells, and low concentrations of Wnt3a has a protective effect against oxidative damage. Level of evidence: Level 2.

Biochemical and microscopy evidence on adverse effects of nitroxidized human serum albumin

Earlier researches have pointed about the accumulation of peroxynitrite modified proteins and their aggregates in the etiopathogenesis of many age-related neurodegenerative and several autoimmune diseases. Human serum albumin (HSA) is present in abundance in plasma and is susceptible to modification by peroxynitrite. In this study, HSA modified with peroxynitrite (nitroxidized-HSA) formed aggregate besides other gross structural changes. Aggregation or assembly of aberrant proteins is responsible for increase in production of reactive species and is often correlated with toxicity in neurodegenerative diseases. However, lack of literature on the cytotoxicity of aggregated nitroxidized-HSA led us to explore its toxicity using human peripheral blood lymphocytes. Elevated protein carbonyl coupled with decreased protein thiol, and release of antioxidant enzymes and lactate dehydrogenase (LDH) were observed upon incubation of lymphocytes with nitroxidized-HSA. Trypan blue exclusion and MTT assays indicated nitroxidized-HSA induced injury/death of lymphocytes. This may be attributed to the observed reactive oxygen species generation during the interaction of nitroxidized-HSA with lymphocytes. Moreover, the analysis of the cellular morphology by dual staining, fluorescence and confocal microscopy further confirms the cytotoxicity of nitroxidized-HSA. Since various age-related degenerative diseases are characterized by deposition of protein aggregates, the demonstrated toxicity of nitroxidized-HSA may be an important driver in the pathophysiology of neurodegenerative diseases.

Estrés nitroxidativo pulmonar y sistémico en pacientes en ventilación mecánica con sepsis sin lesión pulmonar aguda y en pacientes en ventilación mecánica sin sepsis / Pulmonary and systemic nitroxidative stress in mechanically ventilated patients with sepsis without acute lung injury and in mechanically ventilated patients without sepsis

Propósito: Durante la sepsis y la ventilación mecánica, se genera estrés oxidativo por activación de las células pulmonares endoteliales e inflamatorias y producción de especies reactivas del oxígeno (ERO). Nuestro principal objetivo fue estudiar la producción pulmonar y sistémica de óxido nítrico (•NO) y oxidantes derivados del •NO que generan estrés nitroxidativo y su relación con la lesión pulmonar aguda (LPA) en pacientes en ventilación mecánica sépticos y no sépticos. Métodos: estudiamos 69 pacientes ventilados mecánicamente, de estos 36 pacientes con sepsis y 33 pacientes sin sepsis. Los pacientes fueron estudiados dentro de las primeras 48 horas de ingreso a unidad de cuidado intensivo (UCI). La producción de estrés nitroxidativo se comparó entre los pacientes con sepsis y los pacientes ventilados mecánicamente sin sepsis (VM). Ocho pacientes de quirófano sin enfermedad pulmonar sirvieron como grupo de control sano (GCBQ). Se analizaron nitrito más nitrato (NOx - ), 3-nitrotirosina (3-NT) y malondialdehído (MDA) en líquido de lavado bronquioloalveolar (LBA). En plasma se midió NOx - (n=69). Adicionalmente en plasma se midió 3-NT, MDA, y alfa tocoferol (α-TOH). Resultados: NO x - , 3-NT, MDA en LBA y NOx - y α-TOH en plasma fueron mayores en pacientes con sepsis que en los pacientes con VM sin sepsis (todos p <0,05). Tanto los pacientes con sepsis como VM tenían concentración de NOx - en LBA mayor que el grupo de control sano (p <0,001). En los pacientes con sepsis, los pacientes que fallecieron en la UCI tuvieron concentraciones mayores de NOx - en LBA que los sobrevivientes en la UCI, 80 (70 - 127) µM en comparación con 31 (15 - 47) µM, respectivamente, p <0,001. Los pacientes con síndrome de distress respiratorio agudo (SDRA) en el grupo sepsis tuvieron mayor concentración de NOx - en LBA. Conclusiones: Durante las fases tempranas de la sepsis y la ventilación Sepsis y Estrés Nitroxidativo Pulmonar 20 mecánica hay aumento del estrés nitroxidativo pulmonar y sistémico debido a un aumento de la producción de •NO que conduce a oxidantes secundarios derivados del •NO, los que promueven la nitración de proteínas y la peroxidación de lípidos. Esto se asocia con SDRA/LPA y aumento de la mortalidad en UCI

