A chromatographic tool for preparing combinatorial quinone–thiol conjugate libraries

Quinones are well established as key players in the production of reactive oxygen species within cellular environments. Many factors govern their cytotoxicity but most studies have been restricted to a few, core, derivatives. A new strategy for the in situ production of quinone derivatives has been developed such that libraries of diverse functionality can be rapidly created without recourse to extensive synthetic procedures. The approach relies upon nucleophilic addition by reduced thiol derivatives to the quinone core within a pre-culture assay mixture and provides a generic strategy that exploits the large reservoir of commercial thiols currently available. A readily accessible chromatographic method has been developed that allows the derivatisation process to be easily monitored and the purity of the resulting one pot preparation to be assessed. The viability of the combinatorial approach has been fully validated through comparison with a range of quinone-S-conjugates prepared using conventional bench synthesis. The latter have been fully characterised.     

Role of Endogenous Glutathione in the Oxidation of Dopamine

Abstract: Intrastriatal injection of dopamine causes the selective degeneration of tyrosine hydroxylase-containing terminals and an increase in content of cysteinylcatechols, an index of dopamine oxidation. Both of these effects can be attenuated by coadministration of antioxidants such as glutathione. Therefore, we investigated the effects of decreased endogenous glutathione on the neurotoxic potential of dopamine. We observed that pretreatment with either l -buthionine sulfoximine, a specific inhibitor of glutathione synthesis, or diethyl maleate, which forms adducts with glutathione, caused significant decreases in endogenous glutathione levels at the time of dopamine injection. Pretreatment with l -buthionine sulfoximine potentiated the formation of protein cysteinyl-dopamine after intrastriatal injection of 1.0 µmol of dopamine. We also observed that intrastriatal injection of 1.0 µmol of dopamine decreased striatal glutathione content in all pretreatment conditions. However, injection of a low dose (0.05 µmol of dopamine) caused a decrease in striatal glutathione levels only in the l -buthionine sulfoximine-pretreated rats. Diethyl maleate pretreatment was not effective in potentiating either cysteinyl-catechol formation or glutathione loss after dopamine injection. We conclude that dopamine contributes to cellular oxidative stress and that this can be exacerbated, or at least unmasked, if glutathione synthesis is compromised.     

Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism

The recent knowledge on mitochondria as the substantial source of reactive oxygen species, namely superoxide and hydrogen peroxide efflux from mitochondria, is reviewed, as well as nitric oxide and subsequent peroxynitrite generation in mitochondria and their effects. The reactive oxygen species formation in extramitochondrial locations, in peroxisomes, by cytochrome P450, and NADPH oxidase reaction, is also briefly discussed. Conditions are pointed out under which mitochondria represent the major ROS source for the cell: higher percentage of non-phosphorylating and coupled mitochondria, in vivo oxygen levels leading to increased intensity of the reverse electron transport in the respiratory chain, and nitric oxide effects on the redox state of cytochromes. We formulate hypotheses on the crucial role of ROS generated in mitochondria for the whole cell and organism, in concert with extramitochondrial ROS and antioxidant defense. We hypothesize that a sudden decline of mitochondrial ROS production converts cells or their microenvironment into a “ROS sink” represented by the instantly released excessive capacity of ROS-detoxification mechanisms. A partial but immediate decline of mitochondrial ROS production may be triggered by activation of mitochondrial uncoupling, specifically by activation of recruited or constitutively present uncoupling proteins such as UCP2, which may counterbalance the mild oxidative stress.     

