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1.
Soybean lipoxygenase-mediated cooxidation of reduced glutathione (GSH) and concomitant superoxide generation was examined. The oxidation of GSH was dependent on the concentration of linoleic acid (LA), GSH, and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 0.42 mM LA, 2 mM GSH, and 50 pmole of enzyme/mL. The GSH oxidation was linear up to 10 minutes and exhibited a pH optimum of 9.0. The reaction displayed a Km of 1.49 mM for GSH and Vmax of 1.35 ± 0.02 μmoles/min/nmole of enzyme. Besides LA, arachidonic and γ-linolenic acids also supported the lipoxygenase-mediated GSH oxidation. Hydrogen peroxide and 13-hydroperoxylinoleic acid supported GSH cooxidation, but to a very limited extent. Oxidized glutathione (GSSG) was identified as the major product of the reaction based on the depletion of nicotinamide-adenine dinucleotide 3′-phosphate (NADPH) in the presence of glutathione reductase. The GSH oxidation was accompanied by the reduction of ferricytochrome c, which can be completely abolished by superoxide dismutase (SOD), suggesting the generation of superoxide anion radicals. Under optimal conditions, the rate of superoxide generation (measured as the SOD-inhibitable reduction of ferricytochrome c) was 10 ± 1.0 nmole/min/nmole of enzyme. These results clearly suggest that lipoxygenase is capable of oxidizing GSH to GSSG and simultaneously generating superoxide anion radicals, which may contribute to oxidative stress in cells under certain conditions.  相似文献   

2.
The antioxidant capacity of human plasma was determined by following the oxidation kinetics of the lipid-soluble fluorescent marker BODIPY using 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) as the lipophilic radical initiator. The results are expressed as a total antioxidant performance (TAP) value based on the inhibition of BODIPY oxidation, as determined by the appearance of green fluorescence, with respect to a control sample (phosphatidylcholine with or without delipidized human serum). The suitability of the assay was evaluated on the basis of its precision, reproducibility, and specificity. The intra- and interassay coefficients of variation both were less than 5%. The addition of a representative substrate of plasma peroxidation, phosphatidylcholine, up to 750mug/ml did not induce significant changes in the TAP value. Also, BODIPY photooxidation was not observed during the experimental time course (220min). The TAP values of 6 plasma samples from healthy donors were measured and correlated with the main plasma water- and lipid-soluble antioxidants (uric acid and ascorbic acid, alpha-tocopherol, and carotenoids) and lipid profiles. Significant correlations were found between TAP and uric acid (R=0.97, P<0.05) and cholesterol-adjusted alpha-tocopherol (R=0.93, P<0.01). The results confirm that the TAP assay is suitable to measure the antioxidant activity of plasma antioxidants localized in both the lipophilic and hydrophilic compartments.  相似文献   

3.
This work investigated the oxidative injury to human red blood cells (RBCs) by the exposure to exogenous malondialdehyde (MDA), in a physiological environment. When a 10% RBC suspension was incubated in autologous plasma, in the presence of 50 λμM MDA, 30% of MDA entered into the cells. A time-course study showed that MDA caused early (30-120 λmin) and delayed (3-18 λh) effects. MDA caused a fast depletion of reduced glutathione, and loss of the glucose-6-phosphate dehydrogenase activity, followed by a decrease of HbO 2 . Accumulation of methemoglobin, and formation of small amounts of hemichrome were later evident. Also, an HbO 2 -derived fluorescent product was measured in the membrane. The redox unbalance was followed by structural and functional damage to the membrane, evident as the formation of conjugated diene lipid hydroperoxides, concurrent with a sharp accumulation of MDA, consumption of membrane vitamin E, and egress of K + ions. SDS--PAGE of membrane proteins showed formation of high molecular weight aggregates. In spite of the marked oxidative alterations, the incubation plasma prevented a substantial hemolysis, even after a 18 λh incubation. On the contrary, the exposure of RBCs to 50 λμM MDA in glucose-containing phosphate saline buffer, resulted in a 16% hemolysis within 6 λh. These results indicate that the exposure to MDA causes a rapid intracellular oxidative stress and potentiates oxidative cascades on RBCs, resulting in their dysfunction.  相似文献   

