Reactive Oxidative Species Levels Research Articles

The Ccz1 mediates the autophagic clearance of damaged mitochondria in response to oxidative stress in Candida albicans

Autophagy plays a critical role in response to numerous cellular stresses, such as nutrient deprivation, hypoxia, starvation and organelle damage. The disruption of autophagy pathway affects multiple aspects of cellular stress response. Here we for the first time identified Ccz1 as an essential component for autophagy in Candida albicans. Our experiments demonstrated that loss of CCZ1 gene led to vacuolar fragmentation and disruption of the autophagy pathway. Our results also suggested that Ccz1 functioned in oxidative stress. In the ccz1Δ/Δ mutant, the levels of reactive oxidative species (ROS) sharply increased under H2O2 treatment. Further studies demonstrated that breakdown of the autophagic clearance pathway led to the accumulation of oxidative stress-damaged mitochondria, and consequently elevated cellular ROS levels in the ccz1Δ/Δ mutant. Furthermore, deletion of CCZ1 led to a significant defect in filamentous development at both 30°C and 37°C. The disruption of CCZ1 gene led to decreased capacity of macrophage killing and increased sensitivity to the macrophages. In addition, the ccz1Δ/Δ mutant exhibited attenuated virulence and decreased fungal burdens in the mouse systemic infection model, indicating that CCZ1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of autophagy-related gene in C. albicans.

Bhlhe40 Represses PGC-1α Activity on Metabolic Gene Promoters in Myogenic Cells

PGC-1α is a transcriptional coactivator promoting oxidative metabolism in many tissues. Its expression in skeletal muscle (SKM) is induced by hypoxia and reactive oxidative species (ROS) generated during exercise, suggesting that PGC-1α might mediate the cross talk between oxidative metabolism and cellular responses to hypoxia and ROS. Here we found that PGC-1α directly interacted with Bhlhe40, a basic helix-loop-helix (bHLH) transcriptional repressor induced by hypoxia, and protects SKM from ROS damage, and they cooccupied PGC-1α-targeted gene promoters/enhancers, which in turn repressed PGC-1α transactivational activity. Bhlhe40 repressed PGC-1α activity through recruiting histone deacetylases (HDACs) and preventing the relief of PGC-1α intramolecular repression caused by its own intrinsic suppressor domain. Knockdown of Bhlhe40 mRNA increased levels of ROS, fatty acid oxidation, mitochondrial DNA, and expression of PGC-1α target genes. Similar effects were also observed when the Bhlhe40-mediated repression was rescued by a dominantly active form of the PGC-1α-interacting domain (PID) from Bhlhe40. We further found that Bhlhe40-mediated repression can be largely relieved by exercise, in which its recruitment to PGC-1α-targeted cis elements was significantly reduced. These observations suggest that Bhlhe40 is a novel regulator of PGC-1α activity repressing oxidative metabolism gene expression and mitochondrion biogenesis in sedentary SKM.

Rescue effect of lipid emulsion on bupivacaine-induced cardiac toxicity in cardiomyocytes.

The aim of the present study was to investigate the mechanism underlying the rescue effect of lipid emulsion on bupivacaine (BPV)‑induced cardiomyocyte toxicity. The inhibitory effects of BPV on H9c2 myoblast cell proliferation were investigated using an MTT assay. The H9c2 myoblast cells were treated with either 1 mM BPV or 1% lipid emulsion (LE) alone, or co‑treated with both of the drugs. Cell apoptosis was detected using both Annexin V/propidium iodide staining and a terminal deoxynucleotidyl transferase dUTP assay. The protein expression levels of apoptosis-associated proteins were quantified using western blot analysis, and the mRNA expression levels were quantified by reverse transcription‑quantitative polymerase chain reaction. The expression levels of reactive oxidative species, malondialdehyde, superoxide dismutase, and catalase were quantified using the optical density values obtained from a spectrophotometer. In addition, the mechanism underlying the mitochondrial function of the H9c2 myoblast cells was investigated using both JC‑1 staining, and cyclosporin A and atractyloside treatment. The results indicated that the H9c2 myoblast cells treated with BPV exhibited significantly higher levels of apoptosis. Furthermore, BPV treatment increased the levels of oxidative stress, and caused mitochondrial dysfunction within the H9c2 myoblast cells. LE treatment reversed the effects of BPV treatment in the H9c2 myoblast cells.

Berberis libanotica Ehrenb extract shows anti-neoplastic effects on prostate cancer stem/progenitor cells.

Cancer stem cells (CSCs), including those of advanced prostate cancer, are a suggested reason for tumor resistance toward conventional tumor therapy. Therefore, new therapeutic agents are urgently needed for targeting CSCs. Despite the minimal understanding of their modes of action, natural products and herbal therapies have been commonly used in the prevention and treatment of many cancers. Berberis libanotica Ehrenb (BLE) is a plant rich in alkaloids which may possess anti-cancer activity and a high potential for eliminating CSCs. We tested the effect of BLE on prostate cancer cells and our data indicated that this extract induced significant reduction in cell viability and inhibited the proliferation of human prostate cancer cell lines (DU145, PC3 and 22Rv1) in a dose- and time-dependent manner. BLE extract induced a perturbation of the cell cycle, leading to a G0-G1 arrest. Furthermore, we noted 50% cell death, characterized by the production of high levels of reactive oxidative species (ROS). Inhibition of cellular migration and invasion was also achieved upon treatment with BLE extract, suggesting a role in inhibiting metastasis. Interestingly, BLE extract had a major effect on CSCs. Cells were grown in a 3D sphere-formation assay to enrich for a population of cancer stem/progenitor cells. Our results showed a significant reduction in sphere formation ability. Three rounds of treatment with BLE extract were sufficient to eradicate the self-renewal ability of highly resistant CSCs. In conclusion, our results suggest a high therapeutic potential of BLE extract in targeting prostate cancer and its CSCs.

Resveratrol supplementation restores high-fat diet-induced insulin secretion dysfunction by increasing mitochondrial function in islet.

Resveratrol (RSV), a natural compound, is known for its effects on energy homeostasis. Here we investigated the effects of RSV and possible mechanism in insulin secretion of high-fat diet rats. Rats were randomly divided into three groups as follows: NC group (animals were fed ad libitum with normal chow for 8 weeks), HF group (animals were fed ad libitum with high-fat diet for 8 weeks), and HFR group (animals were treated with high-fat diet and administered with RSV for 8 weeks). Insulin secretion ability of rats was assessed by hyperglycemic clamp. Mitochondrial biogenesis genes, mitochondrial respiratory chain activities, reactive oxidative species (ROS), and several mitochondrial antioxidant enzyme activities were evaluated in islet. We found that HF group rats clearly showed low insulin secretion and mitochondrial complex dysfunction. Expression of silent mating type information regulation 2 homolog- 1 (SIRT1) and related mitochondrial biogenesis were significantly decreased. However, RSV administration group (HFR) showed a marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated SIRT1 protein expression and antioxidant enzyme activities, resulting in increased mitochondrial respiratory chain activities and decreased ROS level. This study suggests that RSV may increase islet mitochondrial complex activities and antioxidant function to restore insulin secretion dysfunction induced by high-fat diet.

Na⁺/H⁺ exchanger 1 participates in tobacco disease defence against Phytophthora parasitica var. nicotianae by affecting vacuolar pH and priming the antioxidative system.

Despite the importance of NHX1 (Na(+)/H(+) exchanger 1) in plant salt tolerance, little is known about its other functions. In this study, intriguingly, it was found that NHX1 participated in plant disease defence against Phytophthora parasitica var. nicotianae (Ppn) in Nicotiana benthamiana. NbNHX1 was originally isolated from N. benthamiana, and characterized. The subcellular localization of NbNHX1 with its C-terminus fused with green fluorescent protein indicated that NbNHX1 localized primarily to the tonoplast. Tobacco rattle virus-induced NbNHX1 silencing led to reduced H(+) efflux from the vacuole to cytoplasts, and decreased Ppn resistance in N. benthamiana. After attack by Ppn, NbNHX1-silenced plants exhibited impaired ability to scavenge reactive oxidative species (ROS) induced by the pathogen. Pea early browning virus-mediated ectopic expression of SeNHX1 (from Salicornia europaea) or AtNHX1 (from Arabidopsis thaliana) both conferred enhanced Ppn resistance to N. benthamiana, with a lower H2O2 concentration after Ppn inoculation. Further investigation of the role of NHX1 demonstrated that transient overexpression of NbNHX1 improved the vacuolar pH and cellular ROS level in N. benthamiana, which was coupled with an enlarged NAD(P) (H) pool and higher expression of ROS-responsive genes. In contrast, NbNHX1 silencing led to a lower pH in the vacuole and a lower cellular ROS level in N. benthamiana, which was coupled with a decreased NAD(P) (H) pool and decreased expression of ROS-responsive genes. These results suggest that NHX1 is involved in plant disease defence; and regulation of vacuolar pH by NHX1, affecting the cellular oxidation state, primes the antioxidative system which is associated with Ppn resistance in tobacco.

Interleukin-1β Reduces L-type Ca2+ Current through Protein Kinase Cϵ Activation in Mouse Heart

Inflammation is now widely recognized as a key component of heart disease. Patients suffering from arrhythmias and heart failure have increased levels of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β). Evidence suggests that these cytokines are important mediators of cardiac remodeling; however, their effects on ion channels and arrhythmogenesis remain incompletely understood. The L-type Ca(2+) current (ICaL) is a major determinant of the plateau phase of cardiac action potential and has a critical excitation-contraction coupling role. Thus, altering its properties could have detrimental effects on cardiac electrical and contractile functions. Accordingly, the objective of this study was to elucidate the effect of TNFα and IL-1β on ICaL, while exploring the underlying regulatory mechanisms. Neonatal mouse ventricular myocytes were treated with a pathophysiological concentration (30 pg/ml) of TNFα and IL-1β for 24 h. Voltage-clamp recordings showed that TNFα had no effect on ICaL, whereas IL-1β decreased the current density by 36%. Although both IL-1β- and TNFα-treated myocytes showed significant increase in reactive oxidative species (ROS), Western blot experiments revealed that only IL-1β increased PKCϵ membrane translocation. The antioxidant N-acetyl-L-cysteine normalized ROS levels and restored ICaL density. Furthermore, the PKCϵ translocation inhibitor ϵ-V1-2 blocked the effect of IL-1β on ICaL. The reduction of ICaL by IL-1β was also seen in cultured adult ventricular myocytes. Overall, chronic IL-1β treatment decreased ICaL density in cardiomyocytes. These effects implicated ROS signaling and PKCϵ activation. These findings could contribute to explain the role of IL-1β in the development of arrhythmia and heart failure.

Ceramides, mitochondrial fission, and reactive oxygen species a novel mechanism of obesity‐induced insulin resistance (LB167)

Reactive oxidative species (ROS) is a known byproduct of mitochondrial dysfunction and is believed to contribute to inflammation signaling commonly found in insulin resistance. Ceramide is a sphingolipid that serves as an important second messenger in an increasing number of stress‐related pathways. Ceramide has long been known to have morphological and physiological effects on mitochondria, which could potentially alter levels of ROS. The focus of this study is to determine whether increased ceramide levels result in ROS generation and decreased mitochondrial respiration in myotubes via altered mitochondrial morphology. C2C12 Skeletal muscle cells were used to assess the effect of ceramide on ROS generation and electron microsopy was used to image the morphological changes. Ceramide synthesis can be stimulated by treatment with exogenous fatty acid, thus myotubes were simultaneously treated with exogenous fatty acid following SPT2 knockdown (SPT is the rate limiting enzyme in ceramide synthesis), ROS increased within the myotubes and cellular respiration dropped. To verify the disruptive effect of ceramide on respiration caused by a change of mitochondrial morphology, cells were treated with a fission inhibitor (Mdivi‐1) prior to the addition of exogenous fatty acid. The resulting inhibition of mitochondrial fission via Mdivi‐1 helped protect the myotubes from ceramide‐induced metabolic disruption, including maintained mitochondrial respiration, and reduced ROS levels. Images taken with the confocal microscope verify that cells with increased levels of ceramide undergo mitochondrial fission while cells treated with Mdivi‐1 are protected from morphological changes. Altogether, these data suggest a critical role for mitochondrial fission as a mediator of ceramide‐induced metabolic disruption.

Bisdemethoxycurcumin suppresses MCF-7 cells proliferation by inducing ROS accumulation and modulating senescence-related pathways

BackgroundBisdemethoxycurcumin (BDMC) is a natural derivative of curcumin present in the phenolic components extracted from the dried rhizome of Curcuma longa L. BDMC demonstrated potential chemotherapeutic activities but the underlying mechanisms have not been fully clarified. In the present study, the role of reactive oxidative species (ROS) in the anti-cancer effects of BDMC was investigated. MethodsMCF-7 cells were exposed to BDMC, and then the cell proliferation, colony formation ability and cell cycle profile were analyzed. Cellular ROS level was determined by flow cytometry and fluorescent microscope observation using specific fluorescent probes. Mitochondrial membrane potential (ψm) was assessed using JC-1. In addition, effects of BDMC on senescence-related molecules were analyzed by western blot assay. ResultsBDMC significantly inhibited MCF-7 breast cancer cell proliferation, while a rapid rise of the intracellular ROS level accompanied with a reduction of Δψm were observed. In addition, BDMC activated the pro-apoptotic protein p53 and its downstream effector p21 as well as the cell cycle regulatory proteins p16 and its downstream effector retinoblastoma protein (Rb). All of these BDMC-induced effects were counteracted with the pre-incubation of the antioxidant N-acetylcysteine (NAC). ConclusionsThese results suggested that BDMC-induced ROS accumulation may contribute to its inhibitory effect on MCF-7 cell viability through regulation of p53/p21 and p16/Rb pathways.

Broad Suppression of NADPH Oxidase Activity Exacerbates Ischemia/Reperfusion Injury Through Inadvertent Downregulation of Hypoxia-inducible Factor-1α and Upregulation of Peroxisome Proliferator–activated Receptor-α

NADPH oxidase (Nox) 2 and Nox4 are major components of the Nox family which purposefully produce reactive oxidative species, namely O2(-) and H2O2, in the heart. The isoform-specific contribution of Nox2 and Nox4 to ischemia/reperfusion (I/R) injury is poorly understood. We investigated the role of Nox2 and Nox4 in mediating oxidative stress and myocardial injury during I/R using loss-of-function mouse models. Systemic (s) Nox2 knockout (KO), sNox4 KO, and cardiac-specific (c) Nox4 KO mice were subjected to I/R (30 minutes/24 hours, respectively). Both myocardial infarct size/area at risk and O2(-) production were lower in sNox2 KO, sNox4 KO, and cNox4 KO than in wild-type mice. Unexpectedly, however, the myocardial infarct size/area at risk was greater, despite less O2(-) production, in sNox2 KO+cNox4 KO (double-KO) mice and transgenic mice (Tg) with cardiac-specific expression of dominant-negative Nox, which suppresses both Nox2 and Nox4, than in wild-type or single KO mice. Hypoxia-inducible factor-1α was downregulated whereas peroxisome proliferator-activated receptor-α was upregulated in Tg-dominant-negative Nox mice. A cross with mice deficient in prolyl hydroxylase 2, which hydroxylates hypoxia-inducible factor-1α, rescued the I/R injury and prevented upregulation of peroxisome proliferator-activated receptor-α in Tg-dominant-negative Nox mice. A cross with peroxisome proliferator-activated receptor-α KO mice also attenuated the injury in Tg- dominant-negative Nox mice. Both Nox2 and Nox4 contribute to the increase in reactive oxidative species and injury by I/R. However, low levels of reactive oxidative species produced by either Nox2 or Nox4 regulate hypoxia-inducible factor-1α and peroxisome proliferator-activated receptor-α, thereby protecting the heart against I/R, suggesting that Noxs also act as a physiological sensor for myocardial adaptation.

Evidence of mitochondrial dysfunction and impaired ROS detoxifying machinery in Fanconi Anemia cells

Fanconi Anemia (FA) is a rare genetic disorder associated with a bone-marrow failure, cancer predisposition and hypersensitivity to DNA crosslinking agents. Majority of the 15 FA genes and encoded proteins characterized so far are integrated into DNA repair pathways, however, other important functions cannot be excluded. FA cells are sensitive to oxidants, and accumulation of oxidized proteins has been characterized for several FA subgroups. Clinical phenotypes of both FA and other closely related diseases suggest altered functions of mitochondria, organelles responsible for cellular energetic metabolism, and also serving as an important producer and the most susceptible target from reactive oxidative species (ROS). In this study, we have shown that elevated level of mitochondrial ROS in FA cells is in parallel with the decrease of mitochondrial membrane potential, the decrease of ATP production, impaired oxygen uptake and pathological changes in the morphology of mitochondria. This is accompanied by inactivation of enzymes that are essential for the energy production (F1F0ATPase and cytochrome C oxidase) and detoxification of ROS (superoxide dismutase, SOD1). In turn, overexpression of SOD1 could rescue oxygen consumption rate in FA-deficient cells. Importantly, the depletion of mitochondria improved survival rate of mitomycin C treated FA cells suggesting that hypersensitivity of FA cells to chemotherapeutic drugs could be in part due to the mitochondria-mediated oxidative stress. On the basis of our results, we propose that deficiency in FA genes lead to disabling mitochondrial ROS-scavenging machinery further affecting mitochondrial functions and suppressing cell respiration.

Activation of Wnt/β-catenin Pathway by Exogenous Wnt1 Protects SH-SY5Y Cells Against 6-Hydroxydopamine Toxicity

Wnt1, initially described as a modulator of embryonic development, has recently been discovered to exert cytoprotective effects in cellular models of several diseases, including Parkinson's disease (PD). We, therefore, examined the neuroprotective effects of exogenous Wnt1 on dopaminergic SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA). Here, we show that 10-500μM 6-OHDA treatment decreased cell viability and increased lactate dehydrogenase (LDH) leakage. SH-SY5Y cells treated with 100 μM 6-OHDA for 24 h showed reduced Wnt/β-catenin activity, decreased mitochondrial transmembrane potential, elevated levels of reactive oxidative species (ROS) and phosphatidylserine (PS) extraversion, increased levels of Chop and Bip/GRP78 and reduced level of p-Akt (Ser473). In contrast, exogenous Wnt1 attenuated 6-OHDA-induced changes. These results suggest that activation of the Wnt/β-catenin pathway by exogenous Wnt1 protects against 6-OHDA-induced changes by restoring mitochondria and endoplasmic reticulum (ER) function.

662 Molecular Mechanisms in Interleukin-6-induced Vascular Endothelial Growth Factor-C Expression in Lymphatic Endothelial Cells

Material and Methods: Mouse embryonic fibroblasts (MEFs) were isolated and cultured from E11-E13 embryos of control, Sos1-, Sos2or Sos1/2-null mice. To by-pass the lethality of embryos lacking Sos1, tamoxifen-induced Sos1-null mice were generated. Using our available Sos2-mutant mice, double knockout Sos1/Sos2 animals were also produced. Complete Sos1 depletion in MEFs was achieved after a nine day treatment of cultures with tamoxifen. To exclude tamoxifen off-target effects, control MEFs were also tamoxifen-treated. Cell signaling studies of our knockout MEFs were performed using PDGF or EGF as agonists. The ultrastructure of all experimental groups including control and Sos-deficient MEFs was examined by electron microscopy. Finally, flow cytometry analyses were employed to measure the level of oxidative stress in the MEFs. Results and Discussion: Light microscopy observations showed cytoplasmic swellings or inclusions in Sos1and double Sos1/Sos2-null MEFs. In contrast, tamoxifen-treated control MEFs did not exhibit these alterations. Ultrastructural analyses clearly demonstrated the occurrence of mitophagy in the cytoplasm of Sos1and Sos1/2-depleted MEFs, being more noticeable in the latest. Consistent with this, flow cytometry results seem to suggest an increase in the level of intracellular ROS (Reactive Oxidative Species) in mutant MEFs. Somewhat surprisingly, the absence of Sos proteins did not result in downregulation of Ras-MAPK signaling. In fact, quantitative measurements of RasMAPK activity in Sos-deficient MEFs showed unexpectedly enhanced levels in comparison to control fibroblasts. Conclusions: In conclusion, Sos proteins appear to be essential for normal homeostasis in MEFs. Additionally, the enhanced signal of Ras-MAPK pathway observed after depletion of Sos proteins suggests the occurrence of still undefined compensatory mechanisms. This work was supported by grants from FIS (PS09/01979), RTICC (RD06/0020/000), JCyL (SA044A08 and GR93), and Samuel SolorzanoBarruso Foundation.

664 Molecular Mechanisms in Simvastatin-induced HCT116 Colorectal Cancer Cell Apoptosis

Material and Methods: Mouse embryonic fibroblasts (MEFs) were isolated and cultured from E11-E13 embryos of control, Sos1-, Sos2or Sos1/2-null mice. To by-pass the lethality of embryos lacking Sos1, tamoxifen-induced Sos1-null mice were generated. Using our available Sos2-mutant mice, double knockout Sos1/Sos2 animals were also produced. Complete Sos1 depletion in MEFs was achieved after a nine day treatment of cultures with tamoxifen. To exclude tamoxifen off-target effects, control MEFs were also tamoxifen-treated. Cell signaling studies of our knockout MEFs were performed using PDGF or EGF as agonists. The ultrastructure of all experimental groups including control and Sos-deficient MEFs was examined by electron microscopy. Finally, flow cytometry analyses were employed to measure the level of oxidative stress in the MEFs. Results and Discussion: Light microscopy observations showed cytoplasmic swellings or inclusions in Sos1and double Sos1/Sos2-null MEFs. In contrast, tamoxifen-treated control MEFs did not exhibit these alterations. Ultrastructural analyses clearly demonstrated the occurrence of mitophagy in the cytoplasm of Sos1and Sos1/2-depleted MEFs, being more noticeable in the latest. Consistent with this, flow cytometry results seem to suggest an increase in the level of intracellular ROS (Reactive Oxidative Species) in mutant MEFs. Somewhat surprisingly, the absence of Sos proteins did not result in downregulation of Ras-MAPK signaling. In fact, quantitative measurements of RasMAPK activity in Sos-deficient MEFs showed unexpectedly enhanced levels in comparison to control fibroblasts. Conclusions: In conclusion, Sos proteins appear to be essential for normal homeostasis in MEFs. Additionally, the enhanced signal of Ras-MAPK pathway observed after depletion of Sos proteins suggests the occurrence of still undefined compensatory mechanisms. This work was supported by grants from FIS (PS09/01979), RTICC (RD06/0020/000), JCyL (SA044A08 and GR93), and Samuel SolorzanoBarruso Foundation.

The biphasic redox sensing of SENP3 accounts for the HIF-1 transcriptional activity shift by oxidative stress

To investigate the mechanisms underlying the biphasic redox regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity under different levels of oxidative stress caused by reactive oxidative species (ROS). HeLa cells were exposed to different concentrations of H(2)O(2) as a simple model for mild and severe oxidative stress. Luciferase reporter assay and/or quantitative real-time PCR were used to investigate the transcriptional activity. Immunoblot was used to detect protein expression. Chromatin immunoprecipitation assay was used to detect HIF-1/DNA binding. The interaction of p300 with HIF-1α or with SENP3, and the SUMO2/3 conjugation states of p300 were examined by coimmunoprecipitation. HIF-1 transcriptional activity in HeLa cells was enhanced by low doses (0.05-0.5 mmol/L) of H(2)O(2), but suppressed by high doses (0.75-8.0 mmol/L) of H(2)O(2). The amount of co-activator p300 bound to HIF-1α in HeLa cells was increased under mild oxidative stress, but decreased under severe oxidative stress. The ROS levels differentially modified cysteines 243 and 532 in the cysteine protease SENP3, regulating the interaction of SENP3 with p300 to cause different SUMOylation of p300, thus shifting HIF-1 transcriptional activity. The shift of HIF-1 transactivation by ROS is correlated with and dependent on the biphasic redox sensing of SENP3 that leads to the differential SENP3/p300 interaction and the consequent fluctuation in the p300 SUMOylation status.

The redox-sensitive transcription factor Nrf2 regulates murine hematopoietic stem cell survival independently of ROS levels

AbstractSeveral studies have found that high levels of reactive oxidative species (ROS) are associated with stem cell dysfunction. In the present study, we investigated the role of nuclear factor erythroid-2–related factor 2 (Nrf2), a master regulator of the antioxidant response, and found that it is required for hematopoietic stem progenitor cell (HSPC) survival and myeloid development. Although the loss of Nrf2 leads to increased ROS in most tissues, basal ROS levels in Nrf2-deficient (Nrf2−/−) BM were not elevated compared with wild-type. Nrf2−/− HSPCs, however, had increased rates of spontaneous apoptosis and showed decreased survival when exposed to oxidative stress. Nrf2−/− BM demonstrated defective stem cell function, as evidenced by reduced chimerism after transplantation that was not rescued by treatment with the antioxidant N-acetyl cysteine. Gene-expression profiling revealed that the levels of prosurvival cytokines were reduced in Nrf2−/− HSPCs. Treatment with the cytokine G-CSF improved HSPC survival after exposure to oxidative stress and rescued the transplantation defect in Nrf2−/− cells despite increases in ROS induced by cytokine signaling. These findings demonstrate a critical role for Nrf2 in hematopoiesis and stem cell survival that is independent of ROS levels.

Oxidative stress in synapse development and function

Oxidative stress, caused by increased levels of reactive oxidative species (ROS), is considered a major contributor to the aging process. How oxidative stress may bring about changes to structures and function in the aging brain is poorly understood. Oxidative stress activates a number of cellular responses, including activation of the Jun-N-terminal kinase (JNK) pathway and autophagy. In addition to their pathological role, ROS also act as signaling molecules. ROS such as nitric oxide have a well-known role in learning and memory. In addition, activation of JNK and its transcriptional effector AP-1 are well-known mediators of synaptic function and growth. Both are essential mediators of physiological correlates of learning and memory such as long-term potentiation. JNK and AP-1 are potently activated and regulated by oxidative stress and mediate protective cellular responses such as autophagy. Recent work at the Drosophila neuromuscular junction implicates autophagy as a regulator of synaptic growth via activation of the JNK signaling pathway. We here outline a framework predicating oxidative stress as a major regulator of synaptic function and growth by the activation of JNK/AP-1 and autophagy. Such responses, we suggest, may underpin some forms of synaptic growth responses and synaptic aging.

English

Elevated reactive oxidative species (ROS) are cytotoxic, and chronic elevated levels of ROS have been implicated in multiple diseases as well as cellular transformation and tumor progression. However, the potential for a transient and minimally toxic episode of ROS exposure, or a minimal threshold dose of ROS, to initiate disease or cellular transformation is unclear. We examined both transcriptional and phospho-proteomic responses of murine embryonic stem (ES) cells to a single brief exposure of minimally toxic hydrogen peroxide (H(2)O(2)). The cellular response was distinct from those induced by either an acute exposure to H(2)O(2) or the topoisomerase II poison etoposide. Analysis of tumorigenesis-related transcripts revealed a significant up-regulation of oncogenes and down-regulation of tumor suppressors. Analysis of the phospho-proteomic response demonstrated insulin-signaling induction, including insulin receptor Y972 hypophosphorylation, similar to insulin-resistance mouse models and observed in diabetic patients. In addition, ES cells were more resistant to ROS than differentiated cells, and retained their transcriptional self-renewal signature, suggesting stem cells have a higher potential for ROS-mediated mutagenesis and proliferation in vivo. These results are a direct demonstration that even brief and non-toxic exposures to ROS may induce transduction of insulin resistance and transformation signaling in stem cells leading to diabetes and cancer.

Ambient fecal reactive oxidative species (ROS) buffering capacity as a function of geography and habitual diet: A comparison of rural and urban Guatemalan subjects

BackgroundIn the ambient state, fecal matrix has a powerful capacity to buffer ROS induction in an in situ assay system.ObjectivesTo examine factors influencing the buffering capacity of stools across differing dietary habits, with emphasis on dietary fiber (DF), and between sexes.MethodsAmbient stool samples were collected on 2 nonconsecutive days in 20 rural women (RW) and 20 urban women (UW), with concurrent 24‐h recalls, and on 3 consecutive days in 27 urban men (UM). An assay for Fe‐induced adducts of salicylic acid in mg/mL units gauged fecal buffering. Stool non‐heme Fe was measured by a colorimetric test.>ResultsRW consumed 2.5 X the DF as UW: 40±21 vs 16±8 g, p<0.001, but ROS generation was identical across areas: 0.077±0.015 vs 0.078±0.019 mg/mL, p=0.86. UM had ROS levels 150% higher than women: 0.20±0.11 mg/mL, p<0.0001. Fecal non‐heme Fe associated poorly with ROS production, r=−0.05 (p=0.71), and failed to explain sex difference in buffering capacity.>ConclusionHigh and low DF intakes have no influence on ROS buffering in women, but sex strongly determines ambient oxidative quenching in stools. The latter could explain gender disparities for certain intestinal diseases.Funded by Hildegard Grunow Foundation

Relating Mitochondrial Flickering to Whole-Cell Oscillations in Cardiac Mitochondrial Networks

The membrane potential of individual mitochondria has been shown to “flicker” randomly, in conjunction with the release of reactive oxidative species (ROS), such as superoxide flashes demonstrated recently in experiments. Under conditions of laser-induced oxidative stress, mitochondrial depolarization waves and oscillations, attributed to ROS-induced ROS release (RIRR) have been observed in cardiac myocytes and simulated in computer models. However, how asynchronous single mitochondrial flickering transitions to organized mitochondrial waves and oscillations is unknown. In this study, we developed a simplified, agent-based model of mitochondrial networks. In a single mitochondrion, superoxide production was modeled as a bistable process, where low oxygen triggers a higher percentage of oxygen shunted to superoxide. Mitochondrial channels (IMAC and mPTP) open stochastically in response to superoxide inside the matrix and cytoplasm (RIRR), causing flickering in the membrane potential and release of superoxide into the cytoplasm. In a globally coupled network simulating a well-mixed population of isolated mitochondria in a cuvette, we recapitulated experimental findings from isolated heart mitochondria showing that membrane potential oscillated when oxygen reached a critically low level. The oscillations were a self-organizing behavior arising from synchronization of flickering mitochondria at low oxygen levels. Using the same mitochondrial model in a locally coupled network to simulate the mitochondrial network of a myocyte, we found that the frequency of randomly flickering increased as ROS levels increased. At a critical level, a phase transition occurred in which self-organizing clusters of depolarized mitochondria propagated through the network, resulting in ROS waves and whole-cell oscillations. Our model predicts that this phase transition can be induced through either excessive laser light or hypoxic conditions.