Thioredoxin reductase-1 knock down does not result in thioredoxin-1 oxidation

The active site of thioredoxin-1 (Trx1) is oxidized in cells with increased reactive oxygen species (ROS) and is reduced by thioredoxin reductase-1 (TrxR1). The purpose of the present study was to determine the extent to which the redox state of Trx1 is sensitive to changes in these opposing reactions. Trx1 redox state and ROS generation were measured in cells exposed to the TrxR1 inhibitors aurothioglucose (ATG) and monomethylarsonous acid (MMA(III)) and in cells depleted of TrxR1 activity by siRNA knock down. The results showed that all three treatments inhibited TrxR1 activity to similar extents (90% inhibition), but that only MMA(III) exposure resulted in oxidation of Trx1. Similarly, ROS levels were elevated in response to MMA(III), but not in response to ATG or TrxR1 siRNA. Therefore, TrxR1 inhibition alone was not sufficient to oxidize Trx1, suggesting that Trx1-independent pathways should be considered when evaluating pharmacological and toxicological mechanisms involving TrxR1 inhibition.     

氧化应激与幽门螺杆菌感染相关性胃炎

幽门螺杆菌(Helicobacter pylori,H.pylori)是一种选择性定植于胃上皮细胞的革兰氏阴性菌,是一种广泛传染的病原菌,也是诱导产生慢性胃炎的主要因素之一。近年来研究表明幽门螺杆菌感染诱导机体产生氧化应激反应,并通过各种逃逸机制避免被杀灭。幽门螺杆菌能不断刺激中性粒细胞和巨噬细胞表达活性氧和活性氮,导致体内活性氧和活性氮的过度积累,致使细胞的凋亡和胃粘膜损伤的加剧,这是导致胃炎发生及加重的重要因素。本文对幽门螺杆感染引起氧化应激反应的研究进展作简要综述。     

A polyphenolic flavonoid glabridin: Oxidative stress response in multidrug-resistant Staphylococcus aureus

Glabridin a polyphenolic flavonoid from Glycyrrhiza glabra is known to possess several therapeutic properties. In the present study, we report for the first time the in vitro antibacterial activity (MIC values ranging from 3.12 to 25 μg/mL) of glabridin against multidrug-resistant clinical isolates of S. aureus by inducing oxidative stress. Increased levels of H₂O₂ and NO were observed in a dose-dependent manner after treatment of glabridin that further affected macromolecules such as DNA, lipids, and proteins. Surprisingly, glabridin was found to possess antioxidant properties when used at lower concentrations using three different methods including DPPH, FRAP, and SOD assays. These observations were further validated through the expression analysis of oxidative stress-responsive genes using qRT-PCR wherein glabridin was observed to up- and down-regulate these genes at lower and higher concentrations, respectively. In in vitro combination experiments, glabridin was found to reduce the MIC of different antibiotics such as norfloxacin, oxacillin, and vancomycin by up to 4-fold, while the MIC of glabridin itself was found to be reduced by up to 8-fold in the presence of antibiotics. A synergistic interaction was observed between norfloxacin and glabridin when used in combination against multidrug-resistant clinical isolate SA 4627 of Staphylococcus aureus at much lower concentrations, indicating the suitability of glabridin in combination therapy.     

Key roles of the Escherichia coli AhpC C-terminus in assembly and catalysis of alkylhydroperoxide reductase,an enzyme essential for the alleviation of oxidative stress

2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H₂O₂. We show that the E. coli AhpC (EcAhpC, 187 residues) forms a decameric ring structure under reduced and close to physiological conditions, composed of five catalytic dimers. Single particle analysis of cryo-electron micrographs of C-terminal truncated (EcAhpC_1 -172 and EcAhpC_1 -182) and mutated forms of EcAhpC reveals the loss of decamer formation, indicating the importance of the very C-terminus of AhpC in dimer to decamer transition. The crystallographic structures of the truncated EcAhpC_1 -172 and EcAhpC_1 -182 demonstrate for the first time that, in contrast to the reduced form, the very C-terminus of the oxidized EcAhpC is oriented away from the AhpC dimer interface and away from the catalytic redox-center, reflecting structural rearrangements during redox-modulation and -oligomerization. Furthermore, using an ensemble of different truncated and mutated EcAhpC protein constructs the importance of the very C-terminus in AhpC activity and in AhpC–AhpF assembly has been demonstrated.     

抗氧化系统研究新进展

氧化还原系统主要由活性氧、自由基、活性氧生成系统和抗氧化系统组成。大量的研究表明,氧化还原系统在机体多种生物学功能中发挥关键的调节作用。抗氧化系统主要包括酶类抗氧化剂和非酶类抗氧化剂。抗氧化系统一方面可以通过调节活性氧的水平影响各种生物学功能,另一方面各种酶类抗氧化剂和非酶类抗氧化剂本身也可以参与多种生化反应,调节机体功能。近年来的研究表明,机体内除了典型的抗氧化酶,如超氧化物歧化酶和过氧化氢酶等,还存在多种抗氧化新型抗氧化酶,如硫氧还蛋白、谷氧还蛋白和金属基质蛋白酶等。在本文中,我们将回顾近年来的一些文献,综述抗氧化系统的研究新进展,旨在为抗氧化系统的深入研究提供理论基础。     

Redox evaluation in sepsis model mice by the in vivo ESR technique using acyl-protected hydroxylamine

In vivo electron spin resonance (ESR) spectroscopy is a noninvasive technique that measures the oxidative stress in living experimental animals. The rate of decay of the ESR signal right after an injection of nitroxyl radical has been measured to evaluate the oxidative stress in animals, although the probe’s disposition could also affect this rate. Because the amount of probes forming the redox pair of hydroxyl amine and its corresponding nitroxyl radical was shown to be nearly constant in most organs or tissues 10 min after the injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in mice, we evaluated the oxidative stress in sepsis model mice induced by lipopolysaccharide (LPS) by intravenously injecting ACP as a precursor of redox probes. The in vivo ESR signal increased up to 7–8 min after the ACP injection and then decreased. Decay of the in vivo signal in LPS-treated mice was significantly slower than that in healthy mice, whereas no significant difference was observed in the rate of change in the total amount of redox probes in the blood and liver between these groups. ESR imaging showed that the in vivo signals observed at the chest and upper abdomen decayed slowly in LPS-treated mice. Suppression of the decay in LPS-treated mice was canceled by the administration of a combination of pegylated superoxide dismutase and catalase, or an inhibitor of nitric oxide synthase, or gadolinium chloride. These results indicate that the LPS-treated mouse is under oxidative stress and that reactive oxygen species, such as superoxide and peroxynitrite, related to macrophages are mainly involved in the oxidative stress.     

Oxidative stress and antioxidant defense responses of Etroplus suratensis to acute temperature fluctuations

Fishes are always exposed to various environmental stresses and the chances of succumbing to such stresses are of great physiological concern. Any change in temperature from the ambient condition can induce various metabolic and physiological changes in the body. The present study evaluates the effects of temperature induced stress on the antioxidant profile of Etroplus suratensis such as superoxide dismutase, catalase, glutathione peroxidase and lipid peroxidation. Fishes of same size were kept in a thermostatized bath at three different temperature regimes viz 16 °C, 27 °C (ambient temperature) and 38 °C for 72 h. These temperatures were selected based on the CT Max (Critical Thermal Maximum) and CT Min (Critical Thermal Minimum) exhibited by E. suratensis. Superoxide dismutase and catalase activity was found maximum in brain and muscle respectively during the 48th hour of exposure in fishes kept at 38 °C. At 16 °C the antioxidant response of glutathione peroxidase was maximum in muscles, whereas the lipid peroxidation rate was found to be high in gills compared to other tissues. The profound increase in the levels of oxidative stress related biomarkers indicate that the thermal stressors severely affected oxidative state of E. suratensis by the second day of experiment. Such down-regulation of redox state accompanied with the induction of oxidative stress cascade may lead to physiological damage in various tissues in fishes, in vivo. However current data indicate that a transition to low and high temperature environment from ambient condition severely affected the levels and profile of the antioxidant markers overtime in E. suratensis.     