4.
Degenerative diseases such as cancer are induced by oxidative genetic damage. Antioxidants can scavenge reactive oxygen species, but to prevent disease, they must do so quickly, before the DNA bases are damaged. In the present study, a novel method was established for evaluating the potency of antioxidants employing 2'-deoxyguanosine as a target and 2,2'-azobis(2-amidinopropane) dihydrochloride as a reactive oxygen generator. The reaction formed one product linearly with time. This product was a novel 8-hydroperoxy-2'-deoxyguanosine (8-OOHdG). Using this system, 81 antioxidants occurring in our diet were assayed for activity to suppress the formation of 8-OOHdG by high-performance liquid chromatography (HPLC). The system was useful for the evaluation of antioxidative potency, compared to another method utilizing 1,1-diphenyl-2-picrylhydrazyl (DPPH). Further, it was enabled to examine the synergism of antioxidants. The formation of 8-OOHdG started only after the antioxidants had been consumed. Ascorbic acid, quercetin, and epigallocatechin gallate together delayed the formation by the sum total of the delay times of each factor alone. The proposed method is simple and easy, and can evaluate which dietary antioxidants inhibit reactive oxygen species more quickly than the DNA bases are damaged.  相似文献   

5.
Endotoxin lipopolysaccharide (LPS) and streptozotocin-induced diabetes are known to cause oxidative stress in vivo. There is some evidence that a sublethal dose of LPS provides protection against subsequent oxidative stress. Because of its wide use as a diabetogenic agent, this study was undertaken to determine if streptozotocin can likewise provide a protective effect against further oxidative stress in rats. Female Sprague–Dawley rats were given streptozotocin (50 mg/kg intraperitoneally once) prior to exposure to either bacterial endotoxin from Salmonella abortus equii (5 mg/kg intraperitoneally) or three additional daily doses of streptozotocin (50 mg/kg intraperitoneally). One week after LPS or streptozotocin treatments, oxidative stress was determined by measuring changes in antioxidant activity (glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, glutathione S-transferase, and γ-glutamyltranspeptidase) and in concentrations of glutathione, nitrite, and thiobarbituric acid reactants in liver, kidney, intestine, and spleen. High levels of some antioxidants in the LPS-control and streptozotocin-control rats, in contrast to normal levels found in diabetes + LPS and multidose-streptozotocin rats, suggest that streptozotocin, like LPS, may confer a protective effect against subsequent oxidative stress. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 143–149, 1998  相似文献   

6.
The aim of this study was to investigate the influences of different stress models on the antioxidant status and lipid peroxidation (LPO) in erythrocytes of rats. Swiss-Albino female rats (3 months old) were used in this study. Rats were randomly divided into the following four groups; control group (C), cold stress group (CS), immobilization stress group (IS) and cold+immobilization stress group (CS+IS). Control group was kept in an animal laboratory (22 ±2°C). Rats in CS group were placed in cold room (5°C) for 15 min/day for 15 days. Rats in IS group were immobilized for 180 min/day for 15 days. Rats in CS+IS group were exposed to both cold and immobilization stresses for 15 days. At the end of experimental periods, the activities of glucose-6-phosphate dehydrogenase (G-6-PD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and concentration of reduced glutathione (GSH) were measured. LPO was determined by measuring the contents of thiobarbituric acid-reactive substances (TBARS). Cu,Zn-SOD activity and TBARS concentration were increased after cold and immobilization stresses, but CAT and GSH-Px activities and GSH levels were decreased. Immobilization stress decreased the activity of G-6-PD. The activities of G-6-PD, CAT and GSH-Px, and the level of GSH were lower in CS+IS group than in the control group. Cu,Zn-SOD activity and TBARS levels were increased in CS+IS group when compared with the control group. From these findings, three stress models are thought to cause oxidative stress.  相似文献   