Purpose: During sepsis and mechanical ventilation oxidative stress is generated by endothelial and inflammatory lung cells. Our main objective was to study pulmonary and systemic •NO (nitric oxide) production and nitroxidative stress in mechanically ventilated septic patients. Methods: we study 69 mechanically ventilated patients, 36 with sepsis and 33 without sepsis within the first 48 hours of ICU admission compared with 33 mechanically ventilated patients without sepsis (MV) plus eight operating room patients without lung disease served as control healthy group (ORCG). Nitrite plus nitrate (NOx - ), 3-nitrotyrosine (3-NT) and malondialdehyde (MDA) in bronchoalveolar lavage fluid (BALF) were analyzed. Additionally, we measured plasma alpha tocopherol (α-TOH), MDA, and 3-NT. Results: BALF NOx - , BALF 3-NT, BALF MDA, and plasma NOx - were higher in the Sepsis than in MV patients (all p<0.05). Both SG and MV patients had higher BALF NOx - than the healthy control group (p<0.001). Sepsis patients had higher plasma NOx - and α TOH than mechanically-ventilated patients without sepsis (all p <0,05). In the Sepsis patients, the ICU non-survivors had higher levels of BALF NOx - than ICU survivors 280(70 - 127) µM versus 31(15 - 47) µM, p< 0.001. Conclusions: We conclude that during early phases of sepsis there is an enhanced lung nitroxidative stress due to an increase of •NO production leading to secondary Sepsis y Estrés Nitroxidativo Pulmonar 21 •NO-derived oxidants, which promote protein nitration and lipid peroxidation. This is associated with ARDS /ALI and increased mortality in ICU

Nitrosative stress in human spermatozoa causes cell death characterized by induction of mitochondrial permeability transition-driven necrosis / 亚洲男科学杂志(英文版)

Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without (untreated control) 3-morpholinosydnonimine (SIN-1), in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition (MPT), externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-1, the sperm viability was significantly reduced compared to untreated control (P < 0.001). Furthermore, the MPT was induced (P < 0.01) and increment in DNA oxidation (P < 0.01), DNA fragmentation (P < 0.01), tyrosine nitration (P < 0.0001) and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control (P < 0.001), this process was observed in <10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.

Identification and quantification of flavonoids in Carica papaya leaf and peroxynitrite-scavenging activity

Objective: To characterize the types, contents, and peroxynitrite-scavenging activities of flavonoids in the leaf of Carica papaya (C. papaya). Methods: Chromatographic and spectroscopic techniques along with high performance liquid chromatography quantitative analysis and peroxynitrite-scavenging assay were performed to isolate and quantify flavonoid compounds in the flavonoid-rich fraction (BuOH fraction) derived from MeOH extract of C. papaya leaves and evaluate their peroxynitrite-scavenging activities. Results: Seven flavonoids were isolated from the leaves of C. papaya, including quer-cetin 3-(2G-rhamnosylrutinoside), kaempferol 3-(2G-rhamnosylrutinoside), quercetin 3-rutinoside, myricetin 3-rhamnoside, kaempferol 3-rutinoside, quercetin, and kaemp-ferol. All of the substances exhibited potent activities on peroxynitrite scavenging (IC50 ≤ 4.15 μmol/L), which were stronger than the positive control, L-penicillamine (6.90μmol/L). The content of kaempferol 3-(2G-rhamnosylrutinoside) was significantly higher than other identified compounds (123.18 mg/g BuOH fraction and 7.23 mg/g MeOH extract). Conclusions: The results of the present study demonstrate the potent antioxidant fla-vonoids of C. papaya leaf, with kaempferol 3-(2G-rhamnosylrutinoside) as the major one.