A novel redox-based switch: LMW-PTP oxidation enhances Grb2 binding and leads to ERK activation

Low molecular weight-PTP has been reported as a redox-sensitive protein during both platelet-derived growth factor and integrin signalling. In response to oxidation the phosphatase undergoes a reversible inactivation, which in turn leads to the increase in tyrosine phosphorylation of its substrates and the properly executed anchorage-dependent proliferation program. Here, we report that an exogenous oxidative stress enhances LMW-PTP tyrosine phosphorylation, through oxidation/inactivation of the enzyme, thus preventing its auto-dephosphorylation activity. In particular, we observed a selective hyper-phosphorylation of Tyr132, that acts as a docking site for the adaptor protein Grb2. The redox-dependent enhancement of Grb2 recruitment to LMW-PTP ultimately leads to an improvement of ERK activation, likely triggering a prosurvival signal against the oxidant environment.     

体内游离谷氨酰胺的抗氧化作用

作为一种条件必需氨基酸,体内存在的丰富的游离谷氨酰胺具有重要的生理调节功能,尤其在抗氧化反应中发挥对机体的保护作用。游离谷氨酰胺可通过氧化供能、调节信号传递过程中凋亡酶的活性、抑制一氧化氮(NO)合成、参与肿瘤坏疽因子d(TNF-a)诱导的细胞毒性作用、参与合成谷胱甘肽,加速细胞增殖等生理过程减轻氧化压力。谷氨酰胺抗氧化作用的发现可以为癌症、移植手术、免疫功能低下等病人的治疗提供依据。     

Zinc deficiency differentially affects redox homeostasis of rice genotypes contrasting in ascorbate level

Zinc (Zn) deficiency is an important mineral disorder affecting rice production, and is associated with the formation of oxidative stress in plant tissue. In this study we investigated processes of oxidative stress formation as affected by ascorbate (AsA) in two pairs of contrasting rice genotypes: (i) two indica lines differing in field tolerance to Zn deficiency and AsA metabolism, i.e. RIL46 (tolerant) and IR74 (sensitive); (ii) the japonica wild-type Nipponbare (tolerant) and the AsA deficient TOS17 mutant line ND6172 (sensitive) having a 20–30% lower AsA level due to the knockout of an AsA biosynthetic gene (OsGME1). Plants were grown hydroponically under +Zn and −Zn conditions for 21 days and samples were investigated after 7, 14, and 21 days of treatment. Tissue Zn concentrations below 20 mg kg⁻¹ in the −Zn treatment induced the formation of visible symptoms of Zn deficiency from day 14 in all genotypes, but especially in the sensitive IR74. Significant increases in lipid peroxidation were observed in the leaves of the sensitive genotypes IR74 and ND6172, and in the roots of IR74, but not in the tolerant genotypes. At day 21, the tolerant genotypes RIL46 and Nipponbare had significantly higher AsA levels in both shoots and roots compared to the sensitive lines. Consistently, higher levels of hydrogen peroxide formation in leaves and roots of the sensitive genotypes were detected using staining methods. Differences in foliar hydrogen peroxide formation between IR74 and RIL46 became apparent on day 7 and between ND6172 and Nipponbare on day 14. Similarly, genotypic differences in hydrogen peroxide formation in the roots were seen on day 21. In conclusion, our data demonstrate that Zn deficiency leads to a redox imbalance in roots and shoots prior to the occurrence of visible symptoms, and that the antioxidant AsA plays an important role in maintaining the redox homeostasis under Zn deficiency.     

Promoted proliferation of an SOD-deficient mutant of Escherichia coli under oxidative stress induced by photoexcited TiO2

An SOD null mutant of Escherichia coli (IM303) and its wild-type strain (MM294) were cultivated with or without sublethal oxidative stress generated from photoexcited TiO2. Concerning maximum specific growth rate of the cells, mum, measured under various conditions, the mum value of IM303 cells increased notably in the presence of TiO2 illuminated with light (I = 12.5 W m(-2)), being about two times higher than that of the cells grown in the absence of TiO2 and light. The mum value of IM303 cells under the oxidative condition restored to a level comparable to that of wild-type MM294 cells, which coincided with the finding that the content of reactive oxygen species lowered in IM303 cells under the oxidative stress. Colony isolation was conducted to obtain the cells prevailing in the early culture phase of IM303 cells in the presence of TiO2 and light. It was found that the isolates exhibited the outgrowing properties with the increased mum values under both the conditions with and without TiO2 and light. It was also indicated that in the culture of typically selected isolate, the cells started to grow with a relatively short lag in a threonine-minus medium.     

Identification of 4-quinolone derivatives as inhibitors of reactive oxygen species production from human umbilical vein endothelial cells

Oxidative stress is widely recognized as being associated with a number of disorders, including metabolic dysfunction and atherosclerosis. A series of substituted 4-quinolone derivatives were prepared and evaluated as inhibitors of reactive oxygen species (ROS) production from human umbilical vein endothelial cells (HUVECs). One compound in particular, 2-({[4-(3-hydroxy-3-methylbutoxy)pyridin-2-yl]oxy}methyl)-3-methylquinolin-4(1H)-one (25b), inhibited ROS production from HUVECs with an IC(50) of 140 nM. This compound also exhibited low CYP2D6 inhibitory activity, high aqueous solubility, and good in vitro metabolic stability. An in vivo pharmacokinetic study of this compound in SD rats revealed high oral bioavailability and a long plasma half-life.     

Systemic oxidative stress in children and teenagers with Down syndrome

Aims

The aim of this study was to evaluate the antioxidant status and oxidative stress biomarkers in the blood of children and teenagers with Down syndrome.

Main methods

The analysis of enzymatic antioxidant defenses, such as the activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione transferase (GST), non-enzymatic antioxidants, such as levels of reduced glutathione (GSH), uric acid (UA) and vitamin E, as well as oxidative damage indicators, such as protein carbonyls (PC) levels and lipoperoxidation (TBARS), of DS individuals (n = 20) compared to healthy controls (n = 18). Except the vitamin E was measured by HPLC, all other markers were measured spectrophotometrically.

Key Findings

Antioxidant enzymes analysis showed significant increases in the SOD (47.2%), CAT (24.7%) and GR (49.6%) activities in DS subjects. No significant difference in GPx activity was detected while GST activity (61.2%) was decreased, and both responses may be consequence of the depletion of GSH (24.9%) levels. There were no significant differences in TBARS levels, while PC levels showed decreased (31.7%) levels compared to healthy controls, which may be related to the increase (16.1%) found in serum UA. Levels of vitamin E showed no significant differences between DS individuals compared to controls.

Significance

The results revealed a systemic pro-oxidant status in DS individuals, evidenced by the increased activity of some important antioxidant enzymes, together with decreased GSH levels in whole blood and elevated UA levels in plasma, probably as an antioxidant compensation related to the redox imbalance in DS individuals.     

Glutathione as an Antioxidant and Regulatory Molecule in Plants Under Abiotic Stress Conditions

The glutathione (GSH)/glutathione disulfide (GSSG) redox couple is involved in several physiologic processes in plants under both optimal and stress conditions. It participates in the maintenance of redox homeostasis in the cells. The redox state of the GSH/GSSG couple is defined by its reducing capacity and the half-cell reduction potential, and differs in the various organs, tissues, cells, and compartments, changing during the growth and development of the plants. When characterizing this redox couple, the synthesis, degradation, oxidation, and transport of GSH and its conjugation with the sulfhydryl groups of other compounds should be considered. Under optimal growth conditions, the high GSH/GSSG ratio results in a reducing environment in the cells which maintains the appropriate structure and activity of protein molecules because of the inhibition of the formation of intermolecular disulfide bridges. In response to abiotic stresses, the GSH/GSSG ratio decreases due to the oxidation of GSH during the detoxification of reactive oxygen species (ROS) and changes in its metabolism. The lower GSH/GSSG ratio activates various defense mechanisms through a redox signalling pathway, which includes several oxidants, antioxidants, and stress hormones. In addition, GSH may control gene expression and the activity of proteins through glutathionylation and thiol-disulfide conversion. This review discusses the size and redox state of the GSH pool, including their regulation, their role in redox signalling and defense processes, and the changes caused by abiotic stress.     

Modification of glycolysis affects cell sensitivity to apoptosis induced by oxidative stress and mediated by mitochondria

The effect of alteration of the glycolytic pathway on cell damage induced by oxidative stress was investigated with dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells that either overexpress cytosolic glycerol-3-phosphate dehydrogenase (CHO/cGPDH cells) or are depleted of the A subunit of lactate dehydrogenase as a result of anti-sense RNA expression (CHO/anti-LDH cells). The extent of oxidative phosphorylation in CHO/anti-LDH and CHO/cGPDH cells was increased and decreased, respectively, relative to that in parental CHO cells, as revealed by measurement of the intracellular content of ATP, the rate of cellular O(2) consumption, the mitochondrial membrane potential (DeltaPsi(m)), and the generation of reactive oxygen species. The sensitivity of these cell lines to cell death induced by the exogenous oxidant tert-butyl hydroperoxide decreased according to the rank order CHO/anti-LDH>CHO>CHO/cGPDH. Exogenous pyruvate markedly increased the sensitivity of CHO/cGPDH cells to oxidant-induced death. The differences among the three cell lines in susceptibility to oxidant-induced death were reflected in the proportion of oxidant-treated cells with a subdiploid DNA content, with a collapsed DeltaPsi(m), and with cytochrome c in the cytosol, indicating that death was mediated by apoptosis. These results demonstrate that the influx of respiratory substrate into mitochondria is an important determinant of cell sensitivity to oxidant-induced apoptosis.     

Protein oxidation in the leaves and roots of cucumber plants (Cucumis sativus L.), mutant MSC16 and wild type

Reactive oxygen species (ROS) may cause irreversible carbonylation of proteins, resulting in structural and/or functional modifications. Carbonylated proteins were analyzed and compared in tissue extracts or purified mitochondria isolated from the leaves and roots of wild-type (WT) or MSC16 mutant cucumber plants. For analysis of the oxidized protein formation and degradation, several techniques were applied: Western blotting, quantitative, spectrophotometric assay of carbonyl concentration and protease activity measurements. Oxidized proteins were tagged with 2,4-dinitrophenylhydrazine (DNPH) and detected with anti-DNP antibodies. Western blots of 1D gels indicated that, in the leaves of both WT and MSC16 plants, certain oxidized proteins have chloroplastic origin. In MSC16 plants, protein oxidation is probably higher in chloroplasts than in mitochondria. Carbonyl concentration is similar in MSC16 and WT leaf extracts, but this may be the result of twice as high protease activity observed in MSC16 leaf extracts and indicates that chloroplastic proteases may effectively remove the oxidized proteins from chloroplasts. In mitochondria of both WT and MSC16 leaves, the levels of oxidized proteins and protease activity are similar. In MSC16 root extracts, the carbonyl concentration is lower and protease activity is similar as compared to WT plants. Nevertheless, in MSC16 root mitochondria, the 30% lower carbonyl concentration, lower band abundance for oxidized proteins and over 50% higher protease activity indicate that mitochondrial proteases are involved in degradation of the oxidatively damaged proteins. In matrix and membrane subfractions, the levels of oxidized proteins are similar in leaf mitochondria or lower in root mitochondria from MSC16 as compared to WT plants. The results show that the oxidized protein degradation network in MSC16 cucumber mutants is well developed, thus becoming a survival factor for plants with mitochondrial dysfunctions.