7.
We investigated the effects of vitamin E and topiramate (TPM) administrations on pentylentetrazol (PTZ)–induced blood and brain toxicity in rats. Forty rats were randomly divided into five equal groups. The first and second groups were used for the control and PTZ groups, respectively. Fifty or 100 mg TPM were administered to rats constituting the third and fourth groups for 7 days, respectively. The TPM and vitamin E combination was given to animals in the fifth group. At the end of 7 days, all groups except the first received a single dose of PTZ. Blood and brain samples were taken at 3 hrs after PTZ administration. Lipid peroxidation levels of plasma, erythrocyte, brain cortex and brain microsomal fraction; nitric oxide levels of serum; and the number of spikes and epileptiform discharges of the EEG were increased by PTZ administration. Plasma and brain vitamin E concentration, erythrocyte glutathione peroxidase (GSH-Px) activity and latency to first spike of the EEG were decreased by PTZ. Plasma lipid peroxidation levels in the third group and plasma and erythrocyte lipid peroxidation levels in the fifth group were decreased compared to the second group, whereas brain vitamin C, vitamin E, erythrocyte GSH-Px and reduced glutathione (GSH) values increased in the fifth group. Brain microsomal GSH levels and EEG records in the third, fourth and fifth groups were restored by the TPM and vitamin E treatment. In conclusion, TPM and vitamin E seems to have protective effects on PTZ-induced blood and brain toxicity by inhibiting free radicals and supporting the antioxidant redox system.  相似文献   

8.
The production of reactive oxygen species (ROS) by aquatic organisms in response to stressful factor is a common feature in aquatic environment. Thus, the evaluation of parameters related to oxidative stress in fish, in first instance those showing the redox potential, is notable to characterize the environmental conditions.Short-term storage of samples is essential when blood must be transported from collection sites to laboratories. Therefore, excessive delays in processing might compromise the reliability of results. The aim of the present study was to assess the effect of short-term storage time at +4 °C on total oxidant status (TOS), total antioxidant capacity (TAC) and oxidative stress index (OSI) in flathead mullet (Mugil cephalus) serum. After blood collection, all sample were analyzed and the assays were repeated after 24, 48 and 72 h from sampling. results showed a significant effect of short-term storage on TOS and TAC (P  0.0001), while the statistical significance of the linear regression study for these parameters was reduced as consequence of storage. These results highlight that the activities of oxidants and antioxidants in flathead mullet serum change during short-term storage at 4 °C and should be assesses as soon as possible from collection.  相似文献   

9.
The antioxidant activities of 5-hydroxyoxindole (1) and newly synthesized 3,5-dihydroxy-3-phenacyl-2-oxindole derivatives against rat liver microsome/tert-butylhydroperoxide system-induced lipid peroxidation and hydrogen peroxide-induced intracellular oxidative stress were investigated. Compound 1 and its derivatives showed significant suppression of lipid peroxidation and an intracellular oxidative stress. The effects of the more lipophilic derivatives tended to be greater than that of the original compound 1. The cytotoxicity of all of the oxindole derivatives on human promyelocytic leukemia HL60 cells was lower than that of 2,6-di(tert-butyl)-4-hydroxytoluene (BHT), a widely used phenolic antioxidant. These results show that compound 1 and its 3-substituted derivatives could be good lead candidates for future novel antioxidant therapeutics.  相似文献   

10.
Recent findings in our laboratory suggested that in citrus cells the salt induction of phospholipid hydroperoxide glutathione peroxidase, an enzyme active in cellular antioxidant defense, is mediated by the accumulation of hydroperoxides. Production of hydroperoxides occurs as a result of non-enzymatic auto-oxidation or via the action of lipoxygenases (LOXs). In an attempt to resolve the role of LOX activity in the accumulation of peroxides we analyzed the expression of this protein under stress conditions and in cells of Citrus sinensis L. differing in sensitivity to salt. Lipoxygenase expression was induced very rapidly only in the salt-tolerant cells and in a transient manner. The induction was specific to salt stress and did not occur with other osmotic-stress-inducing agents, such as polyethylene glycol or mannitol, or under hot or cold conditions, or in the presence of abscisic acid. The induction was eliminated by the antioxidants dithiothreitol and kaempferol, thus once more establishing a correlation between salt and oxidative stresses. Analyses of both in vitro and in vivo products of LOX revealed a specific 9-LOX activity, and a very fast reduction of the hydroperoxides to the corresponding hydroxy derivatives. This suggests that one of the metabolites further downstream in the reductase pathway may play a key role in triggering defense responses against salt stress. Received: 3 February 2000 / Accepted: 13 June 2000  相似文献   

11.
Carbonylation, an oxidative modification of the amino group of arginine and lysine residues caused by reactive oxygen species, has emerged as a new type of oxidative damage. Protein carbonylation has been shown to exert adverse effects on various protein functions. Recently, the role of food components in the attenuation of oxidative stress has been the focus of many studies. Most of these studies focused on the chemical properties of food components. However, it is also important to determine their effects on protein functions via post-translational modifications. In this study, we developed a novel procedure for evaluating the antioxidant capacity of food components. Hydrogen peroxide (H2O2)-induced protein carbonylation in HL-60 cells was quantitatively analyzed by using fluorescent dyes (Cy5–hydrazide dye and IC3–OSu dye), followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and fluorescence determination. Among a panel of food components tested, quinic acid, kaempferol, saponin, squalene, trigonelline, and mangiferin were shown to be capable of suppressing protein carbonylation in HL-60 cells. Our results demonstrated that this fluorescence labeling/SDS–PAGE procedure allows for the detection of oxidative stress-induced protein carbonylation with high sensitivity and quantitative accuracy. This method should be useful for the screening of new antioxidant food components as well as the analysis of their suppression mechanism.  相似文献   

12.
4-Schiff base-7-benzyloxy-coumarins 5a(1)-5h(2) and its derivative 6 were designed and synthesized based on the 7-benzyloxy-coumarin structure as novel antioxidants. The in vitro antioxidant activities screening revealed that 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of compounds 5b(1), 5d(1), 5f(1), 5f(2), 5g(1) and 5g(2), and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) cation (ABTS(+)) radical scavenging activities of compounds 5a(1), 5b(1), 5c(1), 5c(2), 5d(1), 5e(1), 5e(2), 5f(2), 5g(1), 5g(2) and 5h(1) were better than that of the commercial antioxidant butylated hydroxytoluene (BHT), while the superoxide anion radical scavenging activities of 5a(2) and 5g(2) were stronger than that of the commercial antioxidant butylated hydroxyanisole (BHA), and the hydroxyl radical scavenging activity of 5e(1) was much better than that of the common antioxidant ascorbic acid.  相似文献   

13.
14.
NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.  相似文献   

15.
The protective effect of fruits and vegetables against cancer is well established. It is believed that this effect is mediated by antioxidants and decreased oxidative damage to DNA. However, the identity of the antioxidant(s) responsible is not clear. Moreover, a potentially damaging pro-oxidant effect of some antioxidants has been reported. In this study the ex vivo effects of several dietary antioxidants, including quercetin, various catechins, ascorbic acid and &#102 -tocopherol, were investigated, at concentrations up to 200 &#117 &#119 M, using the single cell gel electrophoresis (comet) assay for DNA damage. Lymphocytes from three healthy subjects were pre-incubated with these antioxidants, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 &#117 min exposure to hydrogen peroxide (final concentrations H 2 O 2 : 30, 45, or 60 &#117 &#119 M). Results using this ex vivo cellular assay showed protection by some antioxidants (quercetin, caffeic acid), no effect by some (catechin, epicatechin, catechin gallate, epicatechin gallate) and an apparently damaging effect by others (epigallocatechin, epigallocatechin gallate). Damage may have been caused by production of H 2 O 2 from these polyphenolics. Neither ascorbic acid nor &#102 -tocopherol protected or damaged DNA. Further study of the role of quercetin and caffeic acid in DNA protection is needed.  相似文献   

16.
Non-enzymatic glycation, as the chain reaction between reducing sugars and the free amino groups of proteins, has been shown to correlate with severity of diabetes and its complications. Cyperus rotundus (Cyperaceae) is used both as a food to promote health and as a drug to treat certain diseases. In this study, considering the antioxidative effects of C. rotundus, we examined whether C. rotundus also protects against protein oxidation and glycoxidation. The protein glycation inhibitory activity of hydroalcoholic extract of C. rotundus was evaluated in vitro using a model of fructose-mediated protein glycoxidation. The C. rotundus extract with glycation inhibitory activity also demonstrated antioxidant activity when a ferric reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays as well as metal chelating activity were applied. Fructose (100 mM) increased fluorescence intensity of glycated bovine serum albumin (BSA) in terms of total AGEs during 14 days of exposure. Moreover, fructose caused more protein carbonyl (PCO) formation and also oxidized thiol groups more in glycated than in native BSA. The extract of C. rotundus at different concentrations (25–250 μg/ml) has significantly decreased the formation of AGEs in term of the fluorescence intensity of glycated BSA. Furthermore, we demonstrated the significant effect of C. rotundus extract on preventing oxidative protein damages including effect on PCO formation and thiol oxidation which are believed to form under the glycoxidation process. Our results highlight the protein glycation inhibitory and antioxidant activity of C. rotundus. These results might lead to the possibility of using the plant extract or its purified active components for targeting diabetic complications.  相似文献   

