Tempol protection of spinal cord mitochondria from peroxynitrite-induced oxidative damage

Peroxynitrite (PN)-mediated mitochondrial dysfunction has been implicated in the secondary injury process after traumatic spinal cord injury (SCI). This study investigated the detrimental effects of the PN donor SIN-1 (3-morpholinosydnonimine) on isolated healthy spinal cord mitochondria and the protective effects of tempol, a catalytic scavenger of PN-derived radicals. A 5 min exposure of the mitochondria to SIN-1 caused a dose-dependent decrease in the respiratory control ratio (RCR) that was accompanied by significant increases in complex I-driven states II and IV respiration rates and decreases in states III and V. These impairments occurred together with an increase in mitochondrial protein 3-nitrotyrosine (3-NT), but not in lipid peroxidation (LP)-related 4-hydroxynonenal (4-HNE). Tempol significantly antagonized the respiratory effects of SIN-1 in parallel with an attenuation of 3-NT levels. These results show that the exogenous PN donor, SIN-1, rapidly causes mitochondrial oxidative damage and complex I dysfunction identical to traumatic spinal cord mitochondrial impairment and that this is mainly due to tyrosine nitration. Consistent with that, the protection of mitochondrial respiratory function by tempol is associated with a decrease in 3-NT levels in mitochondrial proteins also similar to the previously reported antioxidant actions of tempol in traumatically-injured spinal cord mitochondria.     

Antioxidant-mediated protective effect of potato peel extract in erythrocytes against oxidative damage

Potato peels are waste by-product of the potato processing industry. They are reportedly rich in polyphenols. Our earlier studies have shown that extracts derived from potato peel (PPE) possess strong antioxidant activity in chemical and biological model systems in vitro, attributable to its polyphenolic content. The main objective of this study was to investigate the ability of PPE to protect erythrocytes against oxidative damage, in vitro. The protection rendered by PPE in erythrocytes was studied in terms of resistance to oxidative damage, morphological alterations as well as membrane structural alterations. The total polyphenolic content in PPE was found to be 3.93 mg/g powder. The major phenolic acids present in PPE were predominantly: gallic acid, caffeic acid, chlorogenic acid and protocatechuic acid. We chose the experimental prooxidant system: FeSO₄ and ascorbic acid to induce lipid peroxidation in rat RBCs and human RBC membranes. PPE was found to inhibit lipid peroxidation with similar effectiveness in both the systems (about 80–85% inhibition by PPE at 2.5 mg/ml). While PPE per se did not cause any morphological alteration in the erythrocytes, under the experimental conditions, PPE significantly inhibited the H₂O₂-induced morphological alterations in rat RBCs as revealed by scanning electron microscopy. Further, PPE was found to offer significant protection to human erythrocyte membrane proteins from oxidative damage induced by ferrous–ascorbate. In conclusion, our results indicate that PPE is capable of protecting erythrocytes against oxidative damage probably by acting as a strong antioxidant.     

Glycyrrhizae Radix attenuates peroxynitrite-induced renal oxidative damage through inhibition of protein nitration

We investigated the protective effects of Glycyrrhizae Radix extract against peroxynitrite (ONOO^-)-induced oxidative stress under in vivo as well as in vitro conditions. The extract showed strong ONOO^- and nitric oxide (NO) scavenging effects under in vitro system, in particular higher activity against ONOO^-. Furthermore, elevations of plasma 3-nitrotyrosine levels, indicative of in vivo ONOO^- generation and NO production, were shown using a rat in vivo ONOO^--generation model of lipopolysaccharide injection plus ischemia-reperfusion. The administration of Glycyrrhizae Radix extract at doses of 30 and 60 mg/kg body weight/day for 30 days significantly reduced the concentrations of 3-nitrotyrosine and NO and decreased inducible NO synthase activity. In addition, the nitrated tyrosine protein level and myeloperoxidase activity in the kidney were significantly lower in rats given Glycyrrhizae Radix extract than in control rats. However, the administration of Glycyrrhizae Radix extract did not result in either significant elevation of glutathione levels or reduction of lipid peroxidation in renal mitochondria. Moreover, the in vivo ONOO^- generation system resulted in renal functional impairment, reflected by increased plasma levels of urea nitrogen and creatinine, whereas the administration of Glycyrrhizae Radix extract reduced these levels significantly, implying that the renal dysfunction induced by ONOO^- was ameliorated. The present study suggests that Glycyrrhizae Radix extract could protect the kidneys against ONOO^- through scavenging ONOO^- and/or its precursor NO, inhibiting protein nitration and improving renal dysfunction caused by ONOO^-.     

