Reactive Oxygen Species Imbalance Research Articles

Nano-sized TiO2 (nTiO2) induces metabolic perturbations in Physarum polycephalum macroplasmodium to counter oxidative stress under dark conditions

Nano-sized TiO2 (nTiO2) exerts an oxidative effect on cells upon exposure to solar or UV irradiation and ecotoxicity of the nTiO2 is an urgent concern. Little information is available regarding the effect of TiO2 on cells under dark conditions. Metabolomics is a unique approach to the discovery of biomarkers of nTiO2 cytotoxicity, and leads to the identification of perturbed metabolic pathways and the mechanism underlying nTiO2 toxicity. In the present study, gas chromatography mass spectrometry (GC/MS)-based metabolomics was performed to investigate the effect of nTiO2 on sensitive cells (P. polycephalum macroplasmodium) under dark conditions. According to the multivariate pattern recognition analysis, at least 60 potential metabolic biomarkers related to sugar metabolism, amino acid metabolism, nucleotide metabolism, polyamine biosynthesis, and secondary metabolites pathways were significantly perturbed by nTiO2. Notably, many metabolic biomarkers and pathways were related to anti-oxidant mechanisms in the living organism, suggesting that nTiO2 may induce oxidative stress, even under dark conditions. This speculation was further validated by the biochemical levels of reactive oxygen species (ROS), 8-hydroxy-2-deoxyguanosine (8-OHdG), and total soluble phenols (TSP). We inferred that the oxidative stress might be related to nTiO2-induced imbalance of cellular ROS. To the best of our knowledge, the present study is the first to investigate the nTiO2-induced metabolic perturbations in slime mold, provide a new perspective of the mechanism underlying nTiO2 toxicity under dark conditions, and show that metabolomics can be employed as a rapid, reliable and powerful tool to investigate the interaction among organisms, the environment, and nanomaterials.

Altered Carbonylation Pattern in Myelodisplastic Syndromes

Abstract INTRODUCTION Myelodisplastic syndrome (MDS) is an aging associated clonal hematopoietic disorder characterized by a bone marrow failure in which immature blood cells do not mature properly. Oxidative stress seems to play a role in its pathogenesis. Reactive oxygen species (ROS) regulation exert a control between self-renewal, proliferation and cell differentiation. Thus, ROS imbalance could lead to disease, especially in bone marrow where cells seems to be vulnerable to oxidative stress. Interestingly, high ROS levels have been observed in MDS, which has been correlated with a diminished survival. Several types of molecules can be directly affected by oxidative damage. However, the oxidation of proteins is of particular concern since it leads to aggregation, unfolding, or conformational changes that may confer a loss of structural or functional activity. As an altered proteome has also been described during MDS, in this study we have analyzed the pattern of oxidized protein associated to MDS. Moreover, we analyzed whether MDS patients could be beneficed by the antioxidant effects of Deferasirox therapy. METHODS Protein carbonylation was analyzed in bone marrow (n=14; 8 MDS and 6 controls) by immunohistochemistry after derivatization of carbonyl compounds with 2,4-dinitrophenylhydrazine (DNPH) and specific antibody detection of the 2,4-dinitrophenyl hydrazones. Protein carbonylation was also studied in expanded erythroblasts during in vitro erythropoiesis (n=9; 5 MDS and 4 controls) by 1-D and 2-D immunoproteomics. p21 mRNA levels were analysed by qRT-PCR using Taqman probes in a Applied Biosystems 7500 RT-PCR System. β-glucuronidase gene was used as endogenous reference to normalize data. Differences in expression mean values between the groups were analyzed using T-student or Mann-Whitney U test. RESULTS Immunohistochemistry assays showed a higher level of carbonylation in MDS samples (Figure 1a). We observed carbonylation in the cytoplasm of myeloid cells, whereas erythroid cells remained negative in both, patients and controls. Interestingly, MDS patients treated with Deferasirox presented lower levels than non-treated patients. Further, to assess carbonylation in the erythroid precursors, we analyzed the pattern of carbonylated proteins in expanded erythroblasts. Again, a higher level was observed in MDS vs . control group, which was reverted after Deferasirox treatment (Figure 1b). Most carbonylated proteins ranged in a size between 40-250 KDa. A highly carbonylated 40 KDa band was detected in all analyzed SMD samples. As seen in the 2D immunoblots, these differences in carbonylation were caused by a limited number of proteins or aggregates, rather than to a large spectrum of proteins (Figure 1c). Finally, as oxidative stress signaling pathways could regulate the cell cycle by p53, we analyzed the expression of p21, a p53 target during stress response. Upregulation of p21 was significantly observed in MDS samples. Remarkably, this effect was reverted upon Deferasirox treatment (Figure 1d). CONCLUSION An altered protein carbonylation pattern of myeloid and erythroid lineages was observed in MDS patients. This boosted oxidative cellular environment seems to activate signaling pathways involving p21. Deferaxirox treatment partially reverted these oxidative lesions at cellular level. *both authors have contributed equally M.L. hold a postdoctoral Fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409) Download : Download high-res image (144KB) Download : Download full-size image Figure . Disclosures No relevant conflicts of interest to declare.

Cellular glutathione levels in HL-60 cells during respiratory burst are not correlated with ultra-weak photon emission

Recently, ultra-weak photon emission (UPE) was developed as a novel tool for measuring oxidative metabolic processes, as its generation is related to reactive oxygen species (ROS). Both an imbalance in ROS or the uncontrolled production of ROS can lead to oxidative stress, which is commonly associated with many diseases. In addition to playing several biological functions, the thiol amino acid glutathione has an important antioxidant function in the body's defense against ROS. Specifically, glutathione is an important endogenous antioxidant that helps maintain oxidant levels. At the cellular level, glutathione is present in its reduced form (GSH) at relatively high concentrations (in the millimolar range) and in its oxidized form (GSSG) at low concentrations (in the micromolar range). Thus, the GSH/GSSG ratio is often used as an indicator of cellular redox state. Here, we used the HL-60 cell line as a model system in order to determine whether UPE is correlated with intracellular GSH and GSSG levels. HL-60 cells were differentiated into neutrophil-like cells and then stimulated to undergo respiratory burst. We then recorded UPE in real time for 9000 seconds and used capillary electrophoresis coupled to mass spectrometry to measure GSH and GSSG levels in cell extracts. We found that although respiratory burst significantly decreased the GSH/GSSG ratio, this change was not significantly correlated with the UPE profile.

Gambogic acid inhibits thioredoxin activity and induces ROS-mediated cell death in castration-resistant prostate cancer.

Advanced prostate cancer (PrCa) is treated with androgen deprivation therapy, and although there is usually a significant initial response, recurrence arises as castrate resistant prostate cancer (CRPC). New approaches are needed to treat this genetically heterogeneous, phenotypically plastic disease. CRPC with combined homozygous alterations to PTEN and TP53 comprise about 30% of clinical samples. We screened eleven traditional Chinese medicines against a panel of androgen-independent Pten/Tp53 null PrCa-derived cell lines and identified gambogic acid (GA) as a highly potent growth inhibitor. Mechanistic analyses revealed that GA disrupted cellular redox homeostasis, observed as elevated reactive oxygen species (ROS), leading to apoptotic and ferroptotic death. Consistent with this, we determined that GA inhibited thioredoxin, a necessary component of cellular anti-oxidative, protein-reducing activity. In other clinically relevant models, GA displayed submicromolar, growth inhibitory activity against a number of genomically-representative, CRPC patient derived xenograft organoid cultures. Inhibition of ROS with N-acetyl-cysteine partially reversed growth inhibition in CRPC organoids, demonstrating ROS imbalance and implying that GA may have additional mechanisms of action. These data suggest that redox imbalances initiated by GA may be useful, especially in combination therapies, for treating the heterogeneity and plasticity that contributes to the therapeutic resistance of CRPC.

New Challenges to Study Heterogeneity in Cancer Redox Metabolism.

Reactive oxygen species (ROS) are important pathophysiological molecules involved in vital cellular processes. They are extremely harmful at high concentrations because they promote the generation of radicals and the oxidation of lipids, proteins, and nucleic acids, which can result in apoptosis. An imbalance of ROS and a disturbance of redox homeostasis are now recognized as a hallmark of complex diseases. Considering that ROS levels are significantly increased in cancer cells due to mitochondrial dysfunction, ROS metabolism has been targeted for the development of efficient treatment strategies, and antioxidants are used as potential chemotherapeutic drugs. However, initial ROS-focused clinical trials in which antioxidants were supplemented to patients provided inconsistent results, i.e., improved treatment or increased malignancy. These different outcomes may result from the highly heterogeneous redox responses of tumors in different patients. Hence, population-based treatment strategies are unsuitable and patient-tailored therapeutic approaches are required for the effective treatment of patients. Moreover, due to the crosstalk between ROS, reducing equivalents [e.g., NAD(P)H] and central metabolism, which is heterogeneous in cancer, finding the best therapeutic target requires the consideration of system-wide approaches that are capable of capturing the complex alterations observed in all of the associated pathways. Systems biology and engineering approaches may be employed to overcome these challenges, together with tools developed in personalized medicine. However, ROS- and redox-based therapies have yet to be addressed by these methodologies in the context of disease treatment. Here, we review the role of ROS and their coupled redox partners in tumorigenesis. Specifically, we highlight some of the challenges in understanding the role of hydrogen peroxide (H2O2), one of the most important ROS in pathophysiology in the progression of cancer. We also discuss its interplay with antioxidant defenses, such as the coupled peroxiredoxin/thioredoxin and glutathione/glutathione peroxidase systems, and its reducing equivalent metabolism. Finally, we highlight the need for system-level and patient-tailored approaches to clarify the roles of these systems and identify therapeutic targets through the use of the tools developed in personalized medicine.

The Combination of Physical Exercise with Muscle-Directed Antioxidants to Counteract Sarcopenia: A Biomedical Rationale for Pleiotropic Treatment with Creatine and Coenzyme Q10.

Sarcopenia represents an increasing public health risk due to the rapid aging of the world's population. It is characterized by both low muscle mass and function and is associated with mobility disorders, increased risk of falls and fractures, loss of independence, disabilities, and increased risk of death. Despite the urgency of the problem, the development of treatments for sarcopenia has lagged. Increased reactive oxygen species (ROS) production and decreased antioxidant (AO) defences seem to be important factors contributing to muscle impairment. Studies have been conducted to verify whether physical exercise and/or AOs could prevent and/or delay sarcopenia through a normalization of the etiologically relevant ROS imbalance. Despite the strong rationale, the results obtained were contradictory, particularly with regard to the effects of the tested AOs. A possible explanation might be that not all the agents included in the general heading of “AOs” could fulfill the requisites to counteract the complex series of events causing/accelerating sarcopenia: the combination of the muscle-directed antioxidants creatine and coenzyme Q10 with physical exercise as a biomedical rationale for pleiotropic prevention and/or treatment of sarcopenia is discussed.

352 - Heme-Driven ROS Production Leads to Increased P38 MAP Kinase Activity and Endothelial Barrier Disruption

Heme is involved in a wide number of biological processes including gas exchange, antioxidant defense, signal transduction, as well as energy production within the mitochondria. It’s concentration within the cell is tightly regulated, and the consequences of underproduction of heme (anemia) are widely recognized. However, the pathophysiological role of high heme concentrations is less studied, but is already implicated in a number of cardiopulmonary diseases. An overabundance of intracellular heme precedes an over production of mitochondrial reactive oxygen species (ROS). ROS imbalance in the cell overwhelms basal antioxidant defenses and leads to various stress responses. Primarily, in this study, we focus on heme-driven effects of cellular signaling on human lung micro vascular endothelial cells (HLMVECs) permeability. We show that the p38 mitogen activated protein kinase (MAP kinase) pathway is activated with a specific response over a time course of heme treatment and that heme driven ROS production and cellular responses leads to endothelial barrier disruptions. In addition, we focus on a few therapeutic targets of heme pathway with potential to alleviate the harmful and cascading effects of ROS: 1) Inhibition of Heme Carrier Protein 1 (HCP-1) that mediates free heme transition into the cell; 2) Inhibition of heme mediated mitochondrial ROS production to study the downstream effects of lowering mitochondrial ROS on HLMVEC permeability; 3) Inhibition of p38 MAP Kinase to study the downstream effects of lowering activity of this pathway counteracting the response of ROS activity. These potential strategies in combating the pathophysiology of high levels of free heme are preventive and solidify our understanding of intracellular heme activity. However, the specific mechanism by which heme/ROS activates stress signaling pathways is a topic for future study and of high therapeutic value.

Evaluating the toxic potential of benzothiazoles with the rainbow trout cell lines, RTgill-W1 and RTL-W1

Benzothiazole (BTHs) are environmental contaminants of emerging concern for which little toxicological information is available. Therefore the toxic potential of twelve BTHs was evaluated with two rainbow trout epithelial cell lines, RTgill-W1 and RTL-W1. The BTHs were benzothiazole (BTH), 3,3′-diethylthia dicarbocyanine iodide (DTDC), C.I. sulphur orange 1 (SO), 2-mercaptobenzothiazole (2MBTH), zinc 2-mercaptobenzothiazole (ZnMBTH), sodium 2-mercaptobenzothiazole (NaMBTH), 2-hydroxy-benzothiazole (OHBTH), 2- aminobenzothiazole (2ABTH), C.I. vat yellow 2 (VY), N,N-dicyclohexyl-2-benzothiazolsulfene amide (NNA), 2,2′-dithiobis (benzothiazole) (DBTH) and 2-(p-aminophenyl)-6-methylbenzothiazole-7-sulfonic acid (MBTHS). All BTHs, except for NNA, DBTH, and MBTHS, caused both cytotoxicity and a transitory elevation in reactive oxygen species (ROS) levels. Yet, neither N-acetyl cysteine (NAC) nor IM-54 inhibited cytotoxicity, suggesting that ROS imbalance did not contribute to cell death. Cell death was not blocked by Necrostatin-1 nor accompanied by DNA laddering, suggesting that neither necroptosis nor apoptosis took place. The comet assay revealed DNA strand breaks after exposures to 2ABTH and OHBTH for 1 day and to BTH for 12 days. In RTL-W1, cytochrome P4501A was induced noticeably by 2ABTH, OHBTH, and MBTHS and weakly by NaMBTH, ZnMBTH, SO, VY, and NNA, suggesting that these BTHs have the potential to alter xenobiotic metabolism and activate the aryl hydrocarbon receptor. In summary, several toxic actions were initiated in vitro by some but not all BTHs, warranting further study of these BTHs in vivo.

ROS homeostasis as a prerequisite for the accomplishment of plant cytokinesis.

Reactive oxygen species (ROS) are emerging players in several biological processes. The present work investigates their potential involvement in plant cytokinesis by the application of reagents disturbing ROS homeostasis in root-tip cells of Triticum turgidum. In particular, the NADPH-oxidase inhibitor diphenylene iodonium, the ROS scavenger N-acetyl-cysteine, and menadione that leads to ROS overproduction were used. The effects on cytokinetic cells were examined using light, fluorescence, and transmission electron microscopy. ROS imbalance had a great impact on the cytokinetic process including the following: (a) formation of atypical "phragmoplasts" incapable of guiding vesicles to the equatorial plane, (b) inhibition of the dictyosomal and/or endosomal vesicle production that provides the developing cell plates with membranous and matrix polysaccharidic material, (c) disturbance of the fusion processes between vesicles arriving on the cell plate plane, (d) disruption of endocytic vesicle production that mediates the removal of the excess membrane material from the developing cell plate, and (e) the persistence of large callose depositions in treated cell plates. Consequently, either elevated or low ROS levels in cytokinetic root-tip cells resulted in a total inhibition of cell plate assembly or the formation of aberrant cell plates, depending on the stage of the affected cytokinetic cells. The latter failed to expand towards cell cortex and hence to give rise to complete daughter cell wall. These data revealed for the first time the necessity of ROS homeostasis for accomplishment of plant cytokinesis, since it seems to be a prerequisite for almost every aspect of this process.

Oxidant Mechanisms in Renal Injury and Disease.

A common link between all forms of acute and chronic kidney injuries, regardless of species, is enhanced generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during injury/disease progression. While low levels of ROS and RNS are required for prosurvival signaling, cell proliferation and growth, and vasoreactivity regulation, an imbalance of ROS and RNS generation and elimination leads to inflammation, cell death, tissue damage, and disease/injury progression. Many aspects of renal oxidative stress still require investigation, including clarification of the mechanisms which prompt ROS/RNS generation and subsequent renal damage. However, we currently have a basic understanding of the major features of oxidative stress pathology and its link to kidney injury/disease, which this review summarizes. The review summarizes the critical sources of oxidative stress in the kidney during injury/disease, including generation of ROS and RNS from mitochondria, NADPH oxidase, and inducible nitric oxide synthase. The review next summarizes the renal antioxidant systems that protect against oxidative stress, including superoxide dismutase and catalase, the glutathione and thioredoxin systems, and others. Next, we describe how oxidative stress affects kidney function and promotes damage in every nephron segment, including the renal vessels, glomeruli, and tubules. Despite the limited success associated with the application of antioxidants for treatment of kidney injury/disease thus far, preventing the generation and accumulation of ROS and RNS provides an ideal target for potential therapeutic treatments. The review discusses the shortcomings of antioxidant treatments previously used and the potential promise of new ones. Antioxid. Redox Signal. 25, 119-146.

Prostate-specific membrane antigen-targeted liposomes specifically deliver the Zn(2+) chelator TPEN inducing oxidative stress in prostate cancer cells.

To evaluate the potential use of zinc chelation for prostate cancer therapy using a new liposomal formulation of the zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). TPEN was encapsulated in nontargeted liposomes or liposomes displaying an aptamer to target prostate cancer cells overexpression prostate-specific membrane antigen. The prostate cancer selectivity and therapeutic efficacy of liposomal (targeted and nontargeted) and free TPEN were evaluated in vitro and in tumor-bearing mice. TPEN chelates zinc and results in reactive oxygen species imbalance leading to cell death. Delivery of TPEN using aptamer-targeted liposomes results in specific delivery to targeted cells. In vivo experiments show that TPEN-loaded, aptamer-targeted liposomes reduce tumor growth in a human prostate cancer xenograft model.

Repetitive ROS Injury Leads to Inactivation of NRF2‐Induced Antioxidant Defense and Mitochondrial Dysfunction in Ischemic Heart Failure

Ischemic heart failure (IHF) is associated with an imbalance of reactive oxygen species (ROS) and antioxidants. In IHF, the failing heart is repeatedly exposed to increased ROS secondary to multiple factors including mitochondrial dysfunction. Repetitive injury, such as occurs in IHF with increased ROS, can have effects much different from a single treatment. Nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2), is the master regulator of antioxidant genes. Nrf2 binds to Kelch like‐ECH‐associated protein (KEAP) 1 under normal conditions. When stressed by ROS, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it activates the antioxidant response element (ARE) pathway. We found that Nrf2 was paradoxically decreased in left ventricles from rats with IHF, as are antioxidant genes. In isolated adult rat cardiac myocytes subjected to repetitive H2O2 treatment, this repetitive ROS exposure decreased Nrf2 expression, nuclear binding activity, and reduced cytoplasmic Nrf2 translocation to the nucleus. Moreover, mitochondria isolated from the stressed cardiac myocyte showed decreased expression of mitochondrial dynamics related proteins, including OPA1, Parkin, and VCP, consistent with our findings with in vivo heart failure. The Nrf2 knockout heterozygous mice had impaired cardiac function. Overall, our data show that repetitive ROS treatment leads to paradoxical inactivation of the Nrf2‐induced antioxidant defense and mitochondrial dysfunction in heart failure.

Targeting Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer by Inducing Epidermal Growth Factor Receptor Degradation via Methionine 790 Oxidation.

Aims: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to treat non-small cell lung cancer (NSCLC) patients with EGFR mutation, but TKI resistance is common. Almost half of the acquired resistance patients are due to additional T790M mutation on EGFR (EGFRT790M), thus overcoming TKI resistance is important. In this study, we aim to investigate the role of reactive oxygen species (ROS) in TKI resistance as well as the molecular and biological effects of EGFRT790M after redox manipulation. Results: The basal ROS levels in EGFRT790M-containing TKI-resistant NSCLC cell lines were substantially high. Sixty-three human lung tumors showed higher NADPH oxidase isoform 2 (NOX2) expression than normal lung tissues, which may contribute to high basal ROS in cancer and poor survival. Interestingly, only NOX3 was upregulated by sanguinarine, a pharmacological agent to elevate ROS, and resulted in EGFR overoxidation, degradation, and apoptosis. By contrast, such responses were lacking in EGFRWT cells. Selective EGFRT790M degradation was manipulated by redox imbalance between NOX3 and methionine reductase A (MsrA). Furthermore, the in vivo tumor suppression effect of sanguinarine, NOX3 upregulation, and EGFR degradation were confirmed. Innovation: We have found a new treatment strategy to overcome TKI resistance by selectively inducing EGFRT790M degradation via specific stimulation of methionine 790 (M790) oxidation. It can be achieved via manipulating redox imbalance between NOX3 and MsrA. Conclusion: Targeting EGFR by elevating ROS and redox imbalance is a potential new strategy to develop a new EGFR inhibitor for TKI-resistant patients with a wide therapeutic window between EGFRT790M and EGFRWT. Antioxid. Redox Signal. 24, 263–279.

NET amyloidogenic backbone in human activated neutrophils.

Activated human neutrophils produce a fibrillar DNA network [neutrophil extracellular traps (NETs)] for entrapping and killing bacteria, fungi, protozoa and viruses. Our results suggest that the neutrophil extracellular traps show a resistant amyloidogenic backbone utilized for addressing reputed proteins and DNA against the non-self. The formation of amyloid fibrils in neutrophils is regulated by the imbalance of reactive oxygen species (ROS) in the cytoplasm. The intensity and source of the ROS signal is determinant for promoting stress-associated responses such as amyloidogenesis and closely related events: autophagy, exosome release, activation of the adrenocorticotrophin hormone/α-melanocyte-stimulating hormone (ACTH/α-MSH) loop and synthesis of specific cytokines. These interconnected responses in human activated neutrophils, that have been evaluated from a morphofunctional and quantitative viewpoint, represent primitive, but potent, innate defence mechanisms. In invertebrates, circulating phagocytic immune cells, when activated, show responses similar to those described previously for activated human neutrophils. Invertebrate cells within endoplasmic reticulum cisternae produce a fibrillar material which is then assembled into an amyloidogenic scaffold utilized to convey melanin close to the invader. These findings, in consideration to the critical role played by NET in the development of several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in immunomediated diseases in which the innate branch of the immune system has a pivotal role.

An Impdh Inhibitor, FF-10501 Possesses Activating Effects on Myeloid Differentiation As Well As Anti-Proliferation

Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders, characterized by peripheral blood cytopenia and risk of leukemic progression. The treatment strategy is subdivided into anti-cancer therapy and the improvement of refractory cytopenia. The therapeutic options are still limited while the number of patients is increasing with growth of the aging population. Interestingly, some of the cytotoxic agents such as alkylating agents, antimetabolites, and antitumor antibiotics have been reported the potency of promoting the differentiation of leukemic cell-lines in cultures. FF-10501, the inhibitor of inosine-5-monophosphate dehydrogenase (IMPDH), which interferes with the synthesis of nucleic acids, is one of candidates for a novel agent for MDS treatment, and now phase I clinical trials for high-risk MDS patients are running in the United States (FF10501US101) and Japan (FF10501JP101). In this study, we evaluated the effects of this agent on myeloid differentiation using human hematopoietic cells. Human samples were collected after informed consent with protocols approved by the Investigational Review Board of Osaka University Hospital.First, FF-10501 inhibited the proliferation of 9 cell-lines of hematological malignancy, including K562, HL-60 and MOLM-13. The erythroid differentiation of K562 human leukemic cells and F36P MDS cells was promoted in the same condition. The induction of differentiation to either granulocytes or monocytes in acute myeloid leukemia cells, MOLM-13 and HL-60 was also observed. These effects were dose-dependent. To elucidate the detailed mechanisms of the FF-10501-induced differentiation, we studied the alternative pathway activated after the IMPDH inhibition. As expected, the production of GMP, GDP and GTP was suppressed while that of ADP was not altered. The accumulation of inosine-5-monophosphate (IMP) was recognized. Among the metabolic changes induced by FF-10501 treatment, we found the elevation in the concentration of inosine and hypoxanthine. Because it is known that reactive oxygen species (ROS) are generated during the degradation of IMP, we examined whether the imbalance of ROS would be associated with the differentiation effect. When ROS scavenger, dimethylthiourea was added into cultures with FF-10501, the erythroid differentiation of K562 and the granulocytic differentiation of MOLM-13 were attenuated. Moreover, the inhibition of p38 also cancelled the effects. These results indicate that overproduction of ROS and the consequent activation of p38 contribute to the promotion of myeloid differentiation of hematological tumor cells by FF-10501.To apply our findings for clinical settings, we examined the effects of FF-10501 using human mononuclear blood cells. When bone marrow cells derived from healthy donors and MDS patients were cultured in colony-forming unit assays, the number of recovered colonies decreased in a dose-dependent manner. Although high dose of FF-10501 (≥ 40 μM) showed strong cytotoxicity, we found that the intermediate dose of FF-10501 promoted generation of erythroid cells from CD34+ hematopoietic stem/progenitor cells. After 3 week-cultures with 4 μM of FF-10501 under erythroid condition (20 ng/ml IL-3, 10 ng/ml Flt3-ligand, 50 ng/ml thrombopoietin, 20 ng/ml stem cell factor and 10 ng/ml erythropoietin) on MS5 stromal cells, the development of glycophorin A+ cells were significantly promoted. Regarding ROS accumulation, we examined cultured cells under the erythroid condition with a fluorescent sensor, Red CC-1. As a result, FF-10501 increased the ROS production.In summary, FF-10501 showed enough anti-proliferative effects to consider the clinical use. Furthermore, in this study we showed that FF-10501 improves normal erythropoiesis. The effect was cancelled by the inhibition of ROS accumulation or MAPK signaling. Our results suggest that FF-10501 could ameliorate myelosuppression during MDS treatment by maintaining myelopoiesis, and the low-dose therapy for low-risk MDS patients might be effective for the improving refractory anemia. DisclosuresMurase:FUJIFILM Corporation: Employment. Komatsu:FUJIFILM Corporation: Employment. Saito:FUJIFILM Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Free Thiol of Transthyretin in Human Plasma Most Accessible to Modification/Oxidation.

Free-thiol(s) in proteins, especially, when located on the surface of the molecule, are susceptible to oxidation/modification, which may cause loss of function or an alteration in the ternary structure. This suggests that the status of thiol group(s) of cysteine residue(s) in a protein, i.e., free-thiol versus an oxidized/modified form, in vivo, could reflect the physiological state of the molecule with respect to susceptibility to oxidative stress. To address this issue, we established an efficient method for isolating proteins that contain free thiol groups from a complex mixture, which permits the amount of free-thiol form(s) to modified/oxidized forms to be estimated. Albumin, which accounts for 55% of the total plasma proteins and has such a free thiol and has been reported to scavenge various reactive oxygen species (ROS) in vivo. The developed method was used to isolate the free form of albumin from fresh plasma. However, contrary to our expectations, transthyretin (TTR), which also has a single free thiol, was found to be the major protein that was the most susceptible to modification/oxidation. In addition, the free-thiol form could be separated from oxidized or modified molecules, permitting the relative abundance of the free-thiol form to be estimated. The findings show that the levels of the free-thiol form of TTR in plasma was significantly lowered after a hydrogen peroxide treatment, even at low concentrations (0.1 mM), suggesting that TTR could be a useful biomarker for monitoring a ROS imbalance in relation to various oxidative stress conditions.

Sphingosines Derived from Marine Sponge as Potential Multi-Target Drug Related to Disorders in Cancer Development.

Haliclona tubifera, marine sponge species abundant in Brazilian coastline, presents only a few papers published in the literature. Recently, we have reported the isolation of two modified C18 sphingoid bases: (2R,3R,6R,7Z)-2-aminooctadec-7-ene-1,3,6-triol and and (2R,3R,6R)-2-aminooctadec-1,3,6-triol. In order to continue our research, in this work aimed at the biological investigation of fractions that led to the isolation of these compounds. We evaluated the cytotoxic effect of marine sponge H. tubifera fractions in glioma (U87) and neuroblastoma (SH-SY5Y) human cell lines. In addition, considering the link between cancer, imbalance of reactive oxygen species and coagulation disorders, we also investigated the in vitro effects on blood coagulation and their redox properties. We showed that the ethyl acetate (EtOAc) fraction, rich in sphingoid bases, had important cytotoxic effects in both cancer cell lines with an IC50 < 15 μg/mL and also can inhibit the production of peroxyl radicals. Interestingly, this fraction increased the recalcification time of human blood, showing anticoagulant properties. The present study indicates the sphingosines fraction as a promising source of chemical prototypes, especially multifunctional drugs in cancer therapy.

Comparison of Glutathione, Cysteine, and Their Redox Potentials in the Plasma of Critically Ill and Healthy Children.

Oxidative stress is known to play a role in critical illness due to an imbalance in reactive oxygen species and reactive nitrogen species, and the body's ability to detoxify pro-oxidants using small molecule anti-oxidants and anti-oxidant enzymes. To compare the concentrations of plasma redox metabolites and redox potentials for the Cys/CySS and GSH/GSSG thiol/disulfide pairs in critically ill children with healthy control children. We performed a prospective clinical observational study of children ages ≤18 years and weight ≥6 kg, who were hospitalized between January 2010 and April 2012 in a 30-bed multidisciplinary medical-surgical pediatric intensive care unit (PICU). We measured the plasma concentrations of Cys, CySS, GSH, and GSSG within the first 24 h of PICU arrival, and we calculated the redox potential for the Cys/CySS (Eh Cys/CySS) and GSH/GSSG (Eh GSH/GSSG) thiol/disulfide pairs in the plasma of 61 critically ill children and 16 healthy control children. Critically ill children have less Cys (p = 0.009), less CySS (p = 0.011), less Total Cys ([Cys] + 2[CySS], p = 0.01), more GSSG (p < 0.001), and more oxidized Eh GSH/GSSG (p < 0.001) compared to healthy children. Our results demonstrate that in the presence of pediatric critical illness, the Total Cys/CySS thiol pool decreases while GSH is likely one component of the cellular redox system that reduces CySS back to Cys, thus maintaining Eh Cys/CySS. The Total Cys pool is more abundant than the Total GSH pool in the plasma of children. Further investigation is needed to elucidate the differences in redox potentials in subgroups of critically ill children, and to determine whether differences in redox metabolite concentrations and redox potentials correlate with severity of critical illness and clinical outcomes.

Identification of total reversible cysteine oxidation in an atherosclerosis model using a modified biotin switch assay.

Oxidative stress due to the imbalance of reactive oxygen species (ROS) and the resulting reversible cysteine oxidation (CysOX) are involved in the early proatherogenic aspect of atherosclerosis. Given that the corresponding redox signaling pathways are still unclear, a modified biotin switch assay was developed to quantify the reversible CysOX in an atherosclerosis model established by using a monocytic cell line treated with platelet releasate. The accumulation of ROS was observed in the model system and validated in human primary monocytes. Through the application of the modified biotin switch assay, we obtained the first reversible CysOX proteome for this model. A total of 75 peptides, corresponding to 53 proteins, were quantified with oxidative modification. The bioinformatics analysis of these CysOX-containing proteins highlighted biological processes including glycolysis, cytoskeleton arrangement, and redox regulation. Moreover, the reversible oxidation of three glycolysis enzymes was observed using this method, and the regulation influence was verified by an enzyme activity assay. NADPH oxidase (NOX) inhibition treatment, in conjunction with the modified biotin switch method, was used to evaluate the global CysOX status. In conclusion, this versatile modified biotin switch assay provides an approach for the quantification of all reversible CysOX and for the study of redox signaling in atherosclerosis as well as in diseases in other biological systems.

New insights into Lyme disease

Lyme borreliosis is transmitted through the bite of a tick that is infected by the bacterial spirochete Borrelia burgdorferi. Clinical manifestation of the disease can lead to heart conditions, neurological disorders, and inflammatory disorders. Oxidative stress has been implicated in the pathogenesis of many human diseases. The aim of this study was to investigate the mechanisms of oxidative stress and intracellular communication in Lyme borreliosis patients. Mitochondrial superoxide and cytosolic ionized calcium was measured in peripheral blood mononuclear cells (PBMCs) of Lyme borreliosis patients and healthy controls. Mitochondrial superoxide levels were significantly higher (p<0.0001) in Lyme borreliosis patients (n=32) as compared to healthy controls (n=30). Significantly low (p<0.0001) levels of cytosolic ionized calcium were also observed in Lyme borreliosis patients (n=11) when compared to healthy controls (n=11). These results indicate that there is an imbalance of reactive oxygen species and cytosolic calcium in Lyme borreliosis patients. The results further suggest that oxidative stress and interrupted intracellular communication may ultimately contribute to a condition of mitochondrial dysfunction in the immune cells of Lyme borreliosis patients.