Synaptosomal response to oxidative stress: Effect of vinpocetine

It has been suggested that reactive oxygen species (ROS) play a role in the neuronal damage occurring in ischemic injury and neurodegenerative disorders and that their neutralization by antioxidant drugs may delay or minimize neurodegeneration. In the present study we examine whether vinpocetine can act as an antioxidant and prevent the formation of ROS and lipid peroxidation in rat brain synaptosomes. After ascorbate/Fe²⁺ treatment a significant increase in oxygen consumption (about 5-fold) and thiobarbituric acid reactive substances (TBARS) formation (about 7-fold) occurred as compared to control conditions. Vinpocetine inhibited the ascorbate/Fe²⁺ stimulated consumption of oxygen and TBARS accumulation, an indicator of lipid peroxidation, in a concentration-dependent manner. The ROS formation was also prevented by vinpocetine. Oxidative stress increased significantly the fluorescence of the probes 2′,7′-dichlorodihydrofluorescein (DCFH₂-DA) (about 6-fold) and dihydrorhodamine (DHR) 123 (about 10-fold), which is indicative of intrasynaptosomal ROS generation. Vinpocetine at 100 μM concentration decreased the fluorescence of DCFH₂-DA and DHR 123 by about 50% and 83%, respectively. We conclude that the antioxidant effect of vinpocetine might contribute to the protective role exerted by the drug in reducing neuronal damage in pathological situations.     

In vitro study of the antioxidant properties of non steroidal anti-inflammatory drugs by chemiluminescence and electron spin resonance (ESR)

Objectives. To determine the antioxidant activities of nonsteroidal anti-inflammatory drugs (NSAIDS), we examined by chemiluminescence (CL) and electron spin resonance (ESR) their scavenging properties towards lipid peroxides, hypochlorous acid and peroxynitrite.

Methods. The antioxidant properties of nimesulide (NIM), 4-hydroxynimesulide (4-HONIM), aceclofenac (ACLO), 4-hydroxyaceclofenac (4-HOA-CLO), diclofenac (DICLO) and indomethacin (INDO) were tested on four different reactive oxygen species (ROS) generating systems: (I) phorbol-myristate acetate (PMA)-activated neutrophils, (II) Fe²⁺/ascorbate-induced lipid peroxidation, (III) HOCl-induced light emission, (IV) the kinetics of ONOO^- decomposition followed by spectrophotometry. ROS production was monitored by luminol-enhanced CL or by ESR using two different spin traps.

Results. At 10 μM, ACLO, NIM, 4-HONIM, 4-HOA-CLO, and DICLO decreased luminol-enhanced CL generated by PMA-activated neutrophils. Inversely, INDO increased the luminol enhanced CL. Interestingly, hydroxylated metabolites were more potent antioxidants than the parent drugs. Furthermore, all drugs tested, excepted ACLO, lowered lipid peroxidation induced by Fe²⁺/ascorbate system. ACLO and DICLO, even at the highest concentration tested (100 μM), did not significantly lower HOCl induced CL, whereas the other drugs were potent scavengers. Finally, all the NSAIDS accelerated decomposition of ONOO^-, suggesting a potential capacity of the molecules to scavenge peroxynitrite.

Conclusion. The NSAIDs possess variable degrees of antioxidant activities, linked to their ability to react with HOCl, lipid peroxides or ONOO^-. These antioxidant activities could offer interesting targeted side-effects in the treatment of joint inflammatory diseases.     

Vinpocetine and α‐tocopherol prevent the increase in DA and oxidative stress induced by 3‐NPA in striatum isolated nerve endings

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3‐nitropropionic acid (3‐NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum‐isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC), and that 3‐NPA increased DA, reactive oxygen species (ROS), DA‐quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3‐NPA‐induced increase in DA, ROS, lipid peroxidation, and DA‐quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α‐tocopherol. Results show that the increase in DA induced by 3‐NPA was inhibited by both 25 μM vinpocetine and 50 μM α‐tocopherol. Vinpocetine, as α‐tocopherol, also inhibited 3‐NPA‐induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA‐RS formation), and DA‐quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3‐NPA, 3‐NPA increases DA‐oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α‐tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA‐rich structures.     

Relations Between Tocopherol Depletion and Coenzyme Q During Lipid Peroxidation in rat Liver Mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe⁺⁺/ascorbate and NADPH/ADP/Fe⁺⁺ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.     

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe²⁺ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl₂ and FeCl₃ on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe²⁺ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl₂/FeCl₃ ratio when PC liposomes deprived of LOOH by triphenyl-phosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl₂. The FeCl₂ concentration at which Fe²⁺ oxidation was maximal, defined as critical Fe²⁺ concentration [Fe²⁺]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl₃, addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe²⁺]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl₃ addition. The experimental data obtained indicate that the kinetics of LOOH-dependent lipid peroxidation depends on the Fe²⁺/Fe³⁺ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl₃ does not affect the LOOH-independent but the LOOH-deendent lipid peroxidation; and suggest that the Feg, endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.     

细菌纳米磁小体的合成机制及应用

综述了近年趋磁细菌纳米磁小体生物合成的分子机制及应用进展。磁小体的合成涉及磁小体膜的形成、铁的吸收和转运、磁小体晶体的矿化、成熟以及磁小体的链状排列等。其中Mam J和Mam K互作并丝状排列,固定磁小体使其链状排列及磁小体膜由细胞质膜内陷而形成是两个令人注目的成就。我们也提出了关于磁小体的生理意义及合成机制的假说:细胞在低氧浓度下由于氧胁迫大量吸收铁,Fe³⁺/Fe²⁺电子对可起到类似O₂/H₂O的作用,产生能量并作为电子受体;Fe3+得到电子还原成的Fe²⁺可引起Fenton反应,此反应产生的活性氧可影响到生物体的正常生理代谢,细胞为降低Fe²⁺浓度,将其与Fe³⁺一同转化为Fe₃O₄颗粒;磁小体的生理功能之一是降低胞内的活性氧。     

An investigation into thee mechanism of citrate---FE-dependent lipid peroxidation

Chelation by citrate was found to promote the autoxidation of Fe²⁺, measured as the disapperance of 1,10-phenanthroline-chelatable Fe²⁺. The autoxidation of citrate---²⁺ could in turn promote the peroxidation of microsomal phospholipid liposomes, as judged by malondialdehyde formation. At low citrate---Fe²⁺ ratios the autoxidation of Fe²⁺ was slow and the formation of malondialdehyde was preceded by a lag phase. The lag phase evidence of this, linear initial rates of lipid peroxidation were obtained via the combination of citrate---Fe²⁺ and citrate---Fe³⁺, optimum activity occurring at a Fe³⁺---Fe²⁺ ratio of 1:1. Evidence is also presented to suggest that the superoxide and the hydrogen peroxide that are formed during the autoxidation of citrate---Fe²⁺ can either stimulate or inhibit lipid peroxidation by affecting the yield of citrate---Fe³⁺ from citrate---Fe²⁺. No evidence was obtained for the participation of the hydroxyl radical in the initiation of lipid peroxidation by citrate---Fe²⁺.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

The biosynthesis of ascorbate protects isolated rat hepatocytes from cumene hydroperoxide-mediated oxidative stress

Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Vinpocetine Attenuates Neointimal Hyperplasia in Diabetic Rat Carotid Arteries after Balloon Injury

Background

Diabetes exacerbates abnormal vascular smooth muscle cell (VSMC) accumulation in response to arterial wall injury. Vinpocetine has been shown to improve vascular remolding; however, little is known about the direct effects of vinpocetine on vascular complications mediated by diabetes. The objective of this study was to determine the effects of vinpocetine on hyperglycemia-facilitated neointimal hyperplasia and explore its possible mechanism.

Materials and Methods

Nondiabetic and diabetic rats were subjected to balloon injury of the carotid artery followed by 3-week treatment with either vinpocetine (10 mg/kg/day) or saline. Morphological analysis and proliferating cell nuclear antigen (PCNA) immunostaining were performed on day 21. Rat VSMCs proliferation was determined with 5-ethynyl-20-deoxyuridine cell proliferation assays. Chemokinesis was monitored with scratch assays, and production of reactive oxygen species (ROS) was assessed using a 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) flow cytometric assay. Apoptosis was detected by annexin V-FITC/PI flow cytometric assay. Cell signaling was assessed by immunblotting.

