Mitochondrial generation of reactive oxygen species is enhanced at the Q<Subscript>o</Subscript> site of the complex III in the myocardium of <Emphasis Type="Italic">Trypanosoma cruzi-</Emphasis>infected mice: beneficial effects of an antioxidant

In this study, we have characterized the cellular source and mechanism for the enhanced generation of reactive oxygen species (ROS) in the myocardium during Trypanosoma cruzi infection. Cardiac mitochondria of infected mice, as compared to normal controls, exhibited 63.3% and 30.8% increase in ROS-specific fluorescence of dihydroethidium (detects O₂ ^•−) and amplex red (detects H₂O₂), respectively. This increase in ROS level in cardiac mitochondria of infected mice was associated with a 59% and 114% increase in the rate of glutamate/malate- (complex I substrates) and succinate- (complex II substrate) supported ROS release, respectively, and up to a 74.9% increase in the rate of electron leakage from the respiratory chain when compared to normal controls. Inhibition studies with normal cardiac mitochondria showed that rotenone induced ROS generation at the Q_Nf-ubisemiquinone site in complex I. In complex III, myxothiazol induced ROS generation from a site located at the Q_o center that was different from the Q_i center of O₂ ^•− generation by antimycin. In cardiac mitochondria of infected mice, the rate of electron leakage at complex I during forward (complex I-to-complex III) and reverse (complex II-to-complex I) electron flow was not enhanced, and complex I was not the main site of increased ROS production in infected myocardium. Instead, defects of complex III proximal to the Q_o site resulted in enhanced electron leakage and ROS formation in cardiac mitochondria of infected mice. Treatment of infected mice with phenyl-α-tert-butyl-nitrone (PBN) improved the respiratory chain function, and, subsequently, decreased the extent of electron leakage and ROS release. In conclusion, we show that impairment of the Q_o site of complex III resulted in increased electron leakage and O₂ ^•− formation in infected myocardium, and was controlled by PBN.     

Icariside II,a novel phosphodiesterase 5 inhibitor,protects against H2O2‐induced PC12 cells death by inhibiting mitochondria‐mediated autophagy

Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress‐induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H₂O₂)‐induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre‐treatment significantly abrogated H₂O₂‐induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H₂O₂‐induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase‐3β (GSK‐3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p‐tyr216‐GSK‐3β level and increased p‐ser9‐GSK‐3β level respectively. The GSK‐3β inhibitor SB216763 abrogated H₂O₂‐induced cell death. Moreover, ICS II significantly inhibited H₂O₂‐induced autophagy by the reducing autophagosomes number and the LC3‐II/LC3‐I ratio, down‐regulating Beclin‐1 expression, and up‐regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3‐methyl adenine (3‐MA) blocked H₂O₂‐induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress‐induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK‐3β/mitochondrial signalling pathways.     

Mitochondrial handling of excess Ca2+ is substrate-dependent with implications for reactive oxygen speciesgeneration

The mitochondrial electron transport chain is the major source of reactive oxygen species (ROS) during cardiac ischemia. Several mechanisms modulate ROS production; one is mitochondrial Ca²⁺ uptake. Here we sought to elucidate the effects of extramitochondrial Ca²⁺ (e[Ca²⁺]) on ROS production (measured as H₂O₂ release) from complexes I and III. Mitochondria isolated from guinea pig hearts were preincubated with increasing concentrations of CaCl₂ and then energized with the complex I substrate Na⁺ pyruvate or the complex II substrate Na⁺ succinate. Mitochondrial H₂O₂ release rates were assessed after giving either rotenone or antimycin A to inhibit complex I or III, respectively. After pyruvate, mitochondria maintained a fully polarized membrane potential (ΔΨ; assessed using rhodamine 123) and were able to generate NADH (assessed using autofluorescence) even with excess e[Ca²⁺] (assessed using CaGreen-5N), whereas they remained partially depolarized and did not generate NADH after succinate. This partial ΔΨ depolarization with succinate was accompanied by a large release in H₂O₂ (assessed using Amplex red/horseradish peroxidase) with later addition of antimycin A. In the presence of excess e[Ca²⁺], adding cyclosporin A to inhibit mitochondrial permeability transition pore opening restored ΔΨ and significantly decreased antimycin A-induced H₂O₂ release. Succinate accumulates during ischemia to become the major substrate utilized by cardiac mitochondria. The inability of mitochondria to maintain a fully polarized ΔΨ under excess e[Ca²⁺] when succinate, but not pyruvate, is the substrate may indicate a permeabilization of the mitochondrial membrane, which enhances H₂O₂ emission from complex III during ischemia.     

