Suppression of reactive oxygen species production enhances neuronal survival in vitro and in vivo in the anoxia-tolerant turtle Trachemys scripta

Hypoxia-ischemia with reperfusion is known to cause reactive oxygen species-related damage in mammalian systems, yet, the anoxia tolerant freshwater turtle is able to survive repeated bouts of anoxia/reoxygenation without apparent damage. Although the physiology of anoxia tolerance has been much studied, the adaptations that permit survival of reoxygenation stress have been largely ignored. In this study, we examine ROS production in the turtle striatum and in primary neuronal cultures, and examine the effects of adenosine (AD) on cell survival and ROS. Hydroxyl radical formation was measured by the conversion of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis; reoxygenation after 1 or 4 h anoxia did not result in increased ROS production compared with basal normoxic levels, nor did H₂O₂ increase after anoxia/reoxygenation in neuronally enriched cell cultures. Blockade of AD receptors increased both ROS production and cell death in vitro , while AD agonists decreased cell death and ROS. As turtle neurons proved surprisingly susceptible to externally imposed ROS stress (H₂O₂), we propose that the suppression of ROS formation, coupled to high antioxidant levels, is necessary for reoxygenation survival. As an evolutionarily selected adaptation, the ability to suppress ROS formation could prove an interesting path to investigate new therapeutic targets in mammals.     

碱胁迫对黑麦草幼苗根系活性氧代谢和渗透溶质积累的影响

为了探讨牧草对碱胁迫的耐受程度,采用营养液砂培方法,研究了不同浓度NaHCO3（0、50、100、150和200 mmol·L^-1）胁迫对黑麦草幼苗根系生长、活性氧代谢和渗透溶质积累的影响。结果表明：NaHCO3胁迫显著抑制黑麦草幼苗根系的生长,其抑制程度随胁迫浓度提高而增强,黑麦草可耐受的最高NaHCO₃浓度约为150 mmol·L^-1。随着NaHCO₃胁迫浓度的增加,黑麦草根中超氧阴离子(O^-·₂)、过氧化氢(H₂O₂)和丙二醛(MDA)含量明显上升,超氧化物歧化酶(SOD)活性和谷胱甘肽(GSH)含量显著下降,过氧化氢酶(CAT)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性及抗坏血酸(ASA)含量先升后降。黑麦草根中Na⁺含量随NaHCO₃浓度增大而增加,K⁺含量和K⁺/Na⁺比降低,可溶性糖含量先升后降,脯氨酸含量则先降后升,游离氨基酸含量呈先升后降再升高变化。表明碱胁迫导致的活性氧代谢失调和Na⁺、K⁺失衡及积累有机溶质进行渗透调节时更多能量的消耗可能是黑麦草根系生长受抑的重要因素。     

Reactive oxygen species are important mediators of taurine release from skeletal muscle cells

The present study illustrates elements ofthe signal cascades involved in the activation of taurine effluxpathways in myotubes derived from skeletal muscle cells. Exposingprimary skeletal muscle cells, loaded with ¹⁴C-taurine, to1) hypotonic media, 2) the phospholipaseA₂ (PLA₂) activator melittin, 3)anoxia, or 4) lysophosphatidyl choline (LPC) causes anincrease in ¹⁴C-taurine release and a concomitantproduction of reactive oxygen species (ROS). The antioxidants butulatedhydroxy toluene and vitamin E inhibit the taurine efflux after cellswelling, anoxia, and addition of LPC. The muscle cells possess twoseparate taurine efflux pathways, i.e., a swelling- andmelittin-induced pathway that requires 5-lipoxygenase activity foractivation and a LPC-induced pathway. The two pathways aredistinguished by their opposing sensitivity toward the anion channelblocker DIDS and cholesterol. These data provide evidence forPLA₂ products and ROS as key mediators of the signalcascade leading to taurine efflux in muscle.

     

Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma

Antioxidants located in both the hydrophilic and lipophilic compartments of plasma are actively involved as a defense system against reactive oxygen species (ROS), which are continuously generated in the body due to both normal metabolism and disease. However, when the production of ROS is not controlled, it leads to cellular lipid, protein, and DNA damage in biological systems. Several assays to measure 'total' antioxidant capacity of plasma have been developed to study the involvement of oxidative stress in pathological conditions and to evaluate the functional bioavailability of dietary antioxidants. Conventional assays to determine antioxidant capacity primarily measure the antioxidant capacity in the aqueous compartment of plasma. Consequently, water-soluble antioxidants such as ascorbic acid, uric acid and protein thiols mainly influence these assays, whereas fat-soluble antioxidants such as tocopherols and carotenoids play only a minor role. However, there are active interactions among antioxidants located in the hydrophilic and lipophilic compartments of plasma. Therefore, new approaches to define the 'true' total antioxidant capacity of plasma should reflect the antioxidant network between water- and fat-soluble antioxidants in plasma. Revelation of the mechanism of action of antioxidants and their true antioxidant potential will help us to optimize the antioxidant defenses in the body.     

活性氧在UV-B诱导的玉米幼苗叶片乙烯产生中的作用

 研究了活性氧在UV-B(280～320 nm)诱导的玉米(Zea mays)幼苗叶片乙烯合成中的作用。结果表明,UV-B促进了玉米幼苗活性氧和乙烯的产生;乙 烯合成抑制剂氨氧乙烯基甘氨酸 (AVG)和氨氧乙酸(AOA)能明显减弱UV-B对玉米幼苗乙烯产生的诱导作用,但对活性氧(ROS)的 产生没有明显影 响;ROS的清除剂不但能抑制UV-B诱导的 ROS的产生,而且还可以抑制UV_B诱导的乙烯的产生,但这种抑制作用可以被外源O₂^.-的供体所逆转。这 说明,乙烯的积累不能作为UV-B胁迫下ROS的诱导的因素,相反,ROS的积累则导致了乙烯的积累;因此,ROS可能参与了UV-B胁迫诱导的乙烯的产生 。质膜NADPH氧化酶的抑制剂二苯碘鎓(DPI)和H₂O₂的特异性清除剂过氧化氢酶（CAT）对UV-B胁迫诱导的乙烯积累 几乎没有影响, 这说明H₂O₂ 可能与UV-B诱导的玉米幼苗叶片乙烯的产生无关, 在UV-B诱导的玉米幼苗叶片乙烯的生物合成过程中O₂^.-起着很重要的作用,相关的O₂^.-不是由 NADPH氧化酶催化产生的。     

Curcumin-induced inhibition of cellular reactive oxygen species generation: Novel therapeutic implications

There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric,Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca²⁺ influx suggest that a rise in cytosolic Ca²⁺ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca²⁺ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.     

Antioxidant activity of Terminalia arjuna bark extract on N-nitrosodiethylamine induced hepatocellular carcinoma in rats

Role of oxidative stress and Na⁺,K⁺-ATPase in the cytotoxicity of hexachlorocyclohexane (HCH) on Ehrlich Ascites tumor (EAT) cells has been studied. HCH caused dose dependent cell death as measured by trypan blue exclusion and lactate dehydrogenase (LDH) leakage from the cells. HCH induced oxidative stress in EAT cells which was characterized by glutathione depletion, lipid peroxidation (LPO), reactive oxygen species (ROS) production and inhibition of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). Protective effect of antioxidants on HCH induced oxidative stress was assessed, among the antioxidants used only quercetin inhibited HCH-induced LPO and ROS production as well as cell death whereas α -tocopherol, ascorbic acid and BHA inhibited LPO but not cell death. Inhibition of membrane bound Na⁺,K⁺-ATPase was a characteristic feature of HCH cytotoxicity in EAT cells. Experimental evidence indicates that HCH-induced cell death involves oxidative stress due to ROS production and membrane perturbation in EAT cells.     

Diabetes,oxidative stress and therapeutic strategies

Background

Diabetes has emerged as a major threat to health worldwide.

Scope of Review

The exact mechanisms underlying the disease are unknown; however, there is growing evidence that excess generation of reactive oxygen species (ROS), largely due to hyperglycemia, causes oxidative stress in a variety of tissues. Oxidative stress results from either an increase in free radical production, or a decrease in endogenous antioxidant defenses, or both. ROS and reactive nitrogen species (RNS) are products of cellular metabolism and are well recognized for their dual role as both deleterious and beneficial species. In type 2 diabetic patients, oxidative stress is closely associated with chronic inflammation. Multiple signaling pathways contribute to the adverse effects of glucotoxicity on cellular functions. There are many endogenous factors (antioxidants, vitamins, antioxidant enzymes, metal ion chelators) that can serve as endogenous modulators of the production and action of ROS. Clinical trials that investigated the effect of antioxidant vitamins on the progression of diabetic complications gave negative or inconclusive results. This lack of efficacy might also result from the fact that they were administered at a time when irreversible alterations in the redox status are already under way. Another strategy to modulate oxidative stress is to exploit the pleiotropic properties of drugs directed primarily at other targets and thus acting as indirect antioxidants.

Major Conclusions

It appears important to develop new compounds that target key vascular ROS producing enzymes and mimic endogenous antioxidants.

General significance

This strategy might prove clinically relevant in preventing the development and/or retarding the progression of diabetes associated with vascular diseases.     