Results

Vinpocetine prevented intimal hyperplasia in carotid arteries in both normal (I/M ratio: 93.83 ± 26.45% versus 143.2 ± 38.18%, P<0.05) and diabetic animals (I/M ratio: 120.5 ± 42.55% versus 233.46 ± 33.98%, P<0.05) when compared to saline. The in vitro study demonstrated that vinpocetine significantly inhibited VSMCs proliferation and chemokinesis as well as ROS generation and apoptotic resistance, which was induced by high glucose (HG) treatment. Vinpocetine significantly abolished HG-induced phosphorylation of Akt and JNK1/2 without affecting their total levels. For downstream targets, HG-induced phosphorylation of IκBα was significantly inhibited by vinpocetine. Vinpocetine also attenuated HG-enhanced expression of PCNA, cyclin D1 and Bcl-2.

Conclusions

Vinpocetine attenuated neointimal formation in diabetic rats and inhibited HG-induced VSMCs proliferation, chemokinesis and apoptotic resistance by preventing ROS activation and affecting MAPK, PI3K/Akt, and NF-κB signaling.     

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: implication in hemodialysis mediated atherogenic LDL modification

Objective: Al³⁺ stimulates Fe²⁺ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al³⁺ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al³⁺ on LDL oxidation.

Materials and methods: Using different LDL modifying systems (Fe²⁺, Cu²⁺, free radical generating compounds, human endothelial cells, hemin/H₂O₂ and HOCl), the influence of Al³⁺ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.

Results: Al³⁺ could stimulate the oxidizability of LDL by Fe²⁺, but not in the other systems tested. Al³⁺ and Fe²⁺ were found to bind to LDL and Al³⁺could compete with Fe²⁺ binding to the lipoprotein. Fluorescence polarization data indicated that Al³⁺ does not affect the phospholipid compartment of LDL.

Conclusions:The results indicate that increased LDL oxidation by Fe²⁺ in presence of Al³⁺ might be due to blockage of Fe²⁺ binding sites on LDL making more free Fe²⁺ available for lipid oxidation.     

Inhibition of Membrane Lipid Peroxidation by a Radical Scavenging Mechanism: a Novel Function for Hydroxyl-Containing Ionophores

In the present study we show that K⁺/H⁺ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe²⁺-citrate and 2,2'-azobis(2-amidinopropane) di-hydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidyl-choline (PC) liposomes containing negatively charged lipids—dicetyl phosphate (DCP) or cardiolipin (CL)—and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na⁺ than for K⁺, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O₂ consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by buty-lated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, vali-nomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (S_eff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe²⁺-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe²⁺-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidyl-choline (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

Antioxidant Action of Benzylisoquinoline Alkaloids

The antioxidant action of a series of benzylisoquinoline alkaloids has been investigated. Laudanosoline, protopapaverine, anonaine, apomorphine, glaucine, boldine, bulbocapnine, tetrahydroberberine and stepholidine produced a dose-dependent inhibition of microsomal lipid peroxidation induced by Fe²⁺/ascorbate, CCl₄/NADPH or by Fe³⁺ADP/NADPH. Apomorphine exerted the highest inhibitory effects in the three systems of induction used, with a potency higher than propyl gallate. Laudanosoline was particularly effective in the first system, while bulbocapnine and anonaine were more potent when CCl₄/NADPH or Fe³⁺ -ADP/NADPH were used as inducers. Laudanosoline, protopapaverine, apomorphine, tetrahydroberberine and stepholidine were also potent inhibitors of nitroblue tetrazolium (NBT) reduction. The presence of a free hydroxyl group or preferably of a catechol group is a feature relevant for inhibition of lipid peroxidation and NBT reduction, nevertheless the antioxidant activity of benzylisoquinoline alkaloids cannot be only ascribed to the formation of phenoxy radicals and other free radical species may be formed during aporphine and tetrahydroprotoberberine oxidation. The influence of this series of compounds on the time course of lipid peroxidation suggests that some of them, like apomorphine and boldine act as chain-breaking antioxidants.     

Trehalose: Protector of antioxidant enzymes or reactive oxygen species scavenger under heat stress?