Serum E3 SUMO-protein ligase NSE2 level and peroxynitrite related to oxidative stress in nephrolithiasis patients

Objective: To prove probable relations between serum E3 SUMO-protein ligase NSE2 (NSMCE2) concentration, peroxynitrite related to oxidative stress in nephrolithiasis patients. Methods: A total of 60 patients with nephrolithiasis and 50 healthy volunteers were involved in this study. Colorimetric method was used to detect blood urea, creatinine, uric acid, protein, albumin, total antioxidant status, total oxidant status, peroxynitrite, nitric oxide and oxidative stress index. Glutathione, NSMCE2 and superoxide dismutase were measured by ELISA. Results: A significant increase in level of peroxynitrite, total oxidant status, NSMCE2 and oxidative stress index in patients was observed, while total antioxidant status and glutathione were significantly decreased. Conclusions: The study concluded that serum NSMCE2 significantly correlated with peroxynitrite and oxidative stress in patients with nephrolithiasis.

The inhibitory effect of PNS on brain 3-NT formation in vitro / 天津医药

Objective To study the inhibitory effect of panax notoginseng saponins (PNS) on 3-nitrotyrosine (3-NT) formation in brain induced by heme/NO2 -/H2O2 or ONOO - pathways in vitro. Methods According to the two major pathways of 3-NT formation in vivo, the models of protein nitration induced by heme/NaNO2/H2O2 or ONOO-system were established, respectively, in vitro. Bovine serum albumin (BSA)/rat plasma protein or rat brain homogenate protein were utilized as reactive substrates in both systems. Samples were divided into blank-control group, 3-NT group and PNS group (including low-, medium-and high-concentration subgroups). In 3-NT group, samples were exposed to heme/NaNO2/H2O2 or ONOO-system, respectively, at 37℃for 30 min, whereas in PNS group, samples were pre-incubated with PNS (at final concentrations of 50 mg/L, 100 mg/L, and 200 mg/L) at 37℃for 5 min before the nitrating system exposure. The 3-NT level in each group was detected by Western blot assy. Results Compared with the blank-control group, both heme/NaNO2/H2O2 and ONOO-system can induce significant 3-NT generation in BSA/rat plasma protein or rat brain homogenate protein (P0.05). Medium- and high-concentrations of PNS pre-treatment markedly inhibited 3-NT accumulation, with maximum effect at the concentration of 200 mg/L (P<0.05). Conclusion Medium- and high-concentrations of PNS can inhibit 3-NT formation in brain tissue mediated by either heme/NO2-/H2O2 or ONOO-pathways, implying that potential neuroprotective action against 3-NT involves pathological conditions, like trauma, stroke, and neurodegenerative diseases.

Serum E3 SUMO-protein ligase NSE2 level and peroxynitrite related to oxidative stress in nephrolithiasis patients

Objective To prove probable relations between serum E3 SUMO-protein ligase NSE2 (NSMCE2) concentration, peroxynitrite related to oxidative stress in nephrolithiasis patients. Methods A total of 60 patients with nephrolithiasis and 50 healthy volunteers were involved in this study. Colorimetric method was used to detect blood urea, creatinine, uric acid, protein, albumin, total antioxidant status, total oxidant status, peroxynitrite, nitric oxide and oxidative stress index. Glutathione, NSMCE2 and superoxide dismutase were measured by ELISA. Results A significant increase in level of peroxynitrite, total oxidant status, NSMCE2 and oxidative stress index in patients was observed, while total antioxidant status and glutathione were significantly decreased. Conclusions The study concluded that serum NSMCE2 significantly correlated with peroxynitrite and oxidative stress in patients with nephrolithiasis.

Identification and quantification of flavonoids in Carica papaya leaf and peroxynitrite-scavenging activity

Objective To characterize the types, contents, and peroxynitrite-scavenging activities of flavonoids in the leaf of Carica papaya (C. papaya). Methods Chromatographic and spectroscopic techniques along with high performance liquid chromatography quantitative analysis and peroxynitrite-scavenging assay were performed to isolate and quantify flavonoid compounds in the flavonoid-rich fraction (BuOH fraction) derived from MeOH extract of C. papaya leaves and evaluate their peroxynitrite-scavenging activities. Results Seven flavonoids were isolated from the leaves of C. papaya, including quercetin 3-(2

Laminar lesions in horses with systemic oxidative stress, committed by experimentally induced or naturally occurring gastrointestinal disorders / Lesões laminares em cavalos com estresse oxidativo sistêmico, acometidos por doenças gastrintestinais experimentalmente induzidas ou de ocorrência natural