Vanadate protects human neuroblastoma SH-SY5Y cells against peroxynitrite-induced cell death

We investigated the effect of vanadate, a tyrosine phosphatase inhibitor, on cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Vanadate prevented cell death induced by 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor; whereas SIN-1-induced cell death was not prevented by neither okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, nor cyclosporin A, an inhibitor of serine/threonine phosphatase 2B. Vanadate did not prevent cell death induced by N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine, a nitric oxide donor. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), did not block the protective effect of vanadate, suggesting that the protective effect of vanadate is independent on PI3-kinase. Vanadate increased tyrosine phosphorylation of several proteins including the focal adhesion protein p130 Crk-associated substrate (p130(cas)). By the treatment with SIN-1, the endogenous association of p130(cas) and Crk was disrupted, and the association was restored by vanadate treatment. These results suggest that disruption of tyrosine phosphorylation signaling may be critical for peroxynitrite-induced cell death, and that vanadate prevents cell death at least in part through the enhancement in tyrosine phosphorylation of the proteins including p130(cas).     

Quercetin metabolites and protection against peroxynitrite-induced oxidative hepatic injury in rats

Quercetin has strong antioxidant potency. Quercetin-3′-O-sulphate (Q3′S) and quercetin-3-O-glucuronide (Q3GA) are the main circulating metabolites after consumption of quercetin-O-glucoside-rich diets by humans. However, information about how these quercetin metabolites function in vivo is limited. Hence, this study evaluated the efficacy of Q3′S and Q3GA for the protection of oxidative injury using in vitro and in vivo experiments. Peroxynitrite-mediated hepatic injury in rats was induced by administration of galactosamine/lipopolysaccharide (GalN/LPS). Twenty-four hours after GalN/LPS treatment, plasma ALT and AST levels δ increased significantly. However, pretreatment with 4^G-α-D-glucopyranosyl rutin, a quercetin glycoside (30 mg/kg body weight), prevented these increases and reduced nitrotyrosine formation, indicating that consumption of quercetin glycosides prevent oxidative hepatotoxicity. Moreover, physiological levels of Q3′S and Q3GA (1 µM) effectively prevented peroxynitrite-induced nitrotyrosine formation in human serum albumin in in vitro experiments. These findings indicate peroxynitrite-induced oxidative hepatotoxicity is protected by the in vivo metabolites of quercetin, Q3′S and Q3GA.     

Lipid peroxidative damage on cadmium exposure and alterations in antioxidantsystem in rat erythrocytes: A study with relation to time

Cadmium induced lipid peroxidation (LPO) and the activity of antioxidantenzymes after the administration of a single dose of CdCl 2 (0.4 mg kg body wt, ip) was studied in rat erythrocytes.Cd intoxication increased erythrocyte LPO along with a decrease insuperoxide dismutase (SOD) up to three days of Cd treatment. Thedecrease in erythrocyte catalase (CAT) activity was marked within9 h of Cd intoxication. After three days of Cd treatment, LPOdecreased towards normal, along with an increase in erythrocyteSOC and CAT activity. Blood glutathione (GSH) decreased significantlywithin 24 h of Cd treatment, followed by an increase towards normal.Erythrocyte glutathione S-transferase (GST) activity increased up to10 days of Cd intoxication, probably in an attempt to reduce Cd toxicity.Serum glutamate pyruvate transaminase (SGPT), serum alkaline phosphatase(SALP) and serum bilirubin increased up to 10 days of Cd intoxication.Blood urea increased significantly up to three days, followed by a decreasetowards normal. The results show that Cd induced LPO was associated with adecrease in antioxidant enzymes and GSH in erythrocytes; as these antioxidantsincrease in erythrocytes with recovery from Cd intoxication, the Cd inducedLPO reversed towards normal. The increase in the SGPT, SALP and serum bilirubincorrelated with LPO. The results suggest that Cd intoxication induces oxidativestress and alters the antioxidant system, resulting in oxidative damage torat erythrocytes. © Rapid Science 1998     

Peroxynitrite activates K+-Cl- cotransport in human erythrocytes

Peroxynitrite was found to induce the release of K+ via the Na+/Cl- cotransport system, as do other oxidants. Since peroxynitrite is formed in vivo, its presence could contribute to a pathological dehydration of red blood cells.     