Trehalose is known to protect membranes and macromolecules. Its accumulation has been implicated in allowing plants to tolerate stress, including heat-shock. However, under heat-shock, it is not clear whether trehalose eliminates reactive oxygen species (ROS) directly or indirectly by protecting antioxidant enzymes. In this study, we initially examined the effects of trehalose on the activities of key antioxidant enzymes, including superoxide dismutases (SODs), ascorbate catalases (CATs), and ascorbate peroxidases (APX) from wheat (Triticum aestivum L.), and then measured the ability of trehalose to scavenge hydrogen peroxide (H₂O₂) and superoxide anions (O₂⁻). Our results indicated that trehalose protected SOD activity slightly. However, it inhibited CAT and APX activities under heat stress, with a little protection of CAT activity (only about 7% promotion) at 22 °C. Moreover, trehalose scavenged H₂O₂ and O₂⁻ greatly in a concentration-dependent manner, reaching the maximal scavenging H₂O₂ rate of 95% and O₂⁻ rate of 78%, respectively, at 50 mM trehalose. These results suggest that trehalose plays a direct role in eliminating H₂O₂ and O₂⁻ in wheat under heat stress.     

Mechanism of the Interaction of Superoxide Ion and Ascorbate with Anthracycline Antibiotics

The interaction of superoxide ion and ascorbate anion with anthracycline antibiotics (adriamycin and aclacinimycin A) as well as with their Fe³⁺ complexes has been studied in aprotic and protic media. It was found that both superoxide and ascorbate reduce anthracyclines to deoxyaglycons via a one-electron transfer mechanism under all conditions studied. The reaction of ascorbate anion with adriamycin and aclacinomycin A in aqueous solution proceeded only in the presence of Fe³⁺ ions; it is supposed that an active catalytic species was Fe³⁺ adriamycin. It is also supposed that the reduction of anthracycline antibiotics by O,⁷ and ascorbate in cells may increase their anticancer effect.     

Cu2+对拟南芥根的局部毒性及诱导DNA损伤和细胞死亡

该文探讨了不同浓度的Cu²⁺胁迫对拟南芥(Arabidopsis thaliana)根生长、活性氧(ROS)积累、抗氧化酶活性、质膜完整性和细胞活性的影响, 通过分根实验初步分析了Cu²⁺毒性效应的影响范围。结果表明, Cu²⁺胁迫可显著抑制拟南芥主根伸长, 诱导ROS积累及DNA损伤, 促发抗氧化酶活性升高, 破坏质膜完整性, 且Cu²⁺浓度越高, 毒性效应越明显, 在高浓度Cu²⁺胁迫下细胞活性显著降低。分析各参数之间的关系, 表明ROS的积累与超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)及抗坏血酸过氧化物酶(APX)的活性呈显著正相关; ROS积累与DNA损伤、质膜完整性、细胞活性之间具有显著的近线性关系。分根实验结果表明, 只有在添加重金属Cu²⁺(75 μmol·L^–1)一侧培养基中的根生长受抑制, 并出现ROS积累、细胞死亡, 暗示Cu²⁺对拟南芥根系的局部毒性效应可能是由于ROS的局部性积累导致受胁迫根系一侧的细胞死亡所引起的。     

Fine and coarse regulation of reactive oxygen species in the salt tolerant mutants of barnyard grass and their wild-type parents under salt stress