Laminitis in horses can be associated with lesions in multiple organs secondary to sepsis. Twenty-one horses suffering from gastrointestinal disorders were used in the experiment; 7 horses with experimentally induced endotoxemia and intestinal ischaemia, and 14 horses suffering from naturally occurring colic syndrome. Tissue samples of lungs, liver, heart, brain, cerebellum and hoof laminar tissue were collected for histopathological and oxidative stress evaluation using nitrotyrosine and superoxide dismutase (SOD2) immunostaining. The horses were divided into two groups: the non-oxidative lesions group (NOLG), with 7 horses showing weak immunostaining in lungs, liver and kidney, and the oxidative lesions group (OLG), with 14 horses showing immunostaining indicating systemic oxidative stress in multiple organs. The horses from OLG showed increase of laminar lesions and SOD2 immunostaining in multiple organs when compared to the horses from the NOLG. No differences were found ln regard to laminar immunostaining by nitrotyrosine and SOD2 between experimental groups. It was concluded that systemic oxidative stress can be associated with the development of laminar lesions, and that the laminar tissue does not respond to oxidative stress with increase of SOD as occurs in other organs.(AU)

A laminite em equinos pode estar associada à lesão em múltiplos órgãos secundária a sepse. Foram utilizados 21 cavalos com afecções gastrintestinais, sendo sete com endotoxemia e isquemia intestinal induzidos experimentalmente, e 14 cavalos com síndrome cólica de origem natural. Amostras teciduais de pulmão, rim, fígado, coração, cérebro e cerebelo e de tecido laminar do casco foram coletadas para avaliação de lesão histopatológica e estresse oxidativo, pela imunomarcação de nitrotirosina e superóxido dismutase (SOD2). Os animais foram divididos em dois grupos: grupo sem lesão oxidativa (NOLG), com sete cavalos com fraca imunomarcação em pulmão, fígado e rim, e grupo lesão oxidativa (OLG), contendo 14 cavalos com imunomarcação indicando estresse oxidativo em múltiplos órgãos. Os cavalos do grupo OLG apresentaram aumento de lesões laminares e imunomarcação para SOD2 em múltiplos órgãos, quando comparados ao NOLG. Não houve diferença sobre a imunomarcação laminar para nitrotirosina e SOD2 entre os grupos experimentais. Conclui-se que o estresse oxidativo sistêmico está associado ao desenvolvimento de lesões laminares, e que o tecido laminar não responde ao estresse oxidativo com aumento de SOD como ocorre nos outros órgãos.(AU)

Analysis of Flavonoid Composition of Korean Herbs in the Family of Compositae and their Utilization for Health

Compositional differences in flavonoids are varied in the big family of Compositae. By summarizing our previous analytical studies and other scientific evidences, new strategy will be possible to further analyze flavonoids and utilize them for human health. The HPLC analytical method has been established in terms of linearity, sensitivity, accuracy, and precision. Herbs of the family of Compositae have considerable amounts of peroxynitrite (ONOO-)-scavenging effects and their phenolic substances. These effects may contribute to the prevention of disease associated with excess production of ONOO-, depending on the high content of flavonoid substances.

Arginase Inhibition Restores Peroxynitrite-Induced Endothelial Dysfunction via L-Arginine-Dependent Endothelial Nitric Oxide Synthase Phosphorylation

PURPOSE: Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay. RESULTS: SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. N(G)-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition. CONCLUSION: These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions.

Comparison of two new on-line methods on screening peroxynitrite scavengers in Radix Scrophulariae / 中国药科大学学报

@#Two simple and efficient methods have been developed for screening and identification of natural peroxynitrite scavengers in Radix Scrophulariae (RS). Method I was based on HPLC-DAD-(luminol-peroxynitrite)-CL techniques combined with Q-TOF MS/MS analysis, while method II was based on the pre-column reaction with peroxynitrite followed by HPLC separation with Q-TOF MS/MS analysis. Five active constituents, P1(decaffeoylacteoside), P9(eoside), I6(6″-O-feruloylharpagide), P11(cis-acteoside)and P13(angoroside), were found to possess potential peroxynitrite-scavenging activity by method I, while P9 and P13 were also screened by method II. Method I requires more complex apparatus, but has advantages on simple detection and high sensitivity. Method II requires simpler apparatus than method I, but with more tedious detection and lower sensitivity. However, the methods established above would provide new ways for rapid detection of natural peroxynitrite-scavenging compounds in RX complex matrices.