Low hydrogen peroxide production in mitochondria of the long‐lived Arctica islandica: underlying mechanisms for slow aging

The observation of an inverse relationship between lifespan and mitochondrial H₂O₂ production rate would represent strong evidence for the disputed oxidative stress theory of aging. Studies on this subject using invertebrates are surprisingly lacking, despite their significance in both taxonomic richness and biomass. Bivalve mollusks represent an interesting taxonomic group to challenge this relationship. They are exposed to environmental constraints such as microbial H₂S, anoxia/reoxygenation, and temperature variations known to elicit oxidative stress. Their mitochondrial electron transport system is also connected to an alternative oxidase that might improve their ability to modulate reactive oxygen species (ROS) yield. Here, we compared H₂O₂ production rates in isolated mantle mitochondria between the longest‐living metazoan—the bivalve Arctica islandica—and two taxonomically related species of comparable size. In an attempt to test mechanisms previously proposed to account for a reduction of ROS production in long‐lived species, we compared oxygen consumption of isolated mitochondria and enzymatic activity of different complexes of the electron transport system in the two species with the greatest difference in longevity. We found that A. islandica mitochondria produced significantly less H₂O₂ than those of the two short‐lived species in nearly all conditions of mitochondrial respiration tested, including forward, reverse, and convergent electron flow. Alternative oxidase activity does not seem to explain these differences. However, our data suggest that reduced complex I and III activity can contribute to the lower ROS production of A. islandica mitochondria, in accordance with previous studies. We further propose that a lower complex II activity could also be involved.     

Pretreatment with H(2) O(2) alleviates aluminum-induced oxidative stress in wheat seedlings

Hydrogen peroxide (H₂O₂) is a key reactive oxygen species (ROS) in signal transduction pathways leading to activation of plant defenses against biotic and abiotic stresses. In this study, we investigated the effects of H₂O₂ pretreatment on aluminum (Al) induced antioxidant responses in root tips of two wheat (Triticum aestivum L.) genotypes, Yangmai‐5 (Al‐sensitive) and Jian‐864 (Al‐tolerant). Al increased accumulation of H₂O₂ and O₂^?? leading to more predominant lipid peroxidation, programmed cell death and root elongation inhibition in Yangmai‐5 than in Jian‐864. However, H₂O₂ pretreatment alleviated Al‐induced deleterious effects in both genotypes. Under Al stress, H₂O₂ pretreatment increased the activities of superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and monodehydroascorbate reductase, glutathione reductase and glutathione peroxidase as well as the levels of ascorbate and glutathione more significantly in Yangmai‐5 than in Jian‐864. Furthermore, H₂O₂ pretreatment also increased the total antioxidant capacity evaluated as the 2, 2‐diphenyl‐1‐picrylhydrazyl‐radical scavenging activity and the ferric reducing/antioxidant power more significantly in Yangmai‐5 than in Jian‐864. Therefore, we conclude that H₂O₂ pretreatment improves wheat Al acclimation during subsequent Al exposure by enhancing the antioxidant defense capacity, which prevents ROS accumulation, and that the enhancement is greater in the Al‐sensitive genotype than in the Al‐tolerant genotype.     