17.
We examined the contribution of apoptosis- and oxidative stress-associated genes to apoptosis induction in trophoblast cells of human fetal membrane tissues undergoing apoptosis during in vitro incubation. RT-PCR analyses demonstrated an increased level of HO-1, Mn-SOD, Cox-2, iNOS, TNFalpha, TNFR1, IL-1beta, IL-6, Bax, Bak, and Bad gene expression, while Bcl-2 mRNA expression level decreased. Western blot analyses demonstrated an increase in iNOS, Cox-2, and HO-1 protein levels; a decrease in pro-caspase-3 and 9, proform-PARP, and Apaf-1 protein levels; a leakage of cytochrome c from the mitochondria. An antioxidative reagent, general and selective Cox-2 inhibitors, and an iNOS inhibitor suppressed in vitro progression of the apoptosis. Furthermore, an NO donor reagent induced apoptosis in primary cultured trophoblast cells. Therefore, we concluded that the induction of apoptosis in the smooth chorion trophoblasts is mediated through oxidative stress induction followed by mitochondria damage, suggesting that iNOS and Cox-2 play an important role in the apoptosis induction in trophoblasts of human fetal membrane tissues.  相似文献   

18.
In this work, we evaluated the antioxidant properties of the eight novel silybin analogues for their capacity to scavenge free radicals including superoxide anion radicals and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals in vitro. Compound 7d demonstrated an excellent antioxidant effect in scavenging superoxide anion free radical with an IC50 value of 26.5 μM, while the IC50 of quercetin (the reference compound) was 38.1 μM. Compounds 7b, 7e, 7h showed certain scavenging activities for both types of free radicals.  相似文献   

19.
New chromeno carbamodithioates (7a-i), have been synthesized from 2, 3-dimethyl-7-(oxiran-2-ylmethoxy)-4H-chromen-4-one (5), carbondisulphide and commercially available acyclic and cyclic secondary amines in acetonitrile with good to excellent yields. The free radical scavenging activity of novel chromone-carbamodithioate analogues was quantitatively estimated by spectrophotometric method. Whereas, molecular docking studies were performed with the active site of cyclooxygenase-2 to identify hydrogen bonding, hydrophobic and ionic interactions between protein and ligands. The compounds 7g and 7h demonstrated potent antioxidant activity with IC50 of 1.405 ± 0.019 mM and 1.382 ± 0.35 mM respectively compared to Ascorbic acid.  相似文献   

20.
The peroxisome proliferators perfluorooctanoic acid (PFOA; 0.02% w/w), perfluorodecanoic acid (PFDA; 0.02%, w/w), nafenopin (0.125%, w/w), clofibrate (0.5%, w/w), and acetylsalicylic acid (ASA; 1%, w/w) were administered to male C57 BL/6 mice in their diet for two weeks. Parameters for Fe3+ ADP, NADPH or ascorbic acid-initiated lipid peroxidation in vitro were measured. Approximately a twofold increase in susceptibility to lipid peroxidation was obtained for all the peroxisome proliferators tested. Cotreatment of mice with the peroxisome proliferator ASA (1%, w/w) and a catalase inhibitor, 3-amino-1,2,4-triazole (AT; 0.4%, w/w) for 7 days resulted in little inhibition of peroxisome proliferation, an elevated level of H2O2 in vivo, and total inhibition of the increased susceptibility to lipid peroxidation in vitro. No increase in lipid peroxidation in vivo was observed. Certain antioxidant enzymes (DT-diaphorase, superoxide dismutase, glutathione transferase, glutathione peroxidase, and glutathione reductase) and components (ubiquinone and α-tocopherol) were also measured. The results showed that there was some induction of these antioxidant enzymes and components by ASA or aminotriazole, except for glutathione peroxidase and superoxide dismutase, which were inhibited. The possible involvement of oxidative stress in the carcinogenicity of peroxisome proliferators is discussed.  相似文献   

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