Natural and synthetic antioxidants: An updated overview

Abstract

The current understanding of the complex role of ROS in the organism and pathological sequelae of oxidative stress points to the necessity of comprehensive studies of antioxidant reactivities and interactions with cellular constituents. Studies of antioxidants performed within the COST B-35 action has concerned the search for new natural antioxidants, synthesis of new antioxidant compounds and evaluation and elucidation of mechanisms of action of both natural and synthetic antioxidants. Representative studies presented in the review concern antioxidant properties of various kinds of tea, the search for new antioxidants of herbal origin, modification of tocopherols and their use in combination with selenium and properties of two promising groups of synthetic antioxidants: derivatives of stobadine and derivatives of 1,4-dihydropyridine.     

硝普钠缓解镧引起的黑麦草幼苗生长抑制和根系氧化损伤

采用水培方法,研究了外源一氧化氮（NO）供体硝普钠（SNP）对300 μmol·L^-1 LaCl₃胁迫下黑麦草幼苗生长和根系活性氧代谢的影响。结果表明：在LaCl₃胁迫下,喷施50 μmol·L^-1 SNP能够抑制镧（La）从黑麦草根系向地上部的转运,缓解La对幼苗生长的抑制作用;提高幼苗根系超氧化物歧化酶和抗坏血酸过氧化物酶活性,降低过氧化物酶活性及谷胱甘肽、脯氨酸、H₂O₂、丙二醛含量、超氧阴离子产生速率和质膜相对透性,对过氧化氢酶活性和抗坏血酸含量无显著影响。表明NO可通过活性氧代谢的调节,缓解高浓度La胁迫对黑麦草幼苗生长的抑制作用。     

Control of ascorbic acid efflux in rat luteal cells: role of intracellular calcium and oxygen radicals

In luteal cells, prostaglandin (PG)F_2a mobilizes intracellular calcium concentration ([Ca]_i), generates reactive oxygen species (ROS), depletes ascorbic acid (AA) levels, inhibits steroidogenesis, and ultimately induces cell death. We investigated the hypothesis that [Ca]_i mobilization stimulates ROS, which results in depletion of cellular AA in rat luteal cells. We used a self-referencing AA-selective electrode that noninvasively measures AA flux at the extended boundary layer of single cells and fluorescence microscopy with fura 2 and dichlorofluorescein diacetate (DCF-DA) to measure [Ca]_i and ROS, respectively. Menadione, a generator of intracellular superoxide radical (), PGF_2a, and calcium ionophore were shown to increase [Ca]_i and stimulate intracellular ROS. With calcium ionophore and PGF_2a, but not menadione, the generation of ROS was dependent on extracellular calcium influx. In unstimulated cells there was a net efflux of AA of 121.5 ± 20.3 fmol · cm^–1 · s^–1 (mean ± SE, n = 8), but in the absence of extracellular calcium the efflux was significantly reduced (10.3 ± 4.9 fmol · cm^–1 · s^–1; n = 5, P < 0.05). PGF_2a and menadione stimulated AA efflux, but calcium ionophore had no significant effect. These data suggest two AA regulatory mechanisms: Under basal conditions, AA efflux is calcium dependent and may represent recycling and maintenance of an antioxidant AA gradient at the plasma membrane. Under luteolytic hormone and/or oxidative stress, AA efflux is stimulated that is independent of extracellular calcium influx or generation of ROS. Although site-specific mobilization of calcium pools and ROS cannot be ruled out, the release of AA by PGF_2a-stimulated luteal cells may occur through other signaling pathways. luteolysis; apoptosis; self-referencing microelectrode     

Anoxia-Reoxygenation Regulates Mitochondrial Dynamics through the Hypoxia Response Pathway,SKN-1/Nrf,and Stomatin-Like Protein STL-1/SLP-2

Many aerobic organisms encounter oxygen-deprived environments and thus must have adaptive mechanisms to survive such stress. It is important to understand how mitochondria respond to oxygen deprivation given the critical role they play in using oxygen to generate cellular energy. Here we examine mitochondrial stress response in C. elegans, which adapt to extreme oxygen deprivation (anoxia, less than 0.1% oxygen) by entering into a reversible suspended animation state of locomotory arrest. We show that neuronal mitochondria undergo DRP-1-dependent fission in response to anoxia and undergo refusion upon reoxygenation. The hypoxia response pathway, including EGL-9 and HIF-1, is not required for anoxia-induced fission, but does regulate mitochondrial reconstitution during reoxygenation. Mutants for egl-9 exhibit a rapid refusion of mitochondria and a rapid behavioral recovery from suspended animation during reoxygenation; both phenotypes require HIF-1. Mitochondria are significantly larger in egl-9 mutants after reoxygenation, a phenotype similar to stress-induced mitochondria hyperfusion (SIMH). Anoxia results in mitochondrial oxidative stress, and the oxidative response factor SKN-1/Nrf is required for both rapid mitochondrial refusion and rapid behavioral recovery during reoxygenation. In response to anoxia, SKN-1 promotes the expression of the mitochondrial resident protein Stomatin-like 1 (STL-1), which helps facilitate mitochondrial dynamics following anoxia. Our results suggest the existence of a conserved anoxic stress response involving changes in mitochondrial fission and fusion.     