Role of spinal peroxynitrite in remifentanil-induced hyperalgesia in rats / 中华麻醉学杂志

Objective To evaluate the role of spinal peroxynitrite in remifentanil-induced hyperalgesia in rats.Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 2-3 months, were randomly divided into 4 groups (n =8 each) using a random number table: control group (group C), remifentanil group (group R), hydrogen-rich saline group (group C + H), and remifentanil + hydrogen-rich saline group (group R+H).In group C, normal saline was infused for 60 min at a rate of 0.1 ml · kg-1 · min-1.In group R, remifentanil was infused for 60 min at a rate of 1.0 μg · kg-1 · min-1.In group R+H, remifentanil was infused for 60 min at a rate of 1.0 μg · kg-1 · min-1 ,and hydrogen-rich saline 10 ml/kg was intraperitoneally injected at 10 min before iv infusion.At 24 h before iv infusion and 6, 24 and 48 h after iv infusion (T0-3) , the mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured.All the rats were sacrificed after the last measurement of pain thresholds, the lumbar segment (L4-6) of the spinal cord was removed for determination of the nitrated manganese superoxide dismutase (MnSOD) expression (by immunoprecipitation and Western blot analysis), 3-nitrotyrosine (3-NT) expression (by Western blot) and spinal MnSOD activity (by spectrophotometer).Results Compared with group C, the MWT was significantly decreased, and TWL was shortened at T1-T3, the expression of spinal 3-NT and nitrated MnSOD was up-regulated, and MnSOD activity was decreased in R and R+H groups, and no significant change was found in the parameters mentioned above in group C+H.Compared with group R, the MWT was significantly increased, and TWL was prolonged at T1-T3, the expression of spinal 3-NT and nitrated MnSOD was down-regulated, and MnSOD activity was increased in group R + H.Conclusion Spinal peroxynitrite is involved in the mechanism underlying remifentanil-induced hyperalgesia through inhibiting the activity of MnSOD in rats.

Phytochemical Analysis of the Phenolic Fat-Suppressing Substances in the Leaves of Lactuca raddeana in 3T3-L1 Adipocytes

Lactuca raddeana (Compositae) is used to treat obesity and complications due to diabetes. The five phenolic compounds including chlorogenic acid, chicoric acid, luteolin 7-O-glucoside, luteolin 7-O-glucuronide, luteolin were qualitatively identified by LC-ESI-MS analysis. The contents were quantitatively determined by HPLC, under the condition of a Capcell Pak C18 column (5 microm, 250 mm x 4.6 mm i.d.) and a gradient elution of 0.05% trifluoroacetic acid (TFA) and 0.05% TFA in MeOH-H2O (60 : 40). The contents of chicoric acid (100.99 mg/g extract) and luteolin 7-O-glucoside (101. 69 mg/g extract) were high, while those of other three phenolic substances were very low. The 3T3-L1 adipocyte cells treated with chicoric acid and luteolin 7-O-glucuronide significantly suppressed the accumulation of fat, suggesting they are effective against obesity. Since high level of peroxynitrite (ONOO) causes cardiovascular disease in obese patients, its scavenging activity was also studied.

Acetilação radicalar de amino ácidos, peptídeos e nucleobases pelos sistemas biacetilo/peroxinitrito e metilglioxal/peroxinitrito / Radical acetylation of aminoacids, peptides, and nucleobases by the biacetyl or methylglyoxal/peroxynitrite systems

Biacetilo (2,3-butanediona) é um contaminante de comida e cigarro, também implicado na hepatoxicidade do álcool e em doenças pulmonares. O metilglioxal (MG), um α-oxoaldeído reativo frequentemente associado ao diabetes e envelhecimento, é produto da fragmentação oxidativa de trioses fosfato, acetona e aminoacetona. Por sua vez, peroxinitrito - um potente oxidante, agente nitrante e nucleófilo formado in vivo pela reação controlada por difusão do ânion radical superóxido com o radical óxido nítrico (k ~1010 M-1s-1) é capaz de se adicionar a CO2 e compostos carbonílicos, gerando produtos potencialmente tóxicos ou sinalizadores celulares. Aminoácidos, peptídeos e nucleobases podem ser acetilados nos grupos amina e na porção desoxiribose. Relativamente ao tratamento com peroxinitrito isolado, níveis superiores de 3-nitrotirosina foram detectados quando tirosina foi tratada com peroxinitrito/biacetilo ou metilglioxal. Ambos os grupos amina de lisina (Lys) ou um deles de derivados de lisina bloqueados e um deles (Ac-Lys-OMe, Z-Lys-OMe) foram acetilados pelo sistema biacetilo ou metilglioxal/peroxinitrito. Em tetrapeptídeos sintéticos contendo lisina como aminoácido amino-terminal (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), a lisina foi acetilada pelo sistemas dicarbonilico/peroxinitrito no grupo α-amina (em maior extensão) e/ou no ε-amina (em menor extensão). No conjunto, estes resultados podem ser interpretados à luz do mecanismo proposto para a reação de compostos α-dicarbonílicos com peroxinitrito, o qual envolve sequencialmente: (i) adição nucleofílica de peroxinitrito à carbonila; (ii) homólise do aduto peroxinitroso formado, liberando •NO2 e um radical oxila do reagente carbonílico; (iii) β-clivagem do radical oxila a um ácido carboxílico (ácido acético no caso de biacetilo e ácido fórmico, a partir de metilglioxal) e radical acetila; (iv) captação do radical acetila pelo oxigênio molecular dissolvido dando acetato, ou por aminoácido ou nucleobase...

Diacetyl (2,3-butanedione) is a food and cigarette contaminant recently implicated in alcohol hepatotoxicity and lung disease. In turn, methylglyoxal (MG) is an α-oxoaldehyde frequently associated with diabetes and aging that is putatively formed by the oxidative fragmentation of trioses phosphate, acetone and aminoacetone. Peroxynitrite - a potent oxidant, nitrating agent and nucleophile formed in vivo by the diffusion-controlled reaction of superoxide radical with nitric oxide (k ~1010 M-1s-1) - is able to form adducts with carbon dioxide and carbonyl compounds. When initially present in the reaction mixtures before addition of ONOO-, amino acids, peptides and nucleobases undergo acetylation at the amino group and purine moieties in the presence of biacetyl or methylglyoxal. Higher levels of 3-nitrotyrosine nitration were measured when peroxynitrite/biacetyl or metilglioxal was added to tyrosine, in comparison with peroxynitrite alone. Both amino groups of L-lysine or one of the amino groups of L-lysine derivatives (Z-Lys-OH and Ac-Lys-OH) were acetylated by biacetyl and methylglyoxal/peroxynitrite system. Using tetrapeptides containing lysine at the terminal amino acid (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), the lysine residue was acetylated at both or either α-amino (major adduct) and ε-amino group (minor adduct). Altogether these data can be interpreted by the mechanism proposed to describe the reaction of α-dicarbonyls with peroxynitrite as follows: (i) nucleophilic addition of peroxynitrite to the carbonyl group of the reagent; (ii) homolysis of the formed peroxynitroso carbonyl adduct to •NO2 and a carbonyloxyl radical; (iii) β-cleavage of the oxyl radical to acetyl radical plus acetic acid (from diacetyl) or formic acid (from methylglyoxal); (iv) competitive scavenging of the acetyl radical by dissolved molecular oxygen and by added amino acid, peptide or nucleobase, ultimately yielding acetate or acetylated biomolecule. If occurring in vivo...

Antioxidant effects of crude extracts from Baccharis species: inhibition of myeloperoxidase activity, protection against lipid peroxidation, and action as oxidative species scavenger

The objective of this study was to show a comparison of the antioxidant properties of aqueous and ethanolic extracts obtained from Baccharis articulata (Lam.) Pers., Baccharis trimera (Less.) DC., Baccharis spicata (Lam.) Baill. and Baccharis usterii Heering, Asteraceae, by several techniques covering a range of oxidant species and of biotargets. We have investigated the ability of the plant extracts to scavenge DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical, action against lipid peroxidation of membranes including rat liver microsomes and soy bean phosphatidylcholine liposomes by ascorbyl radical and peroxynitrite. Hydroxyl radical scavenger activity was measured monitoring the deoxyribose oxidation. The hypochlorous acid scavenger activity was also evaluated by the prevention of protein carbonylation and finally the myeloperoxidase (MPO) activity inhibition. The results obtained suggest that the Baccharis extracts studied present a significant antioxidant activity scavenging free radicals and protecting biomolecules from the oxidation. We can suggest that the supposed therapeutic efficacy of this plant could be due, in part, to these properties.

Free radical reactions of a naturally occurring flavone baicalein and possible mechanisms towards its membrane protective properties.

Baicalein (5, 6, 7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavone present in some of the medicinal plants is known for its potential therapeutic effects, such as cardioprotective, anticancer and anti-inflammatory properties. However, detailed role and mechanisms behind its protective properties against different generators for oxidative stress have not been examined. In the present study, we investigated the possible protective ability of baicalein against the membrane damage caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS) and the mechanisms involved using pulse radiolysis technique. Baicalein offered efficient protection even at a concentration of 10 M towards membrane damage caused by lipid peroxidation induced by the -radiation, peroxyl radicals, ascorbate-Fe2+ and peroxynitrite in rat liver mitochondria and heart homogenate. To elucidate its reaction mechanisms with biologically relevant radicals, transient absorption spectroscopy employing pulse radiolysis technique was used. Baicalein showed fairly high rate constants (3.7 × 109, 1.3 × 109 and 8.0 × 108 dm3 mol-1 s-1 for hydroxyl, azidyl and alkylchloroperoxyl radicals, respectively), suggesting that baicalein can act as an effective scavenger of these radicals. In each case, the phenoxyl radical of baicalein was generated. Thus, it was evident that the phenolic moiety of baicalein was responsible for the free radical scavenging process. Baicalein also reacts with linoleic acid peroxyl radical (LOO·), indicating its ability to act as a chain breaking antioxidant. Peroxynitrite-mediated radicals were shown to be reactive towards baicalein and the bimolecular rate constants were 2.5 × 107 and 3 × 108 dm3 mol-1 s-1 for ·NO2 and CO3·- radicals, respectively. In conclusion, our results revealed the potential of baicalein in protecting mitochondrial membrane against oxidative damage induced by the four different agents. We propose that the protective effect is mediated via scavenging of primary and secondary radicals generated during oxidative stress.

The L-Arginine-Nitric Oxide-Peroxynitrite pathway (LANOP pathway): does it protect or worsen the course of Chagas disease? / Vía L-Arginina-Óxido Nítrico-Peroxinitrito (Vía-LAONP): ¿protege o empeora el curso de la enfermedad de Chagas?

Many different immunological processes have been described in the Chagas infection, some of them associated with the Chagas disease. In this scenario, the L-Arginine-Nitric oxide (NO) – Peroxynitrite (NOOO-) pathway (LANOP pathway) appears as an essential component of that process. The relationship is well known between cytokines that can induce Oxide Nitric Synthase (iNOS) genes, such as TNF-a and IFN-g, and other molecules that can inhibit their expression (TGF-b, IL-10 and others), which are involved in both acute and chronic stages of the disease pathogenesis. However the participation of the LANOP pathway seems complex, given that evidence shows different roles for it during the course of the infection. In this article, the authors review the immunological and inflammatory response leading to the activation of the LANOP pathway during the Chagas infection, and the role this via plays, including different effects, protector or deleterious, observed in parallel during the development of the infection.

En la infección chagásica se han descrito diversos procesos inmunológicos, algunos de los cuales se han asociado con la enfermedad. En este escenario, aparece la vía L-Arginina-Óxido Nítrico (NO) - Peroxinitrito (ONOO-) (Vía-LAONP) como un componente esencial de estos procesos. Se conoce la asociación entre citocinas inductoras de los genes de la Sintasa del Óxido Nítrico (iNOS) tales como TNF-a e IFN-g, así como moléculas que inhiben su expresión (TGF-b e IL-10 entre otras), involucradas en la patogénesis tanto de la fase aguda como crónica de la enfermedad. No obstante, la participación de la vía-LAONP parece ser compleja, una vez que las evidencias señalan papeles diferentes de ésta durante el curso de la infección. Por tanto, los autores revisan la respuesta inmunológica e inflamatoria que da lugar a la activación de la vía-LAONP durante la infección chagásica, y el papel que ésta desempeña, incluyendo efectos diversos, tanto protectores como deletéreos, que han sido observados en paralelo durante el curso de la misma.