Detoxification function of aldose/aldehyde reductase during drought and ultraviolet-B (280–320 nm) stresses

Plants may activate similar defence systems to reduce cellular damages caused by different stress conditions. In the present experiments, the formation of lipid peroxidation products [thiobarbituric acid reactive species (TBARS)] was significant during both drought and ultraviolet (UV)‐B stresses, whereas the formation of reactive oxygen species (ROS) was a more delayed response to UV‐B than to drought. H₂O₂ was detected during both stresses, whereas ^·OH radical production was a more characteristic response to drought. The present characterization of transgenic tobacco plants revealed a common role for aldose/aldehyde reductase (ALR) in the detoxification of lipid peroxidation products under water depletion and UV‐B irradiation. As the result of the increased synthesis of ALR enzyme, the transformed plants were more tolerant to both stress conditions, exhibiting reduced loss of photosynthetic function and decreased accumulation of TBARS and H₂O₂ as compared to control (SR1) plants. When plants had been exposed to mild, non‐lethal drought and were then watered again to recover, they were more tolerant to a subsequent stress by UV‐B. This was characteristic to both transgenic and wild‐type plants. However, this drought‐induced cross‐tolerance to UV‐B stress of SR1 tobacco did not reach the enhancement achieved by the overexpression of ALR.     

Role of mitochondria in ultraviolet-induced oxidative stress

The biological effects of ultraviolet radiation (UV), such as DNA damage, mutagenesis, cellular aging, and carcinogenesis, are in part mediated by reactive oxygen species (ROS). The major intracellular ROS intermediate is hydrogen peroxide, which is synthesized from superoxide anion ((*)O(2)(-)) and further metabolized into the highly reactive hydroxyl radical. In this study, we examined the involvement of mitochondria in the UV-induced H(2)O(2) accumulation in a keratinocyte cell line HaCaT. Respiratory chain blockers (cyanide-p-trifluoromethoxy-phenylhydrazone and oligomycin) and the complex II inhibitor (theonyltrifluoroacetone) prevented H(2)O(2) accumulation after UV. Antimycin A that inhibits electron flow from mitochondrial complex III to complex IV increased the UV-induced H(2)O(2) synthesis. The same effect was seen after incubation with rotenone, which blocks electron flow from NADH-reductase (complex I) to ubiquinone. UV irradiation did not affect mitochondrial transmembrane potential (DeltaPsi(m)). These data indicate that UV-induced ROS are produced at complex III via complex II (succinate-Q-reductase).     

Ambivalent effects of diazoxide on mitochondrial ROS production at respiratory chain complexes I and III

Background

Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein (H₂DCF) but had no direct impact on the H₂O₂ production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM).

Methods

H₂O₂ generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors.

Results

We show that diazoxide reduces ROS production by mitochondrial complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Q_o site of complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na⁺ or K⁺ excluding a role for putative mitochondrial K_ATP-channels.

General significance

A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of complex II promotes transient generation of signaling ROS at complex III (during preconditioning) or attenuates the production of deleterious ROS at complex I (during ischemia and reperfusion).     

Effect of Short-Term Caloric Restriction on H2O2 Production and Oxidative DNA Damage in Rat Liver Mitochondria and Location of the Free Radical Source

Oxygen free radicals (ROS) of mitochondrial origin seem to be involved in aging. Whereas in other tissues complexes I or III of the respiratory chain contain the ROS generators, in this study we find that rat liver mitochondria generate oxygen radicals at complexes I, II, and III. Short-term (6 weeks) caloric restriction significantly decreased H₂O₂ production in rat liver mitochondria. This decrease in ROS production was located at complex I because it occurred with complex I-linked substrates (pyruvate/malate), but did not reach statistical significance with the complex II-linked substrate succinate. The mechanism responsible for the lowered ROS production was not a decrease in oxygen consumption. Instead, the mitochondria of caloric-restricted animals released less ROS per unit electron flow. This was due to a decrease in the degree of reduction of the complex I generator. Furthermore, oxidative damage to mitochondrial and nuclear DNA was also decreased in the liver by short-term caloric restriction. The results agree with the idea that caloric restriction delays aging, at least in part, by decreasing the rate of mitochondrial ROS generation and thus the rate of attack to molecules, like DNA, highly relevant for the accumulation of age-dependent changes.     

Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS‐dependent mitochondrial signalling pathway

Oxidative stress is an important molecular mechanism underlying lung fibrosis. The mitochondrion is a major organelle for oxidative stress in cells. Therefore, blocking the mitochondrial signalling pathway may be the best therapeutic manoeuver to ameliorate lung fibrosis. Astaxanthin (AST) is an excellent antioxidant, but no study has addressed the pathway of AST against pulmonary oxidative stress and free radicals by the mitochondrion‐mediated signalling pathway. In this study, we investigated the antioxidative effects of AST against H₂O₂‐ or bleomycin (BLM)‐induced mitochondrial dysfunction and reactive oxygen species (ROS) production in alveolar epithelial cells type II (AECs‐II) in vivo and in vitro. Our data show that AST blocks H₂O₂‐ or BLM‐induced ROS generation and dose‐dependent apoptosis in AECs‐II, as characterized by changes in cell and mitochondria morphology, translocation of apoptotic proteins, inhibition of cytochrome c (Cyt c) release, and the activation of caspase‐9, caspase‐3, Nrf‐2 and other cytoprotective genes. These data suggest that AST inhibits apoptosis in AECs‐II cells through the ROS‐dependent mitochondrial signalling pathway and may be of potential therapeutic value in lung fibrosis treatment.     

On the mechanism of hydrogen peroxide-, superoxide-, and ultraviolet light-induced DNA cleavages of inactive bleomycin-iron (III) complex

The ³²P-labeled DNA cleavage experiments showed that the biological activity of the bleomycin(BLM)-Fe(III)OH^? complex is evidently induced by addition of H₂O₂ and KO₂, or by irradiation of UV light. Hydrogen peroxide contributes to the conversion from the inactive BLM-Fe(III)OH^? complex to the active BLM-Fe(III)O₂H^? complex, and UV light to the reduction of the BLM-Fe(III)OH^? complex to the BLM-Fe(II) complex. The proposed hypothetical mechanism for cyclic function of BLM-iron complex is similar to that of certain heme-oxygenases and heme-oxidases.     

Opening of the mitoKATP channel and decoupling of mitochondrial complex II and III contribute to the suppression of myocardial reperfusion hyperoxygenation

Diazoxide, a mitochondrial ATP-sensitive potassium (mitoK_ATP) channel opener, protects the heart from ischemia–reperfusion injury. Diazoxide also inhibits mitochondrial complex II-dependent respiration in addition to its preconditioning effect. However, there are no prior studies of the role of diazoxide on post-ischemic myocardial oxygenation. In the current study, we determined the effect of diazoxide on the suppression of post-ischemic myocardial tissue hyperoxygenation in vivo, superoxide (O₂ ^??) generation in isolated mitochondria, and impairment of the interaction between complex II and complex III in purified mitochondrial proteins. It was observed that diazoxide totally suppressed the post-ischemic myocardial hyperoxygenation. With succinate but not glutamate/malate as the substrate, diazoxide significantly increased ubisemiquinone-dependent O₂ ^?? generation, which was not blocked by 5-HD and glibenclamide. Using a model system, the super complex of succinate-cytochrome c reductase (SCR) hosting complex II and complex III, we also observed that diazoxide impaired complex II and its interaction with complex III with no effect on complex III. UV–visible spectral analysis revealed that diazoxide decreased succinate-mediated ferricytochrome b reduction in SCR. In conclusion, our results demonstrated that diazoxide suppressed the in vivo post-ischemic myocardial hyperoxygenation through opening the mitoK_ATP channel and ubisemiquinone-dependent O₂ ^?? generation via inhibiting mitochondrial complex II-dependent respiration.     

Oxidative pathways in response to polyunsaturated aldehydes in the marine diatom Skeletonema marinoi (Bacillariophyceae)