The growth of the wild-type and three salt tolerant mutants of barnyard grass ( Echinochloa crusgalli L.) under salt stress was investigated in relation to oxidative stress and activities of the antioxidant enzymes superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), phenol peroxidase (POD: EC 1.11.1.7), glutathione reductase (GR: EC 1.8.1.7) and ascorbate peroxidase (APX: EC 1.11.1.1). The three mutants ( fows B17, B19 and B21) grew significantly better than the wild-type under salt stress (200 m M NaCl) but some salt sensitive individuals were still detectable in the populations of the mutants though in smaller numbers compared with the wild-type. The salt sensitive plants had slower growth rates, higher rates of lipid peroxidation and higher levels of reactive oxygen species (ROS) in their leaves compared with the more tolerant plants from the same genotype. These sensitivity responses were maximized when the plants were grown under high light intensity suggesting that the chloroplast could be a main source of ROS under salt stress. However, the salt sensitivity did not correlate with reduced K ⁺/Na ⁺ ratios or enhanced Na ⁺ uptake indicating that the sensitivity responses may be mainly because of accumulation of ROS rather than ion toxicity. SOD activities did not correlate to salt tolerance. Salt stress resulted in up to 10-fold increase in CAT activity in the sensitive plants but lower activities were found in the tolerant ones. In contrast, the activities of POD, APX and GR were down regulated in the sensitive plants compared with the tolerant ones. A correlation between plant growth, accumulation of ROS and differential modulation of antioxidant enzymes is discussed. We conclude that loss of activities of POD, APX and GR causes loss of fine regulation of ROS levels and hence the plants experience oxidative stress although they have high CAT activities.     

The oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2

Changes in antioxidant metabolism because of the effect of salinity stress (0, 80, 160 or 240 m M NaCl) on protective enzyme activities under ambient (350 μmol mol⁻¹) and elevated (700 μmol mol⁻¹) CO₂ concentrations were investigated in two barley cultivars ( Hordeum vulgare L., cvs Alpha and Iranis). Electrolyte leakage, peroxidation, antioxidant enzyme activities [superoxide dismutase (SOD), EC 1.15.1.1; ascorbate peroxidase (APX), EC 1.11.1.11; catalase (CAT), EC 1.11.1.6; dehydroascorbate reductase (DHAR), EC 1.8.5.1; monodehydroascorbate reductase (MDHAR), EC 1.6.5.4; glutathione reductase (GR), EC 1.6.4.2] and their isoenzymatic profiles were determined. Under salinity and ambient CO₂, upregulation of antioxidant enzymes such as SOD, APX, CAT, DHAR and GR occurred. However, this upregulation was not enough to counteract all ROS formation as both ion leakage and lipid peroxidation came into play. The higher constitutive SOD and CAT activities together with a higher contribution of Cu,Zn-SOD 1 detected in Iranis might possibly contribute and make this cultivar more salt-tolerant than Alpha. Elevated CO₂ alone had no effect on the constitutive levels of antioxidant enzymes in Iranis, whereas in Alpha it induced an increase in SOD, CAT and MDHAR together with a decrease of DHAR and GR. Under combined conditions of elevated CO₂ and salinity the oxidative damage recorded was lower, above all in Alpha, together with a lower upregulation of the antioxidant system. So it can be concluded that elevated CO₂ mitigates the oxidative stress caused by salinity, involving lower ROS generation and a better maintenance of redox homeostasis as a consequence of higher assimilation rates and lower photorespiration, being the response dependent on the cultivar analysed.     

Antioxidant Activities of Some Extracts of Thymus zygis

The antioxidant activities of methanol and ethyl ether extracts obtained from Thymus zygis, collected during the flowering or non-flowering period, were evaluated and compared. To investigate this potential, extracts were tested on their capacity to react with diphenyl-picrylhydrazyl (DPPH) in a homogeneous medium, and to inhibit Fe²⁺/ascorbate-induced membrane lipid peroxidation, as estimated by the formation of thiobar-bituric acid-reactive substances (TBARS). Although methanol extracts reduce DPPH radicals more efficiently than ethyl ether extracts, suggesting a potent radical scavenger activity, the ethyl ether extracts were found to be most active in inhibiting lipid peroxidation in sarcoplasmic reticulum (SR) membranes. In addition, both extracts present peroxyl and superoxide radical scavenging activities. Peroxyl radicals were generated by the water soluble 2, 2A-azobis(2-amidinopropane) dihydrochloride (AAPH) azoinitiator, and the scavenging activities of the extracts were measured by the inhibition of cis-parinaric acid (PnA) fluorescence decay in SR. Superoxide radicals were generated either by an enzymatic or a non-enzymatic system, and the scavenger ability was evaluated by the inhibition of nitrob-lue tetrazolium reduction. Methanolic extracts are more potent as scavengers of peroxyl and super oxide radicals than the ethyl ether extracts. Apparently, there is a relationship between antioxidant potency and the total phenolic groups content in each extract.