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。     

Differential contribution of mitochondria, NADPH oxidases, and glycolysis to region-specific oxidant stress in the anoxic-reoxygenated embryonic heart

The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-L-arginine and N-iminoethyl-L-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.     

Cellular redox regulation,signaling, and stress response in plants

Cellular and organellar redox states, which are characterized by the balance between oxidant and antioxidant pool sizes, play signaling roles in the regulation of gene expression and protein function in a wide variety of plant physiological processes including stress acclimation. Reactive oxygen species (ROS) and ascorbic acid (AsA) are the most abundant oxidants and antioxidants, respectively, in plant cells; therefore, the metabolism of these redox compounds must be strictly and spatiotemporally controlled. In this review, we provided an overview of our previous studies as well as recent advances in (1) the molecular mechanisms and regulation of AsA biosynthesis, (2) the molecular and genetic properties of ascorbate peroxidases, and (3) stress acclimation via ROS-derived oxidative/redox signaling pathways, and discussed future perspectives in this field.     

Deliberately added and "cryptic" antioxidants in three artificial diets for insects

Characteristics of both deliberately added and "cryptic" antioxidants were assayed from hydrophilic and lipophilic extracts from artificial diets for plant bugs, lepidopteran larvae, and green lacewings. Cryptic antioxidants are defined as substances naturally existing in diet ingredients but not deliberately added because of their antioxidant potential. Diets were tested after 1) being freshly produced, 2) stored for 48 h at 4 degrees C, or 3) held for 48 h under rearing room conditions at 27 degrees C. Tests included 1) a general assay of antioxidant capacity known as the ferric-reducing antioxidant power (FRAP) assay. 2) a cation radical-scavenging assay, 3) an ascorbic acid assay, and 4) an assay of inhibition of lipid peroxidation. In all assays, the lepidopteran diet had the highest values for protection against reactive oxygen species (ROS). The lepidopteran diet (with 0.17-0.23-mg equivalents of gallic acid equals total phenolic compounds per gram of diet) had three- to four-fold higher concentrations of phenolic compounds than did either the plant bug diet or the lacewing diet. Unexpectedly, the plant bug and the lacewing diets caused more lipid peroxidation than did the positive controls. This was attributed to the high concentrations of iron in these diets (mainly from chicken eggs), causing an ascorbate-ferric ion-induced lipid peroxidation. Diet storage, measured after 2 d at 27 or 4-6 degrees C, caused no significant declines in overall antioxidant potential. However, storage did lead to decline in ascorbic acid. The FRAP assay offered the best potential as a general, routine test of the potential of various insect diets to resist the destructive effects of ROS. The importance of addressing issues of protection against ROS in insect diets is discussed.     

Mitochondrial reactive oxygen species and Ca2+ signaling

Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with Ca²⁺ is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([Ca²⁺]_i) signals. Several Ca²⁺ transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing Ca²⁺ from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [Ca²⁺]_i signals by Ca²⁺ uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to Ca²⁺ release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial Ca²⁺ uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [Ca²⁺]_i signals. Together, the data reviewed here indicate that Ca²⁺-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [Ca²⁺]_i signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed. mitochondria; calcium     

Protective role of new nitrogen compounds on ROS/RNS-mediated damage to PC12 cells

Reactive oxygen (ROS) and nitrogen (RNS) species are known to be involved in many degenerative diseases. This study reports four new nitrogen compounds from organic synthesis, identified as FMA4, FMA7, FMA762 and FMA796, which differ mainly by the number of hydroxyl groups within their phenolic unit. Their potential role as antioxidants was evaluated in PC12 cells by assessing their protection against oxidative and nitrosative insults. The four compounds, and particularly FMA762 and FMA796, were able to protect cells against lipid peroxidation and intracellular ROS/RNS formation to a great extent. Their protective effects were likely mediated by their free radicals scavenging ability, as they appeared to be involved neither in the induction of natural antioxidant enzymes like GSH-PX and SOD, nor in the inhibition of NOS. Nevertheless, these results suggest a promising potential for these compounds as ROS/RNS scavengers in pathologies where oxidative/nitrosative stress are involved.