The effects of rosiglitazone on oxidative stress and lipid profile in left ventricular muscles of diabetic rats

We investigated the effect of rosiglitazone (RSG), a high-affinity ligand for the peroxisome proliferator-activated receptor gamma which mediates insulin-sensitizing actions, on the lipid profile and oxidative status in streptozotocin (STZ)-induced Type 2 diabetes mellitus (DM) rats. Wistar albino male rats were randomly divided into an untreated control group (C), a C + RSG group which was treated with RSG (4 mg kg(-1)) two times a day by gavage, a diabetic group (D) that was treated with a single intraperitoneal injection of STZ (45 mgkg(-1)), D + RSG group which were treated with RSG two times a day by gavage, respectively. Lipid profiles, HbA(1c) and blood glucose levels in the circulation and malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) levels in left ventricular muscle were measured. Treatment of D rats with RSG resulted in a time-dependent decrease in blood glucose. We found that the lipid profile and HbA(1c) levels in D + RSG group reached the C rat values at the end of the treatment period. There was a statistically significant difference between the C + RSG and C groups in 3-NT levels. In group D, 3-NT and MDA levels were found to be increased when compared with C, C + RSG and D + RSG groups. In the D + RSG group, MDA levels were found to be decreased when compared with C and C + RSG. Our study suggests that the treatment of D rats with RSG for 8 weeks may decrease the oxidative/nitrosative stress in left ventricular tissue of rats. Thus in diabetes-related vascular diseases, RSG treatment may be cardioprotective.     

Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo

The effects of streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, and cefodizime on the enzyme activity of glutathione reductase (GR) were studied using human and rat erythrocyte GR enzymes in in vitro and in vivo studies, respectively. The enzyme was purified 5,342-fold from human erythrocytes in a yield of 29% with 50.75?U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies, and rat erythrocyte hemolysate was used in the in vivo studies. In the in vitro studies, I₅₀ and K_i values were 12.179?mM and 6.5123±4.1139?mM for cefotaxime, and 1.682?mM and 0.7446±0.2216?mM for cefodizime, respectively, showing the inhibition effects on the purified enzyme. Inhibition types were noncompetitive for cefotaxime and competitive for cefodizime. In the in vivo studies, 300?mg/kg cefotaxime and 1000?mg/kg cefodizime when administered to rats inhibited enzyme activity during the first 2?h (p<0.01). Cefotaxime led to increased enzyme activity at 4?h (p<0.05), but neither cefotaxime nor cefodizime had any significant inhibition or activation effects over 6?h (p>0.05).     

Genetic control of amino acid transport in sheep erythrocytes

An inherited amino acid transport deficiency results in low concentrations of glutathione (GSH) in the erythrocytes of certain sheep. Earlier studies based on phenotyping according to GSH concentrations indicated that the gene Tr ^H, which controls normal levels of GSH, behaves as if dominant or incompletely dominant to the allele Tr ^h, which controls the GSH deficiency. The present paper shows that when sheep are classified according to amino acid transport activity, the Tr ^H gene behaves as if codominant to Tr ^h. Erythrocytes from sheep homozygous for the Tr ^H gene exhibit rapid saturable l-alanine influx (apparent K _m ,21.6mm; V _max, 22.4 mmol/liter cells/hr). Cells from sheep homozygous for the Tr ^h gene exhibit slow nonsaturable l-alanine uptake (0.55 mmol/liter cells/hr at 50mm extracellular l-alanine). Cells from heterozygous sheep show saturable l-alanine uptake with a diminished V _max (apparent K _m, 19.1mm; V _max, 12.7 mmol/liter cells/hr). These erythrocytes have a significantly lower GSH concentration than cells from Tr ^H, Tr^H sheep but similar intracellular levels of dibasic amino acids.The authors are grateful to the M.R.C. for a Project Grant.     

牛角花齿蓟马为害对紫花苜蓿AsA、GSH含量及相关代谢酶活性的影响

为了明确非酶抗氧化物质抗坏血酸(AsA)、还原型谷胱甘肽(GSH)及相关代谢酶抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)在紫花苜蓿(Medicago sativa L.)对牛角花齿蓟马Odontothrips loti Haliday为害的抗性中的作用,测定了不同牛角花齿蓟马虫口密度下抗、感蓟马苜蓿无性系R-1、I-1的AsA、GSH含量及APX、GR活性的变化。结果表明:受牛角花齿蓟马为害后,R-1无性系在低虫口密度(1、3头/枝条)下,AsA、GSH含量和GR活性均上升,在高虫口密度(5、7头/枝条)下,AsA含量和GR活性先升高后下降,GSH含量上升后保持稳定;I-1无性系的AsA、GSH含量先升高后下降,GR活性在为害后期呈上升趋势;R-1、I-1无性系的APX活性均先上升后下降,但R-1无性系APX活性的上升速率及下降速率小于I-1无性系。说明AsA、GSH含量及APX、GR活性的升高可能是紫花苜蓿对牛角花齿蓟马诱导抗性的一种表现,但I-1无性系对蓟马为害的应激反应滞后于R-1无性系。在牛角花齿蓟马为害后期,R-1无性系体内的AsA、GSH含量及APX、GR活性仍处于较高水平,也说明了R-1无性系对牛角花齿蓟马为害的抗性较I-1无性系强。     

Water stress-induced oxidative damage and antioxidant responses in micropropagated banana plantlets

Oxidative injury and antioxidant responses were investigated in two banana genotypes (Musa AAA Berangan and Musa AA Mas) subjected to 40 % PEG-induced water stress. PEG treatment resulted in oxidative injury, as expressed in increased lipid peroxidation and reduced membrane stability index, in both cultivars; however, greater oxidative injury was detected in Mas. Under PEG treatment, catalase activity and glutathione reductase activity were enhanced in both cultivars, but were higher in Mas. Ascorbate peroxidase activity was enhanced in Berangan under water stress, but was unaffected in Mas. Meanwhile, superoxide dismutase activity was inhibited in both cultivars under water stress, but higher activity was detected in Berangan. Higher ascorbate peroxidase and superoxide dismutase activities were associated with greater protection against water stress-induced oxidative injury.     

Peroxide-triggered erythrocytes haemolysis as a model for the study of oxidative damage in marine fishes

The haemolysis of sea bass Dicentrarchus labrax red blood cells (RBC) was initiated by tert -butyl-hydroperoxide (t-BHP). The onset of the haemolytic process was accelerated by increasing t-BHP concentration. This process was preceded by a drop in the RBC glutathione content followed by the production of lipid peroxidation products. Also t-BHP induced DNA fragmentation in RBC nuclei as measured by COMET assay. The addition of the antioxidant Trolox C® dose-dependently delayed the onset of both lipid peroxidation and haemolysis, and protected GSH stores against t-BHP-induced depletion. DNA fragmentation was also pre-vented by Trolox C®. These results indicate that t-BHP induces haemolysis in sea bass RBC through the induction of oxidative stress. Such a simple model could prove useful for both fundamental and applied studies on marine fish antioxidant mechanisms.     

Activity of glutathione peroxidase, glutathione reductase, and lipid peroxidation in erythrocytes in workers exposed to lead

The aim of this study was to estimate the activity of glutathione peroxidase (GPx), glutathione reductase (GR), and malondialdehyde (MDA) in erythrocytes in healthy male employees of zinc and lead steelworks who were occupationally exposed to lead over a long period of time (about 15 yr). Workers were divided into two subgroups: the first included employees with low exposure to lead (LL) (n=75) with blood lead level PbB=25–40 μg/dL and the second with high exposure to lead (HL) (n=62) with PbB over 40 μg/dL. Administration workers (n=35) with normal levels of PbB and zinc protoporphyrin in blood (ZPP) in blood were the control group. The activity of GPx significantly increased in LL when compared to the control group (p<0.001) and decreased when compared to the HL group (p=0.036). There were no significant changes in activity of GR in the study population. MDA erythrocyte concentration significantly increased in the HL group compared to the control (p=0.014) and to the LL group (p=0.024). For the people with low exposure to lead (PbB=25–40 μg/dL), the increase of activity of GPx by about 79% in erythrocytes prevented lipid peroxidation and it appears to be the adaptive mechanism against the toxic effect of lead. People with high exposure to lead (with PbB over 40 μg/dL) have shown an increase in MDA concentration in erythrocytes by about 91%, which seems to have resulted from reduced activity of GPx and the lack of increase in activity of GR in blood red cells.     