Polyunsaturated aldehydes (PUA) have recently been shown to induce reactive oxygen species (ROS) and possibly reactive nitrogen species (RNS, e.g., peroxynitrite) in the diatom Skeletonema marinoi (S. marinoi), which produces high amounts of PUA. We now are attempting to acquire better understanding of which reactive molecular species are involved in the oxidative response of S. marinoi to PUA. We used flow cytometry, the dye dihydrorhodamine 123 (DHR) as the main indicator of ROS (but which is also known to partially detect RNS), and different scavengers and inhibitors of both nitric oxide (NO) synthesis and superoxide dismutase activity (SOD). Both the scavengers Tempol (for ROS) and uric acid (UA, for peroxynitrite) induced a lower DHR‐derived green fluorescence in S. marinoi cells exposed to the PUA, suggesting that both reactive species were produced. When PUA‐exposed S. marinoi cells were treated with the NO scavenger 2‐4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), an opposite response was observed, with an increase in DHR‐derived green fluorescence. A higher DHR‐derived green fluorescence was also observed in the presence of sodium tungstate (ST), an inhibitor of NO production via nitrate reductase. In addition, two different SOD inhibitors, 2‐methoxyestradiol (2ME) and sodium diethyldithiocarbamate trihydrate (DETC), had an effect, with DETC inducing the strongest inhibition after 20 min. These results indicate the involvement of O₂^? generation and SOD activity in H₂O₂ formation (with downstream ROS generation dependent from H₂O₂) in response to PUA exposure. This is relevant as it refines the biological impact of PUA and identifies the specific molecules involved in the response. It is speculated that in PUA‐exposed S. marinoi cells, beyond a certain threshold of PUA, the intracellular antioxidant system is no longer able to cope with the excess of ROS, thus resulting in the observed accumulation of both O₂^?? and H₂O₂. This might be particularly relevant for population dynamics at sea, during blooms, when cell lysis increases and PUA are released. It can be envisioned that in the final stages of blooms, higher local PUA concentrations accumulate, which in turn induces intracellular ROS generation that ultimately leads to cell death and bloom decay.     

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.     

Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone

Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide (H₂O₂) in tobacco and tomato, and superoxide (O₂^–?) together with H₂O₂ in Arabidopsis accessions. Accumulation of these reactive oxygen species (ROS) preceded the induction of cell death, and both responses co‐occurred spatially in the periveinal regions of the leaves. Re‐current ozone exposure of the sensitive tobacco cv. Bel W3 in closed chambers or in the field led to an enlargement of existing lesions by priming the border cells for H₂O₂ accumulation. Open top chamber experiments with native herbaceous plants in the field showed that Malva sylvestris L. accumulates O₂^–? at those sites that later exhibit plant cell death. Blocking of ROS accumulation markedly reduced ozone‐induced cell death in tomato, Arabidopsis and M. sylvestris. It is concluded that ozone triggers an in planta generation and accumulation of H₂O₂ and/or O₂^–? depending on the species, accession and cultivar, and that both these reactive oxygen species are involved in the induction of cell death in sensitive crop and native plants.     

Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis

Salicylic acid (SA), a ubiquitous phenolic phytohormone, is involved in many plant physiological processes including stomatal movement. We analysed SA‐induced stomatal closure, production of reactive oxygen species (ROS) and nitric oxide (NO), cytosolic calcium ion ([Ca²⁺]_cyt) oscillations and inward‐rectifying potassium (K⁺_in) channel activity in Arabidopsis. SA‐induced stomatal closure was inhibited by pre‐treatment with catalase (CAT) and superoxide dismutase (SOD), suggesting the involvement of extracellular ROS. A peroxidase inhibitor, SHAM (salicylhydroxamic acid) completely abolished SA‐induced stomatal closure whereas neither an inhibitor of NADPH oxidase (DPI) nor atrbohD atrbohF mutation impairs SA‐induced stomatal closures. 3,3′‐Diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) stainings demonstrated that SA induced H₂O₂ and O₂^– production. Guard cell ROS accumulation was significantly increased by SA, but that ROS was suppressed by exogenous CAT, SOD and SHAM. NO scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO) suppressed the SA‐induced stomatal closure but did not suppress guard cell ROS accumulation whereas SHAM suppressed SA‐induced NO production. SA failed to induce [Ca²⁺]_cyt oscillations in guard cells whereas K⁺_in channel activity was suppressed by SA. These results indicate that SA induces stomatal closure accompanied with extracellular ROS production mediated by SHAM‐sensitive peroxidase, intracellular ROS accumulation and K⁺_in channel inactivation.     

Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress: Involving of mitochondria and energy metabolism

Oxidative stress has been demonstrated to be involved in the etiology of several neurobiological disorders. Sonic hedgehog (Shh), a secreted glycoprotein factor, has been implicated in promoting several aspects of brain remodeling process. Mitochondria may play an important role in controlling fundamental processes in neuroplasticity. However, little evidence is available about the effect and the potential mechanism of Shh on neurite outgrowth in primary cortical neurons under oxidative stress. Here, we revealed that Shh treatment significantly increased the viability of cortical neurons in a dose-dependent manner, which was damaged by hydrogen peroxide (H₂O₂). Shh alleviated the apoptosis rate of H₂O₂-induced neurons. Shh also increased neuritogenesis injuried by H₂O₂ in primary cortical neurons. Moreover, Shh reduced the generation of reactive oxygen species (ROS), increased the activities of SOD and and decreased the productions of MDA. In addition, Shh protected mitochondrial functions, elevated the cellular ATP levels and amelioratesd the impairment of mitochondrial complex II activities of cortical neurons induced by H₂O₂. In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H₂O₂ -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress.     

Caloric restriction influences hydrogen peroxide generation in mitochondrial sub-populations from mouse liver

Calorie restriction (CR) has been shown to decrease H₂O₂ production in liver mitochondria, although it is not known if this is due to uniform changes in all mitochondria or changes in particular mitochondrial sub-populations. To address this issue, liver mitochondria from control and CR mice were fractionated using differential centrifugation at 1,000 g, 3,000 g and 10,000 g into distinct populations labeled as M1, M3 and M10, respectively. Mitochondrial protein levels, respiration and H₂O₂ production were measured in each fraction. CR resulted in a decrease in total protein (mg) in each fraction, although this difference disappeared when adjusted for liver weight (mg protein/g liver weight). No differences in respiration (State 3 or 4) were observed between control and CR mice in any of the mitochondrial fractions. CR decreased H₂O₂ production in all fractions when mitochondria respired on succinate (Succ), succ+antimycin A (Succ+AA) or pyruvate/malate+rotenone (P/M+ROT). Thus, CR decreased reactive oxygen species (ROS) production under conditions which stimulate mitochondrial complex I ROS production under both forward (P/M+ROT) and backward (Succ & Succ+AA) electron flow. The results indicate that CR decreases H₂O₂ production in all liver mitochondrial fractions due to a decrease in capacity for ROS production by complex I of the electron transport chain.     

METAL‐INDUCED REACTIVE OXYGEN SPECIES PRODUCTION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Toxic effects of metals appear to be partly related to the production of reactive oxygen species (ROS), which can cause oxidative damage to cells. The ability of several redox active metals [Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI)], nonredox active metals [Pb(II), Cd(II), Zn(II)], and the metalloid As(III) and As(V) to produce ROS at environmentally relevant metal concentrations was assessed. Cells of the freshwater alga Chlamydomonas reinhardtii P. A. Dang. were exposed to various metal concentrations for 2.5 h. Intracellular ROS accumulation was detected using an oxidation‐sensitive reporter dye, 5‐(and‐6)‐carboxy‐2′,7′‐dihydrodifluorofluorescein diacetate (H₂DFFDA), and changes in the fluorescence signal were quantified by flow cytometry (FCM). In almost all cases, low concentrations of both redox and nonredox active metals enhanced intracellular ROS levels. The hierarchy of maximal ROS induction indicated by the increased number of stained cells compared to the control sample was as follows: Pb(II) > Fe(III) > Cd(II) > Ag(I) > Cu(II) > As(V) > Cr(VI) > Zn(II). As(III) and Cr(III) had no detectable effect. The effective free metal ion concentrations ranged from 10^?6 to 10^?9 M, except in the case of Fe(III), which was effective at 10^?18 M. These metal concentrations did not affect algal photosynthesis. Therefore, a slightly enhanced ROS production is a general and early response to elevated, environmentally relevant metal concentrations.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Abstract

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H₂O₂) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H₂O₂-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H₂O₂-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H₂O₂, and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca^{2 +}) chelator, overexpression of Homer 1a had no significant effects on H₂O₂-induced oxidative stress. These results suggest that Homer 1a has protective effects against H₂O₂-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca^{2 +} homeostasis.