SFRBM - Society for Free Radical Biology and Medicine

Formation of Reactive Sulfite-Derived Free Radicals by the Activation of Human Neutrophils: An ESR Study

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 2 February 2012

Kalina Ranguelova, Annette B. Rice, Abdelahad Khajo, Mathilde Triquigneaux, Stavros Garantziotis, ...

The objective of the present study is to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In the present study sulfite (SO3) and sulfate (SO4) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophilsviasulfite radical chain reaction chemistry. After treatment with (bi)sulfite, PMA-stimulated neutrophils produced DMPO-sulfite anion radical, -superoxide, and -hydroxyl radical adducts. The latter adduct probably resulted, in part, from the conversion of DMPO-sulfate to DMPO-hydroxyl radical adductviaa nucleophilic substitution reaction of the radical adduct. This anion radical (SO4) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.

Highlights

► Sulfite (SO3) and sulfate (SO4) radicals were detected with ESR spin-trapping. ► Sulfite (SO3) radical was detected within human neutrophils. ► Adverse reactions of (bi)sulfite in asthmatics may be related to radical formation.

Fri, 03 Feb 2012 06:02

Deoxyelephantopin impedes mammary adenocarcinoma cell motility through inhibiting calpain-mediated adhesion dynamics and inducing reactive oxygen species and aggresome formation

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 2 February 2012

Wai-Leng Lee, Lie-Fen Shyur

We previously showed that deoxyelephantopin (DET), a plant sesquiterpene lactone, exhibits more profound suppression than paclitaxel (PTX) of lung metastasis of mammary adenocarcinoma TS/A cells in mice. Proteomics studies suggest that DET affects actin cytoskeletal protein networks and downregulates calpain-mediated proteolysis of several actin-associated proteins, while PTX mainly interferes with microtubule proteins. Here, DET was observed to significantly deregulate adhesion formation in TS/A cells likely through inhibition of m-calpain activity. Epithelial growth factor (EGF)-mediated activation of Rho GTPase Rac1 and formation of lamellipodia in TS/A cells were remarkably suppressed by DET treatment. Further, DET impaired vesicular trafficking of EGF and induced protein carbonylation and formation of centrosomal aggregates in TS/A cells. DET-induced reactive oxygen species (ROS) were observed to be the upstream stimulus for the formation of centrosomal ubiquitinated protein aggregates that might subsequently restrict cancer cell motility. PTX, however, caused dramatic morphological changes, interfered with microtubule networking, and moderately inhibited calpain-mediated cytoskeletal and focal adhesion protein cleavage in TS/A cells. This study provides novel mechanistic insights into the pharmacological action of DET against metastatic mammary cell migration and suggests that modulation of oxidative stress might be a potential strategy for treatment of metastatic breast cancer.

Highlights

► Phytoagent deoxyelephantopin (DET) inhibits enzymatic activity of m-calpain ► DET suppresses adhesion and lamellipodia formation in TS/A cells ► DET induces ROS formation, protein carbonylation and centrosomal aggregates ► DET impairs vesicular trafficking of EGF and suppresses Rho GTPase Rac1 activation

Fri, 03 Feb 2012 06:02

Histone carbonylation occurs in proliferating cells

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 2 February 2012

José Luis García-Giménez, Ana María Velázquez Ledesma, Isabel Esmoris, Carlos Romá-Mateo, Pascual Sanz, ...

Chromatin is a dynamic structure formed mainly by DNA and histones and, chemical modifications on these elements regulate its compaction.Histone post-translational modifications (PTMs) have a direct impact on chromatin conformation, controlling important cellular events like cell proliferation and differentiation. Redox related post-translational modifications may have important effects on chromatin structure and function offering a new intriguing area termedRedox Epigenetics.Little is known about histone carbonylation, a PTM that may be related to modifications in the cellular redox environment. The aim of our study was to determine the carbonylation of the different histones during cell proliferation, a moment in cell life where important redox changes take place.Here, we describe changes of histone carbonylation during cell proliferation in NIH3T3 fibroblasts. In addition, we have studied the variations of poly(ADP-ribosyl)ation and phospho-H2AX at the same time, because both modifications are related to DNA damage responses. High levels of carbonylation on specific histones (H1, H1and H3.1 dimers) were found when cells were in an active phase of DNA synthesis. The modification decreased when nuclear proteasome activity was activated. However, these results did not correlate completely with poly(ADP-ribosyl)ation and phospho-H2AX levels. Therefore, histone carbonylation may represent a specific event during cell proliferation.We describe a new methodology named Oxy-2D-TAU Western blot that allowed us to separate and analyze the carbonylation pattern of the different histone variants. In addition we offer a new role for histone carbonylation and their implications in redox epigenetics. Our results suggest that histone carbonylation is involved on histone detoxification during DNA synthesis.

Highlights

► Carbonylation is a histone modification that occurs during cell proliferation. ► Histone carbonylation follows a similar pattern that cellular GSH. ► Histone carbonylation is involved in histone degradation by nuclear proteasome. ► PARP activity is important for histone detoxification.

Fri, 03 Feb 2012 06:02

Early memory deficits precede plaque deposition in APPswe/PS1dE9 mi involvement of oxidative stress and cholinergic dysfunction

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 2 February 2012

Wei Zhang, Miao Bai, Ye Xi, Jian Hao, Liu Liu, ...

A large body of evidence has shown that cognitive deficits occur early prior to amyloid plaque deposition, suggesting that soluble amyloid-beta protein (Aβ) contributes to the development of early cognitive dysfunction in Alzheimer's disease (AD). However, the underlying mechanism(s) through which soluble Aβ exerts its neurotoxicity responsible for cognitive dysfunction in the early-stage of AD remains unclear so far. In this study, we used the pre-plaque APPswe/PS1dE9 mice aged 2.5 and 3.5 months to examine the alterations in cognitive function, oxidative stress, and cholinergic function. We found that only soluble Aβ, but not insoluble Aβ, was detected in these pre-plaque APPswe/PS1dE9 mice. 2.5-month-old APPswe/PS1dE9 mice did not show any significant changes in the measures of cognitive function, oxidative stress, and cholinergic function, whereas 3.5-month-old APPswe/PS1dE9 mice exhibited spatial memory impairment in the Morris water maze, accompanied by significantly decreased acetylcholine (ACh), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) as well as increased malondialdehyde (MDA) and protein carbonyls. In 3.5-month-old pre-plaque APPswe/PS1dE9 mice, correlational analyses revealed that the performance of impaired spatial memory inversely correlated with soluble Aβ, MDA, and protein carbonyls, as well as positively correlated with ACh, ChAT, SOD, and GSH-px; soluble Aβ level inversely correlated with ACh, ChAT, SOD, and GSH-px, as well as positively correlated with MDA and protein carbonyls; ACh level showed a significant positive correlation with ChAT, SOD, and GSH-px, as well as a significant inverse correlation with MDA and protein carbonyls. Collectively, this study provides direct evidence that increased oxidative damage and cholinergic dysfunction may be the early pathological responses to soluble Aβ, and involved in the early memory deficits in the pre-plaque stage of AD. These findings suggest that early antioxidant therapy and improving cholinergic function may be a promising strategy to prevent or delay the onset and progression of AD.

Highlights

► 3.5-month-old APPswe/PS1dE9 mice exhibited spatial memory impairment. ► 3.5-month-old APPswe/PS1dE9 mice had cholinergic dysfunction and oxidative damage. ► Impaired memory correlated with cholinergic dysfunction and oxidative damage. ► Soluble Aβ correlated with ACh, ChAT, SOD, GSH-px, MDA, and protein carbonyls. ► Oxidative damage and cholinergic dysfunction are involved in early memory deficits.

Fri, 03 Feb 2012 06:02

Pharmacological concentrations of ascorbate radio-sensitize glioblastoma multiforme primary cells by increasing oxidative DNA damage and inhibiting G2/M arrest

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 2 February 2012

Patries M. Herst, Kate W.R. Broadley, Jacquie L. Harