Cytoprotective effects of 12-oxo phytodienoic acid,a plant-derived oxylipin jasmonate,on oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells

BackgroundJasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity.MethodsThe cells were pretreated with individual jasmonates for 24 h and exposed to hydrogen peroxide (H₂O₂) for 24 h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA).ResultsAmong the jasmonates, only OPDA suppressed H₂O₂-induced cytotoxicity. OPDA pretreatment also inhibited the H₂O₂-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2.ConclusionsThese results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway.General significancePlant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases.     

Critical role of hydrogen peroxide signaling in the sequential activation of p38 MAPK and eNOS in laminar shear stress

Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways. Using the cone/plate model we found that LSS (12 dyn/cm²) rapidly promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H₂O₂). Physiological concentrations of H₂O₂ (flux of 0.1 nM/min and 15 μM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decreased LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H₂O₂- and LSS-induced eNOS phosphorylation and reduced ^?NO levels. We propose a model in which LSS promotes the formation of signaling levels of H₂O₂, which in turn activate p38α MAPK and then stimulate eNOS, leading to increased ^?NO generation and protection of endothelial function.     

Protective effects of purified safflower extract on myocardial ischemia in vivo and in vitro

Carthamus tinctorius L. (safflower) is one of the most commonly used Chinese herbal medicines to prevent and treat cardiac disease in clinical practice. However, the mechanisms responsible for such protective effects remain largely unknown. In this study, we investigated the anti-myocardial ischemia effects of a purified extract of C. tinctorius (ECT) both in vivo and in vitro. An animal model of myocardial ischemia injury was induced by left anterior descending coronary artery occlusion in adult rats. Pretreatment with ECT (100, 200, 400, 600 mg/kg body wt.) could protect the heart from ischemia injury by limiting infarct size and improving cardiac function. In the in vitro experiment, neonatal rat ventricular myocytes were incubated to test the direct cytoprotective effect of ECT against H₂O₂ exposure. Pretreatment with 100–400 μg/ml ECT prior to H₂O₂ exposure significantly increased cell viability as revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. ECT also markedly attenuated H₂O₂-induced cardiomyocyte apoptosis, as detected by Annexin V and PI double labeling with flow cytometry. The intracellular level of reactive oxygen species (ROS) was shown by 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and ECT pretreatment significantly inhibited H₂O₂-induced ROS increase. We made a preliminary examination of the signaling cascade involved in ECT mediated anti-apoptotic effects. Phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) blocked the cytoprotective effect conferred by ECT. Taken together, our findings provide the first evidence that the cardioprotective effects of ECT in myocardial ischemia operate partially through reducing oxidative stress induced damage and apoptosis. The protection is achieved by scavenging of ROS and mediating the PI3K signaling pathway.     

Oxidative damages induced by short-term exposure to cadmium in bean plants: Protective role of salicylic acid

The role of salicylic acid (SA) in alleviating cadmium (Cd) toxicity was investigated in a hydroponic cultivation system. Short-term exposure of bean (Phaseolus vulgaris) plants to 20 μM Cd inhibited biomass production and intensively increased accumulation of Cd in both roots and leaves. At leaf level, Cd significantly decreased mineral ions, chlorophyll and carotenoids concentrations. Concomitantly, Cd enhanced electrolyte leakage, H₂O₂ content and lipid peroxidation as indicated by malondialdehyde (MDA) accumulation. SA pretreatment decreased the uptake and the transport of Cd, alleviated the Cd-induced inhibition of nutrient absorption and led to a significant increase of chlorophyll and carotenoid content. SA application alleviated the oxidative damages as evidenced by the lowered H₂O₂ and MDA content. SA particularly induced an increase in both CAT and APX activities accompanied by a significant reduction in SOD and POD activities. As important antioxidants, ascorbate and glutathione contents in bean leaves exposed to cadmium were significantly decreased by SA treatment. These results reveal the potentiating effect of salicylic acid in regulating cadmium induced oxidative stress in bean plants.     

Responses of non-protein thiols to Cd exposure in Cd hyperaccumulator Arabis paniculata Franch

We investigated the responses of phytochelatins (PCs), glutathione (GSH) and other non-protein thiols in Cd hyperaccumulator Arabis paniculata after Cd exposure. Applying γ-glutamylcysteine synthetase (γ-ECS) inhibitor, l-buthionine-sulfoximine (BSO), the roles of PCs in Cd tolerance and Cd accumulation in A. paniculata were evaluated. Plants were exposed to four Cd concentrations (0, 50, 100 and 250 μM) for different times (2w or 3w) with and without BSO. Overall, Cd exposure had little impact on plant biomass after 2w or 3w of growth except at the highest Cd level. A. paniculata tolerated ≤100 μM Cd with up to 1127 mg kg^?1 Cd in the shoots and 5624 mg kg^?1 Cd in the roots after 3w of Cd exposure. Cd exposure induced formation of PCs and three unknown thiols in the roots, but none were detected in the shoots. BSO had no significant effect on Cd sensitivity in plants though it reduced Cd accumulation in the roots. In addition, the molar ratio of PCs:Cd, which ranged from 0.7 to 1.3 after exposing to 50–100 μM Cd without BSO in the roots, was close to the value expected for PC-mediated Cd sequestration in plants. Those data indicate that GSH and PCs did not contribute to Cd tolerance in the shoots and Cd transport from the root to shoot in A. paniculata, but they may play an important role in Cd accumulation and Cd complexation in the roots of A. paniculata.     

Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In this study we demonstrated that the exogenous H₂O₂ was able to promote the formation and development of adventitious roots in mung bean seedlings. Treatments with 1–100 mM H₂O₂ for 8–18 h significantly induced the formation and development of adventitious roots. Catalase (CAT) and ascorbic acid, which are H₂O₂ scavengers or inhibitors, eliminated the adventitious root-promoting effects of exogenous H₂O₂. H₂O₂ may have a downstream signaling function in the auxin signaling pathway and be involved in auxin-induced adventitious root formation. 2,3,5-Triiodobenzoic acid (TIBA), an inhibitor of auxin polar transport, strongly inhibited adventitious rooting of mung bean seedlings; however, the inhibiting effects of TIBA on adventitious rooting can be partially reversed by the exogenous IBA or H₂O₂. Diphenylene iodonium (DPI) strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H₂O₂ formation in plant cells. DPI treatment strongly inhibited the formation of adventitious roots in mung bean, but the inhibitory effects of DPI on rooting can be partially reversed by the exogenous H₂O₂ or IBA. This indicates that the formation of adventitious roots was blocked once the generation of H₂O₂ through NADPH oxidase was inhibited, and H₂O₂ mediated the IBA-induced adventitious root formation. Furthermore, a rapid increase in the endogenous level of H₂O₂ was detected during incubation with water 12–36 h after the primary root removal in mung bean seedlings. Three hours after the primary root removal, the generation of endogenous H₂O₂ was markedly induced in IBA-treated seedlings in comparison with water-treated seedlings. This implies that IBA induced overproduction of H₂O₂ in mung bean seedlings, and that IBA promoted adventitious root formation via a pathway involving H₂O₂. Results obtained suggest that H₂O₂ may function as a signaling molecule involved in the formation and development of adventitious roots in mung bean seedlings.     

Membrane potential-related effect of calcium on reactive oxygen species generation in isolated brain mitochondria

The effect of Ca²⁺ applied in high concentrations (50 and 300 µM) was addressed on the generation of reactive oxygen species in isolated mitochondria from guinea-pig brain. The experiments were performed in the presence of ADP, a very effective inhibitor of mitochondrial permeability transition. Moderate increase in H₂O₂ release from mitochondria was induced by Ca²⁺ applied in 50 µM, but not in 300 µM concentration as measured with Amplex red fluorescent assay starting with a delay of 100-150 sec after exposure to Ca²⁺. Parallel measurements of membrane potential (ΔΨm) by safranine fluorescence showed a transient depolarization by Ca²⁺ followed by the recovery of ΔΨm to a value, which was more negative than that observed before addition of Ca²⁺ indicating a relative hyperpolarization. NAD(P)H fluorescence was also increased by Ca²⁺ given in 50 µM concentration. In mitochondria having high ΔΨm in the presence of oligomycin or ATP, the basal rate of release of H₂O₂ was significantly higher than that observed in a medium containing ADP and Ca²⁺ no longer increased but rather decreased the rate of H₂O₂ release. With 300 µM Ca²⁺ only a loss but no tendency of a recovery of ΔΨm was detected and H₂O₂ release was unchanged. It is suggested that in the presence of nucleotides the effect of Ca²⁺ on mitochondrial ROS release is related to changes in ΔΨm; in depolarized mitochondria, in the presence of ADP, moderate increase in H₂O₂ release is induced by calcium, but only in ≤ 100 µM concentration, when after a transient Ca²⁺-induced depolarization mitochondria became more polarized. In highly polarized mitochondria, in the presence of ATP or oligomycin, where no hyperpolarization follows the Ca²⁺-induced depolarization, Ca²⁺ fails to stimulate mitochondrial ROS generation. These effects of calcium (≤ 300 µM) are unrelated to mitochondrial permeability transition.     

Effects of salt stress on ion content,antioxidant enzymes and protein profile in different tissues of Broussonetia papyrifera

Although some plant responses to salinity have been characterized, the precise mechanisms by which salt stress damages plants are still poorly understood especially in woody plants. In the present study, the physiological and biochemical responses of Broussonetia papyrifera, a tree species of the family, Moraceae, to salinity were studied. In vitro-produced plantlets of B. papyrifera were treated with varying levels of NaCl (0, 50, 100 and 150 mM) in hydroponic culture. Changes in ion contents, accumulation of H₂O₂, as well as the activities and isoform profiles of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in the leaves, stems and roots were investigated. Under salt stress, there was higher Na⁺ accumulation in roots than in stems and leaves, and Ca^2 +, Mg^2 + and P^3 + content, as well as K⁺/Na⁺ ratio were affected. NaCl treatment induced an increase in H₂O₂ contents in the tissues of B. papyrifera. The work demonstrated that activities of antioxidant defense enzymes changed in parallel with the increased H₂O₂ and salinity appeared to be associated with differential regulation of distinct SOD and POD isoenzymes. Moreover, SDS-PAGE analysis of total proteins extracted from leaves and roots of control and NaCl-treated plantlets revealed that in the leaves salt stress was associated with decrease or disappearance of some protein bands, and induction of a new protein band after exposure to 100 and 150 mM NaCl. In contrast, NaCl stress had little effect on the protein pattern in the roots. In summary, these findings may provide insight into the mechanisms of the response of woody plants to salt stress.     

IBA-induced changes in antioxidant enzymes during adventitious rooting in mung bean seedlings: The role of H2O2

The changes in antioxidant enzyme activity during the induction of adventitious roots in mung bean seedlings treated with Indole-3-butyric acid (IBA), hydrogen peroxide (H₂O₂), ascorbic acid (ASA) and diphenylene iodonium (DPI) were investigated. As compared with the controls, treatments of seedlings with 10 μM IBA significantly decreased POD activity by 55% and 49.6% at 3 h and 12 h of incubation, respectively, and significantly increased by 49.8% at 36 h of incubation; treatments of seedlings with 10 mM H₂O₂ significantly decreased POD activity by 42%, 60%, 39% and 38% at 3 h, 12 h, 24 h and 48 h of incubation, respectively, the changes in POD activity were coincident with those in IBA-treated seedlings during the 0–12 h incubation period; treatments of seedlings with 2 mM ASA significantly decreased APX activities by 27% only at 3 h of incubation, the varying trend of POD activity was similar to incubation with water; 10 μM DPI treatments significantly decreased POD activity by 42%, 40%, 54% and 28% at 3 h, 6 h, 12 h and 48 h of treatment, respectively. CAT activities remained at relatively stable levels and no major changes occurred from 0 h to 48 h during the incubation phase of adventitious rooting. The results may imply that CAT, an H₂O₂-metabolizing enzyme, is inactivated by H₂O₂ during the formation of adventitious roots. As compared with the controls, IBA treatments significantly decreased APX activities by 48%, 53% and 66% at 3 h, 9 h and 12 h of treatment, respectively; H₂O₂ treatments significantly decreased APX activities by 59%, 51% and 57% at 3 h, 12 h and 36 h of incubation, respectively; ASA treatments significantly decreased APX activities by 37% only at 3 h of incubation; DPI treatments significantly decreased APX activities by 54%, 53% and 63% at 3 h, 6 h and 12 h of incubation, respectively, and significantly increased APX activity by 106% at 24 h. These results indicated that the influence of IBA, H₂O₂, ASA and DPI on the changes in APX activity were the same as on the changes in POD activity. Furthermore, similar trends in the changes of APX activity and POD activity were observed during the induction and initiation rooting phase. This finding implies that APX and POD serve the same functions, possibly related to the level of H₂O₂, during the formation of adventitious roots. The early decrease of POD and APX activities in the initiation phase of IBA- and H₂O₂-treated seedlings may be one mechanism underlying the IBA- and H₂O₂-mediated facilitation of adventitious rooting.     

Effect of atrial natriuretic peptide on reactive oxygen species-induced by hydrogen peroxide in THP-1 monocytes: Role in cell growth,migration and cytokine release

Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present study was to investigate in THP-1 monocytes the ANP effect on hydrogen peroxide (H₂O₂)-induced Reactive Oxygen Species (ROS), cell proliferation and migration. A significant increase of H₂O₂-dependent ROS production was induced by physiological concentration of ANP (10⁻¹⁰ M). The ANP action was partially affected by cell pretreatment with PD98059, an inhibitor of mitogen activated-protein kinases (MAPK) as well as by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) and totally suppressed by diphenylene iodonium (DPI), an inhibitor of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The hormone effect was mimicked by cANF and an ANP/NPR-C signaling pathway was studied using pertussis toxin (PTX). A significant increase of H₂O₂-induced cell migration was observed after ANP (10⁻¹⁰ M) treatment, conversely a decrease of THP-1 proliferation, due to cell death, was found. Both ANP actions were partially prevented by DPI. Moreover, H₂O₂-induced release of IL-9, TNF-α, MIP-1α and MIP-1β was not counteracted by DPI, whereas no effect was observed in any experimental condition for both IL-6 and IL-1β. Our results support the view that ANP can play a key role during the inflammatory process.     

Protective effects of aloe-emodin on scopolamine-induced memory impairment in mice and H2O2-induced cytotoxicity in PC12 cells

Aloe-emodin (AE) is one of the most important active components of Rheum officinale Baill. The present study aimed to investigate that AE could attenuate scopolamine-induced cognitive deficits via inhibiting acetylcholinesterase (AChE) activity and modulating oxidative stress. Kunming (KM) mice were received intraperitoneal injection of scopolamine (2 mg/kg) to induce cognitive impairment. Learning and memory performance were assessed in the Morris water maze (MWM). After behavioral testing, the mice were sacrificed and their hippocampi were removed for biochemical assays (superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), AChE and acetylcholine (ACh)). In vitro, we also performed the AChE activity assay and H₂O₂-induced PC12 cells toxicity assay. After 2 h exposure to 200 μM H₂O₂ in PC12 cells, the cytotoxicity were evaluated by cell viability (MTT), nitric oxide (NO)/lactate dehydrogenase (LDH) release and intracellular reactive oxygen species (ROS) production. Our results confirmed that AE showed significant improvement in cognitive deficit in scopolamine-induced amnesia animal model. Besides, it increased SOD, GPx activities and ACh content, while decreased the level of MDA and AChE activity in AE treated mice. In addition, AE was found to inhibit AChE activity (IC₅₀ = 18.37 μg/ml) in a dose-dependent manner. Furthermore, preincubation of PC12 cells with AE could prevent cytotoxicity induced by H₂O₂ and reduce significantly extracellular release of NO, LDH and intracellular accumulation of ROS. The study indicated that AE could have neuroprotective effects against Alzheimer’s disease (AD) via inhibiting the activity of AChE and modulating oxidative stress.     

Antioxidant response of a novel Streptomyces sp. M3004 isolated from legume rhizosphere to H2O2 and paraquat

This paper aims to investigate the effect of H₂O₂ and paraquat on the activities of superoxide dismutase (SOD) and catalase (CAT), and membrane lipid peroxidation (LPO) levels in newly isolated Streptomyces sp. M3004. SOD activities of Streptomyces sp. M3004, grown in 10 mM and 30 mM H₂O₂, were significantly lower than the control cultures. On the other hand, as an antioxidant enzyme, CAT activity in both H₂O₂ treatment conditions increased significantly compared with the control. These activity values in 10 mM and 30 mM H₂O₂ treatment on the 48th hour of incubation were 3.8- and 6.6-fold higher than the control, respectively. SOD activity decreased significantly with respect to paraquat concentration, which was added at the start of the incubation. CAT activities increased significantly in 1.0 mM and 3.0 mM paraquat treatments compared to control. As an indicative marker of membrane damage, LPO levels of the novel isolate Streptomyces sp. M3004 treated with H₂O₂, and paraquat stress conditions were significantly higher than the control. Nevertheless, compared with the 30 mM H₂O₂ in both treatment conditions, LPO levels in 10 mM H₂O₂ were significantly higher. The decreases in SOD activities in paraquat and H₂O₂ treatment conditions resulted in the increases in the LPO levels although it increases in CAT activities.     

Induction of heme oxygenase-1 by chamomile protects murine macrophages against oxidative stress

AimsProtection of cells from oxidative insult may be possible through direct scavenging of reactive oxygen species, or through stimulation of intracellular antioxidant defense mechanisms by induction of antioxidant gene expression. In this study we investigated the cytoprotective effect of chamomile and elucidated the underlying mechanisms.Main methodsThe cytoprotective effect of chamomile was examined on H₂O₂-induced cellular stress in RAW 264.7 murine macrophages.Key findingsRAW 264.7 murine macrophages treated with chamomile were protected from cell death caused by H₂O₂. Treatment with 50 μM H₂O₂ for 6 h caused significant increase in cellular stress accompanied by cell death in RAW 264.7 macrophages. Pretreatment with chamomile at 10–20 μg/mL for 16 h followed by H₂O₂ treatment protected the macrophages against cell death. Chamomile exposure significantly increased the expression of antioxidant enzymes viz. heme oxygenase-1 (HO-1), peroxiredoxin-1 (Prx-1), and thioredoxin-1 (Trx-1) in a dose-dependent manner, compared with their respective controls. Chamomile increased nuclear translocation of Nrf2 with increased phosphorylated Nrf2 levels, and binding to the antioxidant response element in the nucleus.SignificanceThese molecular findings for the first time provide insights into the mechanisms underlying the induction of phase 2 enzymes through the Keap1-Nrf2 signaling pathway by chamomile, and provide evidence that chamomile possesses antioxidant and cytoprotective properties.     

H2O2 preferentially synergizes with nitroprusside to induce apoptosis associated with superoxide dismutase dysregulation in human melanoma irrespective of p53 status: Antagonism by o-phenanthroline

The pro-oxidant hydrogen peroxide (H₂O₂) is converted to a reactive oxygen species by transition metals like iron. Since mutations in the p53 tumor suppressor gene contribute to drug resistance, we used genetically-matched human C8161 melanoma harbouring wt or DN-R175H mutant p53, to investigate the influence of p53 status on the potentiation of H₂O₂ toxicity by: (a) intact sodium nitroprusside or nitroferricyanide (SNP), (b) its light-exhausted NO-depleted form (lex-SNP), (c) potassium ferricyanide, or (d) ferric ammonium sulphate. Whereas single treatments with SNP or H₂O₂ were partly cytotoxic, preferentially potentiation of H₂O₂ toxicity was evidenced with intact or lex-SNP. No comparable increase of H₂O₂ toxicity was induced by ferricyanide, ferric ammonium sulphate or S-nitroso-N-acetyl penicillamine (SNAP), a known NO donor lacking iron. Immune blotting revealed apoptosis-associated PARP cleavage induced by [SNP + H₂O₂] irrespective of p53 status. This correlated with an eightfold induction of [Mn-SOD; SOD2] in wt p53 melanoma cells, and with a super-induction of the same enzyme reciprocal with loss of [Cu,Zn-SOD; SOD1], in mutant p53 cells. All these changes were antagonized by the anti-oxidant N-acetylcysteine or the iron chelator o-phenanthroline. We hypothesize that superoxide dismutase imbalance and iron-dependent redox changes involving OH species generated from a Fenton reaction between [SNP + H₂O₂], may be important in this anti-tumor activity. Although tumor drug resistance is frequently associated with DN-p53 mutations, our data shows for the first time the preferential ability of SNP to enhance H₂O₂ toxicity, irrespective of p53 status.     

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (H₂O₂). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O₂⁻/H₂O₂ formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O₂⁻/H₂O₂ formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H₂O₂. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O₂⁻/H₂O₂ production by KGDHC, induced a ~ 86% and ~ 84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O₂⁻/H₂O₂ formation by ~ 45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O₂⁻/H₂O₂ production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H₂O₂.     

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5 h/day for 5 days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H₂O₂). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca^2 +-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca^2 +-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.     

MAPKs as a cross point in H2O2 and auxin signaling under combined cadmium and zinc stress in rice roots

Previously, we have reported the role of MAPKs (mitogen-activated protein kinases) under cadmium stress. This work continue to explore the relationship between MAPKs, H₂O₂, auxin signaling, and OsHMA and OsZIP gene expression in rice (Oryza sativa L.) roots under combined cadmium (Cd) and zinc (Zn) stress. Compared with Cd, Cd+Zn reduced Cd levels but increased Zn accumulation in the roots. Three OsMAPK genes were negatively regulated, while two OsHMA and two OsZIP genes were positively regulated by MAPK pathways under Cd+Zn stress. Transgenic rice expressing DR5-GUS exhibited enhanced GUS activity in H₂O₂-, PD (MAPKK inhibitor PD98059)-, or (Cd+Zn)-treated roots, which also exhibited increased H₂O₂ concentrations, whereas GUS staining decreased in roots in response to Cd+Zn+PD, DMTU (N,N′-dimethylthiourea, a H₂O₂ scavenger), or Cd+Zn+DMTU treatment, with reduced H₂O₂ levels. GUS levels were consistent with H₂O₂ levels, suggesting that MAPK pathway-mediated auxin redistribution occurs via H₂O₂, and H₂O₂ functions downstream of MAPK but upstream of auxin signaling pathways. Furthermore, MAPK pathways serve specific functions in regulating the expression of some key genes of auxin signaling (OsYUCCA, OsPIN, OsARF, and OsIAA) under Cd+Zn stress. Overall, MAPK cascades function in the integration of metal transport, H₂O₂ generation, and auxin signaling in rice seedlings grown under Cd+Zn stress.     

Induction in the antioxidative systems and lipid peroxidation in suspension culture roots of Panax ginseng induced by oxygen in bioreactors

Roots of mountain ginseng (Panax ginseng) were exposed to various levels of oxygen (O₂) (30, 40 and 50%) for 15, 30 and 45 days in 5 L (working volume 4 L) airlift bioreactors. Ginsenoside accumulation and dry weight was enhanced up to 40% O₂; but thereafter declined ginsenoside and dry weight of the roots by increasing level of O₂. Gradual increase in H₂O₂ content and lipoxygenase activity (LOX), resulting in cellular damage and oxidative stress as indicated by increased malondialdehyde (MDA) content after 30 and 45 days at all O₂ levels was shown. Increased levels of O₂ (above ambient) resulted in increases in non-protein thiol (NP-SH) and cysteine content. Higher activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), catalase (CAT), guaiacol peroxidase (G-POD), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S transferase (GST) activities indicated that antioxidant enzymes played an important role in protecting the roots from O₂ up to 45 days, except at 50% O₂ where GR, GST and GPx decreased compared to the control. However, after 45 days, SOD activity decreased significantly compared to the control in the O₂-treated roots. This reflects the sensitivity of enzymes to O₂ toxicity. In stress related experiment, roots showed increased synthesis of ginsenosides when 25 and 50 μM H₂O₂ was applied. However, higher dose and increasing treatment inhibited ginsenoside synthesis. The results indicate that plant roots could grow and protect themselves from O₂ stress by coordinated induction of various antioxidant enzymes and metabolite contents. These results suggest that O₂ supplementation is useful for ginsenoside accumulation using 5-L bioreactors.     

Mitochondrial matrix P53 sensitizes cells to oxidative stress

A mitochondrial matrix-specific p53 construct (termed p53-290) in HepG2 cells was utilized to determine the impact of p53 in the mitochondrial matrix following oxidative stress. H₂O₂ exposure reduced cellular proliferation similarly in both p53-290 and vector cells, and p53-290 cells demonstrating decreased cell viability at 1 mM H₂O₂ (~ 85% viable). Mitochondrial DNA (mtDNA) abundance was decreased in a dose-dependent manner in p53-290 cells while no change was observed in vector cells. Oximetric analysis revealed reduced maximal respiration and reserve capacity in p53-290 cells. Our results demonstrate that mitochondrial matrix p53 sensitizes cells to oxidative stress by reducing mtDNA abundance and mitochondrial function.