Reactive Oxygen Species Superoxide Research Articles

Mitochondrial ND5 mutation mediated elevated ROS regulates apoptotic pathway epigenetically in a P53 dependent manner for generating pro-cancerous phenotypes

We have previously observed concomitant events of mutations in mitochondrial and nuclear genes, along with elevated reactive oxygen species (ROS) and differential methylation within the promoters of nuclear genes in tumors and in vitro experiments of tumorigenesis. These observations have made it pertinent to replicate and understand the role of acquired mitochondrial condition in tuning a cell to accomplish a pro-cancerous state. Using a codon optimized vector system for exogenous over-expression and mitochondrial localization; we have characterized here the role of over-expressed wild type mtND5 and one of its non-synonymous somatic mutation, ND5:P265H. The ectopically over-expressed ND5:P265H in mitochondria resulted in a reduced Complex I activity, generation of higher ADP/ATP ratio, reactive oxygen species (ROS) and carbonylation of proteins as compared to mock-transfected cells. Cells over-expressing mtND5 variant produced both peroxide as well as super-oxide ROS; the generation of which was dependent on the functional status of P53; modulating epigenetically the expression of key apoptosis pathway genes. The pro-cancerous phenotypes, of anchorage dependent and independent growth; increased glucose uptake and lactate production, were selectively observed only in P53 non-functional cells over-expressing mutant ND5:P265H. We propose that somatic mutation in mtND5 resulting in down-regulated complex I enzyme activity, elevated ROS and up-regulation of a set of nuclear anti-apoptotic genes epigenetically in the P53 dysfunctional cellular background, has provided a unique understanding of the molecular mechanism of mitochondrial mutation; and the concomitant existence of somatically acquired mitochondrial and nuclear p53 mutations, in cancer progression and promotion.

The human Krebs cycle 2-oxoglutarate dehydrogenase complex creates an additional source of superoxide/hydrogen peroxide from 2-oxoadipate as alternative substrate

Recently, we reported that the human 2-oxoglutarate dehydrogenase (hE1o) component of the 2-oxoglutarate dehydrogenase complex (OGDHc) could produce the reactive oxygen species superoxide and hydrogen peroxide (detected by chemical means) from its substrate 2-oxoglutarate (OG), most likely concurrently with one-electron oxidation by dioxygen of the thiamin diphosphate (ThDP)-derived enamine intermediate to a C2α-centered radical (detected by Electron Paramagnetic Resonance) [Nemeria et al., 2014 [17]; Ambrus et al. 2015 [18]]. We here report that hE1o can also utilize the next higher homologue of OG, 2-oxoadipate (OA) as a substrate according to multiple criteria in our toolbox: (i) Both E1o-specific and overall complex activities (NADH production) were detected using OA as a substrate; (ii) Two post-decarboxylation intermediates were formed by hE1o from OA, the ThDP-enamine and the C2α-hydroxyalkyl-ThDP, with nearly identical rates for OG and OA; (iii) Both OG and OA could reductively acylate lipoyl domain created from dihydrolipoyl succinyltransferase (E2o); (iv) Both OG and OA gave α-ketol carboligaton products with glyoxylate, but with opposite chirality; a finding that could be of utility in chiral synthesis; (v) Dioxygen could oxidize the ThDP-derived enamine from both OG and OA, leading to ThDP-enamine radical and generation of superoxide and H2O2. While the observed oxidation-reduction with dioxygen is only a side reaction of the predominant physiological product glutaryl-CoA, the efficiency of superoxide/ H2O2 production was 7-times larger from OA than from OG, making the reaction of OGDHc with OA one of the important superoxide/ H2O2 producers among 2-oxo acid dehydrogenase complexes in mitochondria.

Simultaneous estimation of scavenging capacities of peach extract for multiple reactive oxygen species by fluorescence fingerprint method

In this study, the potential of using fluorescence fingerprint, also known as fluorescence excitation–emission matrix, for estimating the scavenging capacity of peach extract on reactive oxygen species (ROS) was investigated. Samples from each of the five cultivars (Asama Hakuto, Hakuho, Kawanakajima Hakuto, Natsukko and Ougonto) were freeze-dried and crushed. The scavenging capacities of peach extracts for the target ROS (hydroxyl, superoxide, alkoxyl radicals and singlet oxygen) were measured by electron paramagnetic resonance spin trapping method. Fluorescence fingerprints of the same samples were obtained. Partial least squares regression analysis was carried out to develop prediction models for ROS scavenging capacity. The models were assessed by external validation. Fluorescence fingerprint was found to accurately estimate the scavenging capacity for the alkoxyl and superoxide radicals with the prediction error of 0.06mmoltroloxeq./mL and 0.31mmolα-lipoicacideq./mL with a coefficient of determination of prediction (R2P) of 0.78 and 0.91, respectively.

Radiation-Driven Formation of Reactive Oxygen Species in Oxychlorine-Containing Mars Surface Analogues.

The present study demonstrates that γ-radiolyzed perchlorate-containing Mars soil salt analogues (in a CO2 atmosphere) generate upon H2O wetting the reactive oxygen species (ROS) superoxide radical (O2•-), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH). This study also validates that analogue radiolysis forms oxychlorine species that, in turn, can UV-photolyze to •OH upon UV photolysis. This investigation was made possible by the development of a new assay for inorganic-origin O2•- and H2O2 determination and by the modification of a previous assay for soil •OH. Results show that radiolyzed Mg(ClO4)2 generates H2O2 and •OH; and when included as part of a mixture analogous to the salt composition of samples analyzed at the Mars Phoenix site, the analogue generated O2•-, H2O2, and •OH, with •OH levels 150-fold higher than in the radiolyzed Mg(ClO4)2 samples. Radiolyzed Mars Phoenix site salt analogue that did not contain Mg(ClO4)2 generated only •OH also at 150-fold higher concentration than Mg(ClO4)2 alone. Additionally, UV photolysis of the perchlorate γ radiolysis product chlorite (ClO2-) generated the oxychlorine products trihalide (Cl3-), chlorine dioxide (ClO2•), and hypochlorite (ClO-), with the formation of •OH by UV photolysis of ClO-. While the generation of ROS may have contributed in part to 14CO2 production in the Viking Labeled Release (LR) experiment and O2 (g) release in the Viking Gas Exchange (GEx) experiment, our results indicate that they are not likely to be the major contributor to the LR and GEx results. However, due to their highly reactive nature, they are expected to play a significant role in the alteration of organics on Mars. Additionally, experiments with hypochlorite show that the thermal stability of NaClO is in the range of the thermal stability observed for thermally liable oxidant responsible for the Viking LR results. Key Words: Mars-Oxygen-Salts-Radiation-Habitability. Astrobiology 17, 319-336.

Oncogenic MCT-1 activation promotes YY1-EGFR-MnSOD signaling and tumor progression

Tumor cells often produce high levels of reactive oxygen species (ROS) and display an increased ROS scavenging system. However, the molecular mechanism that balances antioxidative and oxidative stress in cancer cells is unclear. Here, we determined that oncogenic multiple copies in T-cell malignancy 1 (MCT-1) activity promotes the generation of intracellular ROS and mitochondrial superoxide. Overexpression of MCT-1 suppresses p53 accumulation but elevates the manganese-dependent superoxide dismutase (MnSOD) level via the YY1-EGFR signaling cascade, which protects cells against oxidative damage. Conversely, restricting ROS generation and/or targeting YY1 in lung cancer cells effectively inhibits the EGFR-MnSOD signaling pathway and cell invasiveness induced by MCT-1. Significantly, MCT-1 overexpression in lung cancer cells promotes tumor progression, necrosis and angiogenesis, and increases the number of tumor-promoting M2 macrophages and cancer-associated fibroblasts in the microenvironment. Clinical evidence further confirms that high expression of MCT-1 is associated with an increase in YY1, EGFR and MnSOD expression, accompanied by tumor recurrence, poor overall survival and EGFR mutation status in patients with lung cancers. Together, these data indicate that the MCT-1 oncogenic pathway is implicated in oxidative metabolism and lung carcinogenesis.

Tetrabromobisphenol A induces cellular damages in pancreatic β-cells in vitro

ABSTRACTTetrabromobisphenol A (TBBPA) is a well-known organobrominated flame retardant. TBBPA has been detected in the environment. The roles played by environmental pollutants in increasing the prevalence of metabolic syndrome are attracting increasing concern. In the present work, we investigated the effects of TBBPA on rat pancreatic β-cells (the RIN-m5F cell line). RIN-m5F cells were incubated with different concentrations of TBBPA for 48 h, and cell viability and the extent of apoptosis were determined. We also measured the levels of inflammatory cytokines, reactive oxygen species (ROS), mitochondrial adenosine triphosphate (ATP), and cardiolipin, as well as the extent of cytochrome c release from mitochondria. TBBPA reduced the ATP level, induced cardiolipin peroxidation and cytochrome c release, and triggered apoptotic cell death. Moreover, TBBPA increased the levels of inflammatory cytokines (TNF-α and IL-1β), nitric oxide, intracellular ROS, and mitochondrial superoxide. Together, our results indicate that TBBPA damages pancreatic β-cells by triggering mitochondrial dysfunction and inducing apoptosis.

Indoxyl sulfate potentiates endothelial dysfunction via reciprocal role for reactive oxygen species and RhoA/ROCK signaling in 5/6 nephrectomized rats

Accumulative indoxyl sulfate (IS) retained in chronic kidney disease (CKD) can potentiate vascular endothelial dysfunction, and herein, we aim at elucidating the underlying mechanisms from the perspective of possible association between reactive oxygen species (ROS) and RhoA/ROCK pathway. IS-treated nephrectomized rats are administered with antioxidants including NADPH oxidase inhibitor apocynin, SOD analog tempol, and mitochondrion-targeted SOD mimetic mito-TEMPO to scavenge ROS, or ROCK inhibitor fasudil to obstruct RhoA/ROCK pathway. First, we find in response to IS stimulation, antioxidants treatments suppress increased aortic ROCK activity and expression levels. Additionally, ROCK blockade prevent IS-induced increased NADPH oxidase expression (mainly p22phox and p47phox), mitochondrial and intracellular ROS (superoxide and hydrogen peroxide) generation, and decreased Cu/Zn-SOD expression in thoracic aortas. Apocynin, mito-TEMPO, and tempol also reverse these markers of oxidative stress. These results suggest that IS induces excessive ROS production and ROCK activation involving a circuitous relationship in which ROS activate ROCK and ROCK promotes ROS overproduction. Finally, ROS and ROCK depletion attenuate IS-induced decrease in nitric oxide (NO) production and eNOS expression levels, and alleviate impaired vasomotor responses including increased vasocontraction to phenylephrine and decreased vasorelaxation to acetylcholine, thereby preventing cardiovascular complications accompanied by CKD. Taken together, excessive ROS derived from NADPH oxidase and mitochondria coordinate with RhoA/ROCK activation in a form of positive reciprocal relationship to induce endothelial dysfunction through disturbing endothelium-dependent NO signaling upon IS stimulation in CKD status.

Exposure to tetrabromobisphenol A induces cellular dysfunction in osteoblastic MC3T3-E1 cells

ABSTRACTThis study was undertaken to investigate the possible involvement of oxidative stress in tetrabromobisphenol A (TBBPA)-induced toxicity in osteoblastic MC3T3-E1 cells. To examine the potential effect of TBBPA on cultured osteoblastic cells, we measured cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial superoxide, and mitochondrial parameters including adenosine triphosphate (ATP) level, cardiolipin content, cytochrome c release, cyclophilin levels, and differentiation markers in osteoblastic MC3T3-E1 cells. TBBPA exposure for 48 h caused the apoptosis and cytotoxicity of MC3T3-E1 cells. TBBPA also induced ROS and mitochondrial superoxide production in a concentration-dependent manner. These results suggest that TBBPA induces osteoblast apoptosis and ROS production, resulting in bone diseases. Moreover, TBBPA induced cardiolipin peroxidation, cytochrome c release, and decreased ATP levels which induced apoptosis or necrosis. TBBPA decreased the differentiation markers, collagen synthesis, alkaline phosphatase activity, and calcium deposition in cells. Additionally, TBBPA decreased cyclophilin A and B releases. Taken together, these data support the notion that TBBPA inhibits osteoblast function and has detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction.

Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy.

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

Graft-transmissible resistance of cherry pepper (Capsicum annuum var. cerasiforme) to powdery mildew (Leveillula taurica) is associated with elevated superoxide accumulation, NADPH oxidase activity and pathogenesis-related gene expression

We found that resistance to pepper powdery mildew (PM) (Leveillula taurica) develops in a sweet pepper (Capsicum annuum) cultivar (‘Total’) when grafted on a resistant cherry pepper (C. annuum var. cerasiforme) rootstock (cv. Szentesi). Resistance is manifested both towards PM symptoms and pathogen accumulation. In healthy, uninfected plants PM-resistance can be predicted by enhanced accumulation of the reactive oxygen species (ROS) superoxide (O 2 ) and activity of NADPH oxidase, the enzyme mainly responsible for pathogenesis-related superoxide generation. In L. taurica-inoculated PM-resistant ‘Szentesi’ high levels of superoxide and NADPH oxidase activity are sustained even 45 days after inoculation, as opposed to PM-susceptible ‘Total’. This is also true for ‘Total’ grafted on resistant ‘Szentesi’ rootstocks, where PM resistance, enhanced superoxide production and NADPH oxidase activity is likely due to an unknown, graft-transmitted signal. To further elucidate the mechanisms of graft-transmissible PM-resistance we monitored expression of pathogenesis-related (PR) genes in healthy and infected plants. In healthy plants, expression of CaPR-1 is several times higher in leaves of PM-resistant pepper than in sensitive plants, while high expression of CaPR-2 (glucanase) does not entirely correlate with PM-resistance, being detectable only in PM-resistant ‘Szentesi’. However, during advanced stages of PM-pathogenesis (45 DAI) expression of CaPR-1 and CaPR-2 is by far the highest in PM-susceptible ‘Total’. Our results suggest that the direct biochemical cause of graft-transmissible PM-resistance in pepper is the enhanced accumulation of NADPH oxidase-generated superoxide. To our knowledge, this is the first report on the role of ROS (superoxide) in graft-transmissible, pathogen-specific disease resistance.

Platelet CD36 Potentiates Thrombus Formation in Hyperlipidemic Conditions By Activating Redox Sensitive MAP Kinase ERK5

Atherosclerotic plaque instability is a pathological process that can lead to ischemic emergencies, such as myocardial infarction (MI) and stroke. Thrombosis in this context is promoted by circulating oxidized lipids present in LDL particles (oxLDL), which are generated during plaque formation. These particles are recognized by scavenger receptor CD36 present on platelets. CD36 binding to oxLDL lowers the threshold for platelet activation by recruitment and activation of Src kinases Fyn and Lyn, followed by signaling including generation of reactive oxygen species (ROS) through NADPH oxidase (Nox). Although these effectors are important for the prothrombotic properties of CD36, the downstream signaling that links CD36 to classic agonist-induced platelet activation pathways is incompletely defined. We hypothesize that platelet CD36 promotes thrombosis by generating specific ROS to modulate critical redox-sensitive signaling pathways in hyperlipidemia. Platelet MAP kinase ERK5 is a redox sensor that was shown to be required for optimal platelet activation in MI, a condition with greatly elevated ROS. We previously reported that oxLDL-CD36 signaling leads to the generation of the specific ROS superoxide radical anion (O2●-) and that O2●-/hydrogen peroxide (H2O2) are important mediators of platelet aggregation. Additionally, we reported that platelet ERK5 was activated by oxLDL in a CD36-dependent pathway requiring Src kinases, Nox and O2●-/H2O2 to promote platelet activation. We now report the function of platelet ERK5 in this context by performing an ex vivo microfluidic thrombosis assay in which mice whole blood is perfused over immobilized collagen. Platelet adhesion and accumulation were visualized in real time via mepacrine-tagged platelets. We found that stimulating whole blood from wild type C57Bl6 mice with oxLDL promoted platelet adhesion and accumulation by 12.9% compared to buffer stimulation (p=0.033). Whole blood from CD36 null mice showed indistinguishable platelet adhesion and accumulation when stimulated with oxLDL or buffer, suggesting CD36-dependency. We used the platelet ERK5 null mice, which was generated by crossing ERK5flox mice with PF4-cre+ mice (ERK5flox/PF4-cre+), and showed that platelet adhesion and accumulation by oxLDL was abrogated compared to control ERK5flox mice. Additionally, the in vivo relevance of platelet ERK5 activation by CD36 was determined by performing a novel collagen-mediated murine thrombosis assay. This assay is dependent on syngeneic transplantation of the epigastric artery into the carotid artery, where the collagen-rich outer adventitial layer is exposed to blood flow, thus generating a thrombus. Platelets and fibrin accumulation were visualized in real time by Rhodamine 6G and fluorophore-tagged anti-fibrin antibody, respectively. We transplanted bone marrows from donor ERK5flox mice or ERK5flox/PF4-cre+ mice into recipient atherogenic apoE null mice and generated hyperlipidemia by feeding the mice a high fat diet (HFD). ApoE null mice with ERK5flox bone marrows (apoE:ERK5floxBM) on HFD developed rapid platelet accumulation with subsequent thromboembolisms compared to apoE null mice with platelet ERK5-null bone marrows (apoE:ERK5flox/PF4-cre+BM) (p<0.01 at 10 min), demonstrating that this model is sensitive to detect a prothrombotic phenotype in diet-induced hyperlipidemia. Chow fed mice do not show potentiation of platelet accumulation in both apoE chimeras (p=0.56). Additionally, HFD-fed apoE:ERK5floxBM showed accelerated fibrin accumulation compared to apoE:ERK5flox/PF4-cre+BM, suggesting a role for CD36-ERK5 signaling in platelet procoagulant properties. Control diet-fed apoE null chimeras have indistinguishable fibrin accumulation kinetics. Subsequent studies to determine the mechanism for fibrin accumulation (via fluorophore-tagged Annexin V binding) showed that exposure of platelets to oxLDL induced dose-dependent phosphatidylserine (PS) exposure, a mechanism required for platelets to promote coagulation. Buffer or LDL controls had no effect. A CD36 blocking antibody, FA6, was able to abrogate oxLDL-mediated PS exposure compared to control antibody (p=0.041). These findings suggest that platelet CD36 potentiates thrombosis through a specific redox-regulated pathway requiring ERK5 and that ERK5 is a critical modulator of thrombosis in hyperlipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.

Species-specific control of external superoxide levels by the coral holobiont during a natural bleaching event.

The reactive oxygen species superoxide (O2·−) is both beneficial and detrimental to life. Within corals, superoxide may contribute to pathogen resistance but also bleaching, the loss of essential algal symbionts. Yet, the role of superoxide in coral health and physiology is not completely understood owing to a lack of direct in situ observations. By conducting field measurements of superoxide produced by corals during a bleaching event, we show substantial species-specific variation in external superoxide levels, which reflect the balance of production and degradation processes. Extracellular superoxide concentrations are independent of light, algal symbiont abundance and bleaching status, but depend on coral species and bacterial community composition. Furthermore, coral-derived superoxide concentrations ranged from levels below bulk seawater up to ∼120 nM, some of the highest superoxide concentrations observed in marine systems. Overall, these results unveil the ability of corals and/or their microbiomes to regulate superoxide in their immediate surroundings, which suggests species-specific roles of superoxide in coral health and physiology.

Threshold levels of extracellular l-arginine that trigger NOS-mediated ROS/RNS production in cardiac ventricular myocytes.

l-Arginine (L-Arg) is the substrate for nitric oxide synthase (NOS) to produce nitric oxide (NO), a signaling molecule that is key in cardiovascular physiology and pathology. In cardiac myocytes, L-Arg is incorporated from the circulation through the functioning of system-y+ cationic amino acid transporters. Depletion of L-Arg leads to NOS uncoupling, with O2 rather than L-Arg as the terminal electron acceptor, resulting in superoxide formation. The reactive oxygen species (ROS) superoxide (O2˙-), combined with NO, may lead to the production of the reactive nitrogen species (RNS) peroxynitrite (ONOO-), which is recognized as a major contributor to myocardial depression. In this study we aimed to determine the levels of external L-Arg that trigger ROS/RNS production in cardiac myocytes. To this goal, we used a two-step experimental design in which acutely isolated cardiomyocytes were loaded with the dye coelenterazine that greatly increases its fluorescence quantum yield in the presence of ONOO- and O2˙- Cells were then exposed to different concentrations of extracellular L-Arg and changes in fluorescence were followed spectrofluorometrically. It was found that below a threshold value of ~100 µM, decreasing concentrations of L-Arg progressively increased ONOO-/ O2˙--induced fluorescence, an effect that was not mimicked by d-arginine or l-lysine and was fully blocked by the NOS inhibitor l-NAME. These results can be explained by NOS aberrant enzymatic activity and provide an estimate for the levels of circulating L-Arg below which ROS/RNS-mediated harmful effects arise in cardiac muscle.

Perfluorooctanoic acid induces mitochondrial dysfunction in MC3T3-E1 osteoblast cells

ABSTRACTPerfluorooctanoic acid (PFOA), a stable organic perfluorinated compound, is an emerging persistent organic pollutant, found widely in human and wildlife populations. Recent evidence suggests that exposure to environmental toxicants can be associated with higher risks of osteoporosis and fractures. We studied the cellular toxicology of PFOA in MC3T3-E1osteoblast cells. To examine the effect of PFOA, we measured cell viability, reactive oxygen species (ROS), mitochondrial superoxide, and mitochondrial parameters including adenosine triphosphate (ATP) level, mitochondrial membrane potential (MMP), cardiolipin content, and cytochrome c release in MC3T3-E1 cells. Incubating MC3T3-E1 cells in different concentrations of PFOA for 48 h resulted in a concentration-dependent decrease in cell viability and significant inductions of ROS and mitochondrial superoxide. Moreover, PFOA induced MMP collapse, cardiolipin peroxidation, cytochrome c release, and decreased ATP levels, which in turn induced apoptosis or necrosis. When osteoblast differentiation markers were assessed, PFOA treatment caused a significant reduction in alkaline phosphatase activity, collagen synthesis, and mineralization in the cells. In summary, we found an ROS- and mitochondria-mediated pathway for the induction of cell damage by PFOA in MC3T3-E1 cells. Together, our results indicate that mitochondrial toxicity could be a plausible mechanism for the toxic effects of PFOA on osteoblast function.

Small but mighty: how minor components drive major biogeochemical cycles.

Small but mighty: how minor components drive major biogeochemical cycles.

Dark Production of Extracellular Superoxide by the Coral Porites astreoides and Representative Symbionts

The reactive oxygen species (ROS) superoxide has been implicated in both beneficial and detrimental processes in coral biology, ranging from pathogenic disease resistance to coral bleaching. Despite the critical role of ROS in coral health, there is a distinct lack of ROS measurements and thus an incomplete understanding of underpinning ROS sources and production mechanisms within coral systems. Here, we quantified in situ extracellular superoxide concentrations at the surfaces of aquaria-hosted Porites astreoides during a diel cycle. High concentrations of superoxide (~10’s of nM) were present at coral surfaces, and these levels did not change significantly as a function of time of day. These results indicate that the coral holobiont produces extracellular superoxide in the dark, independent of photosynthesis. As a short-lived anion at physiological pH, superoxide has a limited ability to cross intact biological membranes. Further, removing surface mucus layers from the P. astreoides colonies did not impact external superoxide concentrations. We therefore attribute external superoxide derived from the coral holobiont under these conditions to the activity of the coral host epithelium, rather than mucus-derived epibionts or internal sources such as endosymbionts (e.g., Symbiodinium). However, endosymbionts likely contribute to internal ROS levels via extracellular superoxide production. Indeed, common coral symbionts, including multiple strains of Symbiodinium (clades A to D) and the bacterium Endozoicomonas montiporae LMG 24815, produced extracellular superoxide in the dark and at low light levels. Further, representative P. astreoides symbionts, Symbiodinium CCMP2456 (clade A) and E. montiporae, produced similar concentrations of superoxide alone and in combination with each other, in the dark and low light, and regardless of time of day. Overall, these results indicate that healthy, non-stressed P. astreoides and representative symbionts produce superoxide externally, which is decoupled from photosynthetic activity and circadian control. Corals may therefore produce extracellular superoxide constitutively, highlighting an unclear yet potentially beneficial role for superoxide in coral physiology and health.

The Influence of Reactive Oxygen Species on Local Redox Conditions in Oxygenated Natural Waters

Redox conditions in natural waters are a fundamental control on biogeochemical processes and ultimately many ecosystem functions. While the dioxygen/water redox couple controls redox thermodynamics in oxygenated aquatic environments on geological timescales, it is kinetically inert in the extracellular environment on the much shorter timescales on which many biogeochemical processes occur. Instead, electron transfer processes on these timescales are primarily mediated by a relatively small group of trace metals and stable radicals, including the reactive oxygen species superoxide. Such processes are of critical biogeochemical importance because many of these chemical species are scarce nutrients, but may also be toxic at high concentrations. Furthermore, their bioavailability and potentially toxicity is typically strongly influenced by their redox state. In this paper, I examine to what extent redox conditions in oxygenated natural waters are expected to be reflected in the redox states of labile redox-active compounds that readily exchange electrons with the dioxygen/superoxide redox couple, and potentially with each other. Additionally, I present the hypothesis that that the relative importance of the dioxygen/superoxide and superoxide/hydrogen peroxide redox couples exerts a governing control on local redox conditions in oxygenated natural waters on biogeochemically important timescales. Given the recent discovery of widespread extracellular superoxide production by a diverse range of organisms, this suggests the existence of a fundamental mechanism for organisms to tightly regulate local redox conditions in their extracellular environment in oxygenated natural waters.

Protective effect of α-lipoic acid against antimycin A cytotoxicity in MC3T3-E1 osteoblastic cells.

Oxidative stress represents a major cause of cellular damage and death in the process of osteoporosis. Antimycin A (AMA) has been shown to stimulate mitochondrial superoxide anions and reactive oxygen species (ROS). α-Lipoic acid (α-LA) is a naturally occurring essential coenzyme in mitochondrial multienzyme complexes and acts as a key player in mitochondrial energy production. However, whether α-LA affects the cytotoxicity of AMA in osteoblastic cells is unknown. In this study, we investigated the protective effects of α-LA against AMA-induced cytotoxicity using the MC3T3-E1 osteoblast-like cell line. Our results indicated that α-LA treatment attenuated AMA-induced cytotoxicity and LDH release in a dose-dependent manner. Notably, a significant recovery effect of α-LA on mineralization inhibited by AMA was found. Our results also demonstrated that treatment with 50μM AMA leads to a reduction of mitochondrial membrane potential (MMP) and the complex IV dysfunction, which was inhibited by pretreatment with α-LA in a dose-dependent manner. In addition, treatment with α-LA significantly reduced the generation of ROS and mitochondrial superoxide production induced by AMA. In addition, our result suggests that PI3K/Akt and CREB pathways are related to the protective effect of α-LA. Importantly, Hoechst 33258 staining results indicated that pretreatment with α-LA prevented AMA-induced apoptosis. Mechanistically, we found that α-LA prevents MC3T3-E1 cells from apoptosis through attenuating cytochrome C release and reducing the level of cleaved caspase-3.

Preferential uptake of antioxidant carbon nanoparticles by T lymphocytes for immunomodulation.

Autoimmune diseases mediated by a type of white blood cell—T lymphocytes—are currently treated using mainly broad-spectrum immunosuppressants that can lead to adverse side effects. Antioxidants represent an alternative approach for therapy of autoimmune disorders; however, dietary antioxidants are insufficient to play this role. Antioxidant carbon nanoparticles scavenge reactive oxygen species (ROS) with higher efficacy than dietary and endogenous antioxidants. Furthermore, the affinity of carbon nanoparticles for specific cell types represents an emerging tactic for cell-targeted therapy. Here, we report that nontoxic poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), known scavengers of the ROS superoxide (O2•−) and hydroxyl radical, are preferentially internalized by T lymphocytes over other splenic immune cells. We use this selectivity to inhibit T cell activation without affecting major functions of macrophages, antigen-presenting cells that are crucial for T cell activation. We also demonstrate the in vivo effectiveness of PEG-HCCs in reducing T lymphocyte-mediated inflammation in delayed-type hypersensitivity and in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Our results suggest the preferential targeting of PEG-HCCs to T lymphocytes as a novel approach for T lymphocyte immunomodulation in autoimmune diseases without affecting other immune cells.

Myeloid-derived suppressor cells in murine AIDS inhibit B-cell responses in part via soluble mediators including reactive oxygen and nitrogen species, and TGF-β

Monocytic myeloid-derived suppressor cells (M-MDSCs) were increased during LP-BM5 retroviral infection, and were capable of suppressing not only T-cell, but also B-cell responses. In addition to previously demonstrating iNOS- and VISTA-dependent M-MDSC mechanisms, in this paper, we detail how M-MDSCs utilized soluble mediators, including the reactive oxygen and nitrogen species superoxide, peroxynitrite, and nitric oxide, and TGF-β, to suppress B cells in a predominantly contact-independent manner. Suppression was independent of cysteine-depletion and hydrogen peroxide production. When two major mechanisms of suppression (iNOS and VISTA) were eliminated in double knockout mice, M-MDSCs from LP-BM5-infected mice were able to compensate using other, soluble mechanisms in order to maintain suppression of B cells. The IL-10 producing regulatory B-cell compartment was among the targets of M-MDSC-mediated suppression.