过氧亚硝基阴离子诱导离体兔肺动脉舒张反应的特性

实验用离体血管环张力测定技术,观察过氧亚硝基阴离子（ONOO）对离体预收缩的兔肺动脉的舒张反应、肺动脉内皮细胞对舒张反应的影响,并初步探讨其病理意义。结果显示,ONOO可剂量依赖性引起预收缩兔肺动脉舒张。分解的ONOO也有舒张作用,但其效应明显低于ONOO的作用,比较观察表明,ONOO的舒张效应显著低于硝普钠（SNP）和乙酰胆碱（ACh)。与内皮完整2的肺动脉比较,ONOO对去内皮肺动脉的舒张作用明显增加,剂量依赖性舒张反应曲线明显左移。肺动脉对ONOO重复作用时的舒张反应有递减趋势。在本实验条件下,ONOO舒张前后的肺动脉,对ACh的舒张反应无明差异。结果表明,ONOO对肺动脉的舒张作用较弱,此作用受到内皮细胞的抑制性调节并有快速去敏性。     

Selenium modifies glutathione peroxidase activity and glutathione concentration in mice exposed to ozone-provoked oxidative stress

The aim of this study was to show the direct effect of selenium on glutathione peroxidase (GSH-Px) activity and GSH/GSSG concentrations in 3- and 6-month-old mice. An ozone-oxygen mixture was used to provoke an oxygen stress. To measure the Se-effect mice were gavaged with sodium selenite. GSH-Px activity and total glutathione concentrations were determined in serum and in the postnuclear fraction of liver and lungs. Additionally glutathione concentrations were determined in whole blood. Both ozone and selenium, administered separately, reduced GSH-Px activity in lungs of 6-month-old animals, while in young mice an opposite effect of Se was observed. Ozone administered jointly with Se did not influence GSH-Px activity in 6-month-old mice, while in young, 3-month-old mice, a stimulatory effect in lungs was observed. There were no significant changes in GSH-Px activity in the liver of 6-month-old mice, but the stimulatory effect occurred in young mice treated with Se and Se & ozone jointly. In young mice, ozone (also ozone with Se) augmented glutathione concentrations. The response to ozone and selenium strictly depended on age and the antagonism between selenium and ozone was observed only in a few cases.     

Protective effect of glutathione and selenium against alloxan induced lipid peroxidation and loss of antioxidant enzymes in erythrocytes

Alloxan is a diabetogenic drug and is known to induce diabetes through generation of free radicals. The toxic oxygen species can be detoxified by antioxidant enzyme system and thus reduce the deleterious effect of lipid peroxidation. Erythrocytes exposed to alloxan induced lipid peroxidationin vivo as well asin vitro. Although alloxan treatment produced a deleterious effect on antioxidant enzymes, pretreatment with glutathione and selenium led to a recovery of the activities of superoxide dismutase and glutathione peroxidase. However, catalase activity increased on alloxan treatment. Alloxan reduced blood glucose level significantly within 60 min but thereafter a slow and steady rise was observed.     

Levels of selenium in plasma and glutathione peroxidase in erythrocytes and the risk of breast cancer

Plasma selenium and glutathione peroxidase in erythrocytes were analyzed in a case-control study encompassing 441 cases with breast cancer and 191 controls with benign breast disease. No difference in mean serum selenium level between cases and controls on supplementary selenium intake was seen. If only individuals without supplementary intake, 278 cases and 135 controls, were considered a preventive effect was found increasing with selenium level. This finding was significant among women 50 years old or more with Mantel-Haenszel odds ratio=0.16 for individuals with serum selenium >1.21 μmol/L. Also for subjects with serum selenium in the range 1.00–1.21 μmol/L a significant preventive effect was seen with odds ratio=0.38. For women under 50 years of age a nonsignificant preventive effect was seen. Glutathione peroxidase in erythrocytes did not correlate well with serum selenium and was not a marker for the risk of breast cancer.     

Studies on glutathione transport utilizing inside-out vesicle prepared from human erythrocytes

Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Line-weaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25°C, this transport activity was easily lost during the storage of vesicles at 4°C. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation of transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37°C, this transport activity was stable during storage of vesicles at 4°C for several days. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes.