Reactive Oxygen Species

Background. During haemodialysis (HD), polymorphonuclear cells (PMNs) and platelets are activated and release various granule products, including myeloperoxidase (MPO) and platelet factor 4 (PF4). MPO triggers the generation of reactive oxygen species, leading to irreversible protein, carbohydrate and lipid modification. PF4 probably also contributes to oxidative stress. As previously shown, HD-induced PMN degranulation is almost completely abolished during citrate anticoagulation, most probably due to its calcium chelation ability. Methods. In the present study, apart from HDinduced PMN and platelet degranulation, oxidative stress was analysed during three modes of anticoagulation. Heparin, dalteparin and citrate (HD hep , HD dal and HD cit ) were compared in a randomized, crossover fashion in eight chronic HD patients. Multiple blood samples were taken during the third HD session of each modality, from both the afferent and efferent line. Besides the degranulation markers MPO and PF4, various markers of oxidative stress were measured, including oxidized low-density lipoprotein (ox-LDL), malondialdehyde (MDA) and carboxymethyllysine (CML). Results. During HD hep and HD dal , marked degranulation was observed shortly after the start of HD. In contrast, during HD cit , PF4 and MPO levels remained unaltered, suggesting no release at all. After 1 week of HD cit , ox-LDL levels were markedly reduced [median 26% (3-65%), P ¼ 0.01], if compared with HD hep and HD dal . As regards MDA and CML, no differences were found.

Coronary artery disease and its sequelae-ischemia, myocardial infarction, and heart failure-are leading causes of morbidity and mortality in man. Considerable effort has been devoted toward improving functional recovery and reducing the extent of infarction after ischemic episodes. As a step in this direction, it was found that the heart was significantly protected against ischemia -reperfusion injury if it was first preconditioned by brief ischemia or by administering a potassium channel opener. Both of these preconditioning strategies were found to require opening of a K ATP channel, and in 1997 we showed that this pivotal role was mediated by the mitochondrial ATP-sensitive K + channel (mitoK ATP ). This paper will review the evidence showing that opening mitoK ATP is cardioprotective against ischemiareperfusion injury and, moreover, that mitoK ATP plays this role during all three phases of the natural history of ischemia -reperfusion injury preconditioning, ischemia, and reperfusion. We discuss two distinct mechanisms by which mitoK ATP opening protects the heart-increased mitochondrial production of reactive oxygen species (ROS) during the preconditioning phase and regulation of intermembrane space (IMS) volume during the ischemic and reperfusion phases. It is likely that cardioprotection by ischemic preconditioning (IPC) and K ATP channel openers (KCOs) arises from utilization of normal physiological processes. Accordingly, we summarize the results of new studies that focus on the role of mitoK ATP in normal cardiomyocyte physiology. Here, we observe the same two mechanisms at work. In low-energy states, mitoK ATP opening triggers increased mitochondrial ROS production, thereby amplifying a cell signaling pathway leading to gene transcription and cell growth. In high-energy states, mitoK ATP opening prevents the matrix contraction that would otherwise occur during high rates of electron transport. MitoK ATP -mediated volume regulation, in turn, prevents disruption of the structure -function of the IMS and facilitates efficient energy transfers between mitochondria and myofibrillar ATPases. D

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic ...

Oxidative stress is linked with many pathologies ranging from cancer to neurodegenerative disorders and antioxidants have presumably therapeutic value in such diseases. In this review, we categorize different direct and indirect mechanisms by which antioxidants exert their action. These include scavenging and metal chelating effects, mimicking the antioxidant enzymes or upregulation of their expression, activation of nuclear factor erythroid 2-related factor 2 (Nrf2), increasing the activity of sirtuins and inhibition of pro-oxidant enzymes among others. Recent findings on the most frequently investigated antioxidants including polyphenolics, thiolics, spin trapping agents, SOD mimetics, inducers of heme oxygenase-1 and nitric oxide synthase, activators of Nrf2, NADPH oxidase inhibitors and herbal supplements are summarized. Furthermore, the antioxidant effects of drugs that are clinically used for other pharmacological purposes including ACE inhibitors and statins are discussed. Cost-effectiveness and adverse effects of antioxidants are also evaluated. Since antioxidant therapy has failed in many instances, we have classified the reasons that may explain these shortcomings in different categories. Novel approaches to antioxidant therapy, that include mitochondria-targeting drugs, antioxidant gene therapy and approaches for improvement of cell uptake and alteration of subcellular compartment localization are also described. In the end, "shadows" that are shortcomings of antioxidant therapy as well as "lights" that include positive outcomes are addressed. It is concluded that if we learn from failures, invest on agents with higher potential and take advantage of novel emerging approaches, antioxidants could be an asset for the management of certain carefully chosen oxidative stress-related diseases. vii. pulmonary diseases, including chronic obstructive pulmonary disease (COPD) [22] and asthma [23]; viii. chronic kidney disease (CKD) [24]; ix. psychiatric disorders, including depression [25], bipolar disorder [26] and schizophrenia [27].

Percentage of viable sperms has good potential to reflect male fertilizing potential. In the present study, an attempt was made to find out the percentage of viable sperms in normal and abnormal ejaculates and influence of reactive oxygen species in affecting the viability of sperm. Malondialdehyde (MDA) in seminal plasma of normal and abnormal ejaculates was measured by Thiobarbituric Acid method and percentage of viable sperms was assessed by Eosin-Nigrosin staining. The results revealed that the abnormal semen samples have significantly lower number of viable spermatozoa (59.06 +/- 9.63% vs. 68.33 +/- 5.46%, P < 0.05) and higher levels of MDA (2.53 +/- 0.66 vs. 1.66 +/- 0.37 nmol/ml, P < 0.05) as compared to normal. A significant negative correlation was found between seminal MDA level and sperm viability. Based on the results obtained from the present study, we suggest that viability of sperm is an authentic parameter to assess male fertility potential and it can be affect...

A large number of micropropagated Euphorbia millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. The environment inside bioreactor normally used in plant micropropagation is characterised by high relative humidity, poor gaseous exchange between the internal atmosphere of the bioreactor and its surrounding environment, and the accumulation of ethylene, conditions that may induce physiological disorders. A comparison of hyperhydric shoots (HS) with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity in HS plants. The potential role of antioxidant enzymes in protecting hyperhydric shoots from oxidative injury was examined by analyzing enzyme activities and isozyme profiles of hyperhydric and non-hyperhydric leaves of E. millii. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue. After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to Mn-SOD and Cu/Zn-SOD. The activity of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) was proportionally increased in HS tissue compared to normal leaves indicating a crucial role in eliminating toxic H 2 O 2 from plant cells. The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding ascorbate (AA). The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS.

Resveratrol is a natural molecule with antioxidant action. Moreover, resveratrol is also considered to be a molecule with anti-inflammatory action, an effect attributed to suppression of prostaglandin (PG) biosynthesis. The aim of the present study was to investigate the effects of resveratrol, a polyphenol present in most red wines, on reactive oxygen species formation as well as on arachidonic acid (AA) release, cyclooxygenase expression, and PG synthesis in murine resident peritoneal macrophages. Results show that resveratrol exerted a strong inhibitory effect on superoxide radical (O2−) and hydrogen peroxide (H2O2) produced by macrophages stimulated by lipopolysaccharides (LPS) or phorbol esters (PMA). Resveratrol also significantly decreased [3H]AA release induced by LPS and PMA or by exposure to O2− or H2O2. Resveratrol treatment caused a significant impairment of cyclooxygenase-2 (COX-2) induction stimulated by LPS and PMA or by O2− or H2O2 exposure. These effects of resveratrol on [3H]AA release and COX-2 overexpression were correlated with a marked reduction of PG synthesis. Our results indicate that the antioxidant action of resveratrol affects AA mobilization and COX-2 induction.

HDPE Aspergillus tubingensis VRKPT1 Aspergillus flavus VRKPT2 Fourier transformed infra-red spectroscopy Scanning electron microscopy a b s t r a c t High density polyethylene (HDPE) is the most commonly found non-degradable solid waste among the polyethylene. In this present study, HDPE degrading various fungal strains were isolated from the polyethylene waste dumped marine coastal area and screened under in vitro condition. Based on weight loss and FT-IR Spectrophotometric analysis, two fungal strains designated as VRKPT1 and VRKPT2 were found to be efficient in HDPE degradation. Through the sequence analysis of ITS region homology, the isolated fungi were identified as Aspergillus tubingensis VRKPT1 and Aspergillus flavus VRKPT2. The biofilm formation observed under epifluorescent microscope had shown the viability of fungal strains even after one month of incubation. The biodegradation of HDPE film nature was further investigated through SEM analysis. HDPE poses severe environmental threats and hence the ability of fungal isolates was proved to utilize virgin polyethylene as the carbon source without any pre-treatment and pro-oxidant additives.

Neuroglobin and cytoglobin are two recently discovered vertebrate globins, which are expressed at low levels in neuronal tissues and in all tissues investigated so far, respectively. Based on their amino acid sequences, these globins appear to be phylogenetically ancient and to have mutated less during evolution in comparison to the other vertebrate globins, myoglobin and hemoglobin. As with some plant and bacterial globins, neuroglobin and cytoglobin hemes are hexacoordinate in the absence of external ligands, in that the heme iron atom coordinates both a proximal and a distal His residue. While the physiological role of hexacoordinate globins is still largely unclear, neuroglobin appears to participate in the cellular defence against hypoxia. We present the current knowledge on the functional properties of neuroglobin and cytoglobin, and describe a mathematical model to evaluate the role of mammalian retinal neuroglobin in supplying O 2 supply to the mitochondria. As shown, the model argues against a significant such role for neuroglobin, that more likely plays a role to scavenge reactive oxygen and nitrogen species that are generated following brain hypoxia. The O 2 binding properties of cytoglobin, which is upregulated upon hypoxia, are consistent with a role for this protein in O 2 -requiring reactions, such as those catalysed by hydroxylases.

There is a need to develop efficient tools to prevent damage to marine ecosystems due to pollution. Since microalgae play a key role in marine ecosystems, they are considered potentially useful for quick and sensitive toxicity bioassays. In this study an integrative analysis has been carried out of the anti-oxidant enzyme activities of marine microalgae species. Three marine microalgae species (Cylindrotheca closterium, a benthic diatom; Phaeodactylum tricornutum, a diatom which has been used as model organism in toxicity bioassays; and Rhodomonas salina, a cryptophyceae which is considered to present a certain level of heterotrophy) were exposed to selected concentrations of three model pollutants: copper (5 and 10 mg L À 1 ), atrazine (25 and 50 mg L À 1 ) and Irgarol (0.5 and 1.0 mg L À 1 ). These pollutant concentrations are environmentally relevant for coastal ecosystems, and have been selected for checking the efficiency of the reactive oxygen intermediate (ROI) scavenging enzyme system of these organisms. Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APx) and glutathione peroxidase (GPx) activities were measured at the end of 24 h exposure. The integrated biomarker response (IBR) indexin our case for oxidative stresshas been employed to evaluate the ROI-scavenging enzyme system for each species and each treatment. In general, the SOD and CAT enzyme activities measured were higher in exposed populations than in controls, whereas APx and GPx activities showed the opposite trend. These microalgae showed significant responses of oxidative stress biomarkers at environmentally relevant concentrations for the assayed pollutants and short exposure periods, conditions that most other model organisms cannot match. Therefore microalgae present clear advantages over other species for their prospective employment in an "early warning system".

OBJECTIVE To assess the effect of smoking on sperm vitality, sperm DNA integrity, semen reactive oxygen species, and zinc levels in fertile men. METHODS One-hundred sixty men were investigated. They were divided into 2 equal groups: healthy fertile nonsmokers and healthy fertile smokers. They were subjected to history taking, clinical examination, and semen analysis. In their semen, sperm hypo-osmotic swelling test, sperm DNA fragmentation test, seminal reactive oxygen species, and zinc were assessed. RESULTS Compared with fertile nonsmokers, fertile smokers were significantly associated with lower hypo-osmotic swelling test and seminal zinc levels and significantly associated with higher sperm DNA fragmentation percent and seminal reactive oxygen species levels. CONCLUSION Smoking (cigarettes/day and duration) has detrimental effects on sperm motility, viability, DNA fragmentation, seminal zinc levels, and semen reactive oxygen species levels, even in fertile men, and it is directly correlated with cigarette quantity and smoking duration. UROLOGY 80: 822-825, 2012.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associat...

NPR1 is a key regulator of the salicylic acid (SA) dependent pathogen resistance pathway in plants. In this issue of Cell, Mou and Dong demonstrate that Arabidopsis NPR1 undergoes activation from an inactive oligomer to the active monomer as a result of cellular redox changes induced by SA during systemic acquired resistance.

a-Mangostin is a xanthone with antioxidant properties isolated from mangosteen fruit. The reactive oxygen species (ROS) scavenging capacity and the potential protective effect of a-mangostin against the mitochondrial toxin 3-nitropropionic acid (3-NP) in primary cultures of cerebellar granule neurons (CGNs) were studied in the present work. It was found that a-mangostin was able to scavenge in a concentration-dependent way singlet oxygen, superoxide anion and peroxynitrite anion. In contrast, a-mangostin was unable to scavenge hydroxyl radicals and hydrogen peroxide. Furthermore, a-mangostin was able to ameliorate in a concentration-dependent way the neuronal death induced by 3-NP. This protective effect was associated with an amelioration of 3-NP-induced reactive oxygen species formation. It is concluded that a-mangostin is able to scavenge directly several ROS and has a neuroprotective effect against 3-NP in primary cultures of CGNs, which is associated with its ability to ameliorate 3-NP-induced ROS production.

Limited recovery of islets post-cryopreservation influences graft survival and transplantation efficiency during diabetes treatment. As curcumin, a potent antioxidant/radical scavenging compound, protects islets against beta cell toxins, we hypothesized that inclusion of curcumin during cryopreservation or during post-thaw culture or both may rescue islets from cryoinjury. To test the effect of curcumin inclusion on islet recovery murine islets were isolated by the collagenase digestion, cultured for 48 h, cryopreserved using dimethylsulphoxide as cryoprotectantwith or without curcumin (10 μM)and then slow cooled to −40°C before immersing them in liquid nitrogen for 7 days. Following rapid thawing with sucrose gradient and 24 h post-thaw culturein presence or absence of curcumin (10 μM)islet viability and functionality were determined. Islet recovery in curcumin treated groups was significantly higher than in groups where islets were cryopreserved without curcumin. Islets cryopreserved with curcumin also showed more intact islets as well as better morphology as compared to islets cryopreserved without curcumin. Curcumin treated islets also showed significant inhibition of ROS generation as compared to islets cryopreserved without curcumin. Glucose responsiveness and insulin secretion in islets cryopreserved with curcumin was equal to that of the freshly isolated islets as against islets cryopreserved without curcumin. Elevated level of Hsp 70 and HO-1 were observed in islets cryopreserved with curcumin and may contribute to curcumin-induced islet rescue. Hence, we conclude that inclusion of curcumin into cryopreservation medium inhibits ROS generation and corresponding islet damage and dysfunction.

Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells. Similarly, hydrogen peroxide induces VEGF synthesis and recent studies indicate the involvement of ROS in signaling downstream of VEGF stimulation. VEGF synthesis can not only be enhanced by gene transfer of VEGF but also by overexpression of NO synthase genes. Here, we examined the possibility of augmentation of VEGF production by gene transfer of copper/zinc superoxide dismutase (CuZnSOD, SOD1). Overexpression of human SOD1 in mouse NIH 3T3 fibroblasts increased SOD activity, enhanced intracellular generation of H 2 O 2 and significantly stimulated VEGF production as determined by increase in VEGF promoter activity, VEGF mRNA expression and VEGF protein synthesis. The stimulatory effect on VEGF synthesis induced by SOD1 gene transfer was reverted by overexpression of human catalase. The effect of H 2 O 2 produced by engineered cells is mediated by activation of hypoxia-inducible factor response element (HRE) as well as Sp1 recognition site of VEGF promoter. This data suggest the feasibility of stimulation of angiogenesis by overexpression of SOD1. (Mol Cell Biochem 264: [169][170][171][172][173][174][175][176][177][178][179][180][181] 2004)

Your article is protected by copyright and all rights are held exclusively by Springer-Verlag Wien. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to selfarchive your work, please use the accepted author's version for posting to your own website or your institution's repository. You may further deposit the accepted author's version on a funder's repository at a funder's request, provided it is not made publicly available until 12 months after publication.

Apoptotic myocyte cell death, diastolic dysfunction and progressive deterioration in LV pump function, characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG)-transmits danger signals in cardiac muscle cells (CMC). The growth factor adapter protein, p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here, we demonstrate interventions that attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser-36), inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes (ARVM) obtained by enzymatic dissociation were transduced with mutant36 p66ShcA (mu-36) dominant negative expression vector and plated in serum free media (SFM) containing 5 mM or 25 mM glucose. At HG, ARVM exhibit marked increase in reactive oxygen species (ROS) production, upregulation of phospho-Ser-36, collapse of mitochondrial transmembrane potential (Δψm) and increased formation of p66ShcA/cytochrome c complexes. These indices of oxidative stress were accompanied by 40% increase in apoptosis and upregulation of cleaved caspase 3 and the apoptosis related proteins p53 and Bax. To test if p66ShcA functions as a redox sensitive molecular switch in vivo, we examined hearts of male Akita diabtic nonobese (C57BL/6J) mice. Immunoblot analysis detected upregulation of phospho-Ser-36, translocation of p66ShcA to mitochondria and formation of p66ShcA/cytochrome c complexes. Conversely, correction of HG by adenoassociated viral delivery of leptin

The aim of this study was to evaluate, in vitro, the effect of iron on the dissolution of bovine enamel powder, when added to two carbonated beverages.Powdered enamel was produced by griding enamel fragments of bovine incisor in a steel pestle and mortar. Particles between 75 and 106 μm were selected using appropriated meshes. At time zero, the carbonated beverage (Coke® or Sprite Zero®) was added to powdered enamel (1 mg enamel powder/10 μL of beverage) and vortexed for 30 s. The sample was immediately centrifuged (11,000 rpm) for 30 s and the supernatant was removed at 1 min 40 s. This procedure was repeated five times with the beverage containing increasing ferrous sulphate concentrations (1.25, 2.5, 5, 10, 15, 30 and 60 mmol/L). The phosphate released in the medium was analysed spectrophotometrically. Data were analysed using ANOVA and Tukey's test (p < 0.05).When iron at 30 and 60 mmol/L was added to Coke®, a significant reduction in the dissolution of powdered enamel was observed when compared to control (11 and 17%, respectively), while lower iron concentrations did not have any effect on enamel powder dissolution. Regarding Sprite Zero®, iron concentrations up to 10 mmol/L had no significant effect, while higher concentrations significantly increased enamel powder dissolution.The results suggest that iron can interfere with the dissolution of dental enamel powder in the presence of acidic beverages and the type of acid in these beverages seems to modulate this effect.

Alcohol consumption is a predominant etiological factor in the pathogenesis of chronic liver diseases, resulting in fatty liver, alcoholic hepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma (HCC). Although the pathogenesis of alcoholic liver disease (ALD) involves complex and still unclear biological processes, the oxidative metabolites of ethanol such as acetaldehyde and reactive oxygen species (ROS) play a preeminent role in the clinical and pathological spectrum of ALD. Ethanol oxidative metabolism influences intracellular signaling pathways and deranges the transcriptional control of several genes, leading to fat accumulation, fibrogenesis and activation of innate and adaptive immunity. Acetaldehyde is known to be toxic to the liver and alters lipid homeostasis, decreasing peroxisome proliferator-activated receptors and increasing sterol regulatory element binding protein activity via an AMP-activated protein kinase (AMPK)-dependent mechanism. AMPK activation by ROS mod...

Clostridia belong to those bacteria which are considered as obligate anaerobe, e.g. oxygen is harmful or lethal to these bacteria. Nevertheless, it is known that they can survive limited exposure to air, and often eliminate oxygen or reactive derivatives via NAD(P)Hdependent reduction. This system does apparently contribute to survival after oxidative stress, but is insufficient to establish long-term tolerance of aerobic conditions. Here we show that manipulation of the regulatory mechanism of this defence mechanism can trigger aerotolerance in the obligate anaerobe Clostridium acetobutylicum. Deletion of a peroxide repressor (PerR)-homologous protein resulted in prolonged aerotolerance, limited growth under aerobic conditions and rapid consumption of oxygen from an aerobic environment. The mutant strain also revealed higher resistance to H 2O2 and activities of NADH-dependent scavenging of H2O2 and organic peroxides in cell-free extracts increased by at least one order of magnitude. Several genes encoding the putative enzymes were upregulated and identified as members of the clostridial PerR regulon, including the heat shock protein Hsp21, a reverse rubrerythrin which was massively produced and became the most abundant protein in the absence of PerR. This multifunctional protein is proposed to play the crucial role in the oxidative stress defence.

Crude ethanolic extract of the plant Lycopodium clavatum has long been used in complementary and alternative medicine for treating various liver ailments and Alzheimer's disease. It has also been claimed to have potential anti-cancer properties in vivo in mice chronically fed liver carcinogens, p-dimethylamino azobenzene (initiator) and phenobarbital (promoter). Incidentally, crude ethanolic extract of Lycopodium clavatum is a mixture of some 201 alkaloids. In order to ascertain if any major fraction can be attributed to have pronounced anti-cancer effect, we examined this major fraction by eluting the crude extract in petroleum ether:ethyl aetate (17:3 vol/vol;) solvent and tried to understand its underlying mechanism. Studies on morphological changes, cell viability and cytotoxicity by microscopy and FACS, Western blot and immunofluorescence of Bcl-2, Bax, cytochrome c, caspase-3 were conducted. Lycopodine was found to induce chromatin condensation, inter-nucleosomal DNA fragmentation and enhanced cell population in sub-G1 region along with increase in reactive oxygen species generation and mitochondrial membrane potential depolarization, release of cytochrome c and activation of caspase-3 which are the events closely involved in apoptosis. An overall analysis of results showed that Lycopodine considerably inhibited growth of HeLa cells which indicates its potential use in chemotherapy.

Mitochondrial DNA (mtDNA) is located in close proximity of the respiratory chains, which are the main cellular source of reactive oxygen species (ROS). ROS can induce oxidative base lesions in mtDNA and are believed to be an important cause of the mtDNA mutations, which accumulate with aging and in diseased states. However, recent studies indicate that cumulative levels of base substitutions in mtDNA can be very low even in old individuals. Considering the reduced complement of DNA repair pathways available in mitochondria and higher susceptibility of mtDNA to oxidative damage than nDNA, it is presently unclear how mitochondria manage to maintain the integrity of their genetic information in the face of the permanent exposure to ROS. Here we show that oxidative stress can lead to the degradation of mtDNA and that strand breaks and abasic sites prevail over mutagenic base lesions in ROS-damaged mtDNA. Furthermore, we found that inhibition of base excision repair enhanced mtDNA degradation in response to both oxidative and alkylating damage. These observations suggest a novel mechanism for the protection of mtDNA against oxidative insults whereby a higher incidence of lesions to the sugar-phosphate backbone induces degradation of damaged mtDNA and prevents the accumulation of mutagenic base lesions.

Oxalates stimulate alterations in renal epithelial cells and thereby induce calcium oxalate (CaOx) stone formation. Bacillus subtilis YvrK gene encodes for oxalate decarboxylase (OxdC) which degrades oxalate to formate and CO2. The present work is aimed to clone the oxdC gene in a mammalian expression vector pcDNA and transfect into Human Embryonic Kidney 293 (HEK293) cells and evaluate the oxdC expression, cell survival rate and oxalate degrading efficiency. The results indicate cell survival rate of HEK293/pcDNAOXDC cells pre-incubated with oxalate was enhanced by 28%. HEK293/pcDNAOXDC cells expressing OxdC treated with oxalate, significantly restored antioxidant activity, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) generation compared with HEK293/pcDNA. Apoptotic marker caspase 3 downregulation illustrates HEK293/pcDNAOXDC cells were able to survive under oxalate-mediated oxidative stress. The findings suggest HEK293 cells expressing oxdC capa...

Cells respond to a condition of hypo-osmolality by rapid swelling followed by an adaptive response that tends to recover the normal cell volume despite the persistence of the hypo-osmotic condition. This is an active process accomplished by the extrusion of intracellular osmolytes, essentially K ϩ , Cl-, and small organic molecules. This regulatory process operates through a chain of events that essentially consists of a sensor or sensing mechanism to detect changes in cell volume, a signaling cascade to amplify the sensing signal and orient it to activate pathways for osmolyte extrusion, and a memory of the original cell volume, which sets the timing for inactivation of the volume-regulatory process. This article presents a brief overview of recent progress in these different aspects of the volume-regulatory process, including (1) the mechanisms and/or candidate molecules serving the role of volume sensors, (2) the osmosignaling network and the interplay and hierarchy of the different elements in this chain, and (3) the nature and properties of the osmolyte extrusion pathways. Emphasis is placed on some of the main unsolved questions concerning different aspects of the volume-regulatory process. Recent findings regarding the effect of hypo-osmolality on synaptic function are briefly discussed in terms of the possible molecular basis for the neurologic symptoms induced by hyponatremia.

Al though the aetiology of inflammatory bowel disease (IBD) remains unknown, the pathogenesis is gradually being unravelled, seeming to be the result of a combination of environmental, genetic, and immunological factors in which an uncontrolled immune response within the intestinal lumen leads to inflammation in genetically predisposed individuals. Multifactorial evidence suggests that a defect of innate immune response to microbial agents is involved in IBD. This editorial outlines the immunopathogenesis of IBD and their current and future therapy. We present IBD as a result of dysregulated mucosal response in the intestinal wall facilitated by defects in epithelial barrier function and the mucosal immune system with excessive production of cytokines growth factors, adhesion molecules, and reactive oxygen metabolites, resulting in tissue injury. Established and evolving therapies are discussed in the second part of this editorial and at the end of this section we review new therapi...

Cell signaling DNA damage Lipid peroxidation Oxidative stress Reactive oxygen species s u m m a r y Oxygen has a central role in the evolution of complex life on Earth mainly because of the biochemical symmetry of oxygenic photosynthesis and aerobic respiration that can maintain homeostasis within our planet biosphere. Oxygen can also produce toxic molecules, reactive oxygen species (ROS). ROS is a collective term that includes both oxygen radicals and certain oxidizing agents that are easily converted into radicals. They can be produced from both endogenous and exogenous substances. ROS play a dual role in biological systems, since they can be either harmful or beneficial to living systems. They can be considered a double-edged sword because on the one hand oxygen-dependent reactions and aerobic respiration have significant advantages but, on the other, overproduction of ROS has the potential to cause damage.

M., Naarala, J., Juutilainen, J. and Scarfì, M. R. Formation of Reactive Oxygen Species in L929 Cells after Exposure to 900 MHz RF Radiation with and without Co-exposure to 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone. Radiat. Res. 167, 306-311 (2007).

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production.

Less than 20 of the hundreds of carotenoids found in nature are found in the human body. These carotenoids are present in the body from the foods or dietary supplements that humans consume. The body does not synthesize them. Among the carotenoids present in the body, only lutein and its coexistent isomer, zeaxanthin, are found in that portion of the eye where light is focused by the lens, namely, the macula lutea. Numerous studies have shown that lutein and zeaxanthin may provide significant protection against the potential damage caused by light striking this portion of the retina. In the eye, lutein and zeaxanthin have been shown to filter high-energy wavelengths of visible light and act as antioxidants to protect against the formation of reactive oxygen species and subsequent free radicals. Human studies have demonstrated that lutein and zeaxanthin are present in the skin, and animal studies have provided evidence of significant efficacy against light-induced skin damage, especially the ultraviolet wavelengths. Little was known about the protective effects of these carotenoids in human skin until recently. This article reviews the scientific literature pertaining to the effects that lutein and zeaxanthin exhibit in the human eye and skin.

Most of the previous studies dealing with the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the synthesis of inflammatory mediators involved in joint damage have been done in cells culturedin vitro or in blood cells from patients treated for short periods of time. In this work we have evaluated the long-term effect of aceclofenac, a new NSAID, and diclofenac on the production of a series of inflammatory mediators by blood cells from 30 patients with severe knee osteoarthritis. Both aceclofenac and diclofenac significantly inhibited prostaglandin E2 (PGE2) synthesis by blood mononuclear and polymorphonuclear cells after 180 days of treatment. However, no clear effect was noted on leukotriene B4 (LTB4) and platelet activating factor (PAF) production. The generation of O 2− by polymorphonuclear cells, stimulated with FMLP, was decreased after 15 days of treatment with both drugs, but reached normal values after 180 days. Interleukin-1β (IL-1β) production decreased significantly at 180 days with both drugs in the group of high producer patients. In a few (n=3) patients with high basal mononuclear cell tumor necrosis factor α (TNFα) production, this also decreased on treatment for 180 days with the NSAIDs. In the remaining low TNFα-producing patients, TNFα production tended to increase. Interleukin-6 (IL-6) synthesis was not affected by aceclofenac while it was diminished by diclofenac. The decrease in IL-6 in all treated patients was significantly correlated with a worsening of the clinical condition. On the whole, these data could afford a pathogenetic basic for the long-term employment of these drugs in patients with inflammatory conditions.

The British Petroleum (BP) oil spill has raised several ecological and health concerns. As the first response, BP used a chemical dispersant, Corexit-9500, to disperse the crude oil in the Gulf of Mexico to limit shoreline contamination problems. Nevertheless, portions of this oil/Corexit mixture reached the shoreline and still remain in various Gulf shore environments. The use of Corexit itself has become a significant concern since its impacts on human health and environment is unclear.

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P Ͻ .01) increase in hydrogen peroxide production and an associated decrease (P Ͻ .01) in motility parameters. Total motility was significantly (P Ͻ .01) lower in the pres-ence of X-XO compared with the case of the control (29% Ϯ 9% vs 73% Ϯ 1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71% Ϯ 4% vs 30% Ϯ 3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53% Ϯ 3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.

Cisplatin is a broad-spectrum anticancer drug that is also widely used in experimental studies on DNA damage-induced apoptosis. Induction of apoptosis within 24-48 hr requires cisplatin concentrations that are at least one order of magnitude higher than the IC 50 . Here, we show that such high, apoptosis-inducing cisplatin concentrations induce cellular superoxide formation and that apoptosis is inhibited by superoxide scavengers. The same concentration limit and the requirement for superoxide are also true for induction of caspase activation in enucleated cells (cytoplasts), showing that cisplatin-induced apoptosis occurs independently of nuclear DNA damage. In contrast, lower cisplatin concentrations, which do not induce acute apoptosis, are sufficient for induction of DNA damage signaling. We propose that the antiproliferative effects of cisplatin at IC 50 doses involve premature senescence and secondary, nonstress-induced apoptosis. The higher doses currently used in in vitro studies lead to acute, stress-induced apoptosis that involves induction of superoxide but is largely DNA damage-independent. ' 2006 Wiley-Liss, Inc.

Successful defense depends on the ability of the plant to recognize an attacking 'enemy' as early as possible. Early defense responses require enemy-initiated signaling cascades. Their activation ensures an induced response that is quantitative, timely and coordinated with other activities of the host cells. Damage-induced ion imbalances and modulations of channel activities are the first events occurring in the plasma membrane and result in rapid perturbations of the plasma membrane potential (V m ) involving variations of cytosolic Ca 2+ concentrations. Interacting downstream networks of kinases and phytohormones mediate the signal and result in concerted gene activation. Here we review and discuss early events occurring before herbivore attack-related gene expression that are responsible for cascades of events and signal transductions, eventually leading to indirect and direct plant responses.

Reactive oxygen species (ROS) play an important role in cell signaling pathway. Previously, we found that silica induced immediate ROS generation and sequential cellular responses such as kinase activation in Rat2 cells as well as an increase of intracellular calcium concentration in A549 cells. However, the detailed mechanism underlying the immediate ROS generation induced by silica in fibroblast cells remains to be elucidated. Therefore, in the present study, we investigated the mechanism of ROS generation by silica within Rat2 fibroblast cells by examining the effects of a diverse group of inhibitors for the enzymes related with signal transduction events. Inhibitors for protein tyrosine kinase (PTK), phospholipase C (PLC), protein kinase C (PKC) and calmodulin (CaM) kinase II effectively suppressed ROS generation in silica-stimulated Rat2 cells, whereas those for protein kinase A and phospholipase A 2 did not. Diphenyleneiodonium chloride (DPI), an inhibitor for NADPH oxidase was also found to be effective in inhibiting silica-induced ROS generation. These results suggest that PTK, PLC, PKC, CaM kinase II, and NADPH oxidase are all involved in signal transduction pathways for ROS generation in silica-stimulated Rat2 cells.

Sodium hypochlorite and sodium chlorite are commonly used as disinfectants, and understanding the mechanisms of microbial resistance to these compounds is of considerable importance. In this study, the role of oxidative stress and antioxidant enzymes in the sensitivity of the yeast Saccharomyces cerevisiae to hypochlorite and chlorite was studied. Yeast mutants lacking Cu-Zn superoxide dismutase, but not mutants deficient in cytoplasmic and peroxisomal catalase, were hypersensitive to the action of both hypochlorite and chlorite. Both compounds depleted cellular glutathione, induced the production of reactive oxygen species and decreased the viability of the cells. The toxicity of hypochlorite and chlorite was abolished by hypoxic and anoxic conditions and ameliorated by thiol antioxidants and ascorbate. The results demonstrated that the action of hypochlorite and chlorite involves the formation of superoxide and peroxide and that SOD1 is protective, probably by limiting the formation of hydroxyl radicals and damage to proteins.

A relatively simple but sensitive and reliable method ofquantitating the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a few #l is described. In this assay system, ~-phycoerythrin (~-PE) is used as an indicator protein, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble vitamin E analogue) as a control standard. Results are expressed as ORAC units, where 10RAC unit equals the net protection produced by l ~M Trolox. The uniqueness of this assay is that total antioxidant capacity of a sample is estimated by taking the oxidation reaction to completion. At this point all of the nonprotein antioxidants (which include a-tocopherol, vitamin C, E-carotene, uric acid, and bilirubin) and most of the albumin in the sample are oxidized by the peroxyl radical. Results are quantified by measuring the protection produced by antioxidants. This solves many problems associated with kinetics or lag-time measurements. A linear correlation of ORAC value with concentration of serum, Trolox, vitamin C, uric acid, and bovine albumin is demonstrated. The coefficient of variation within a run is found to be about 2% and from run to run about 5%. Trolox, a-tocopherol, vitamin C,/3-carotene, uric acid, and bilirubin completely protect B-PE from oxidation, while bovine albumin protects/3-PE only partially. On a molar basis, the relative peroxyl radical absorbance capacity of Trolox, a-tocopherol acid succinate, uric acid, bilirubin, and vitamin C is l : 1 : 0.92 : 0.84 : 0.52. Bovine albumin per unit weight has a lower peroxyi absorbing capacity than these antioxidants. However, the serum protein fraction, containing some lipid-soluble antioxidants, represents the major contributor to the ORAC value found in whole serum. The minimum amount of vitamin C and uric acid which could still be detectable when added to a serum supernatant fraction is 1.5/~g and 0.59 #g, respectively, which account for about 1% of the total ORAC value of the serum supernatant fraction.

This study aims to elucidate the processes underlying neuroprotection of kaempferol in models of rotenone-induced acute toxicity. We demonstrate that kaempferol, but not quercetin, myricetin or resveratrol, protects SH-SY5Y cells and primary neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species (ROS) levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenyilpiridinium, paraquat) used to reproduce the typical features of Parkinson's disease (PD), but is inefficient against apoptotic stimuli not directly affecting mitochondria (H 2 O 2 , 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in Parkinson's disease. Overall, the data provide further evidence for kaempferol to be identified as an autophagic enhancer with potential therapeutic capacity.

Excessive training (i.e. overtraining, OT) may result in underperformance, which can be characterized by the time needed to re-establish performance (i.e. functional overreaching (FOR), nonfunctional overreaching, OT syndrome). The present study is an initial screening for proteins presenting altered abundance in the red (RG) and white (WG) portions of the gastrocnemius muscle from rats submitted to an OT protocol that induced FOR. In the RG, compared to the nontrained control, FOR demonstrated an increased abundance of proteins normally related to adaptation to endurance training (e.g. proteins of oxidative phosphorylation complexes, proteins related to lipid metabolism, antioxidants, and chaperones). In the WG, spots identified as mitochondrial aconitase and a component of the succinate dehydrogenase complex were downregulated in FOR, as were proteins related to myofibril stabilization; these latter were upregulated in the RG. This initial study shows that skeletal muscles with different fibertype compositions respond differently to an OT period. Also, it is likely that actin-interacting proteins have an important role in muscle adaptation to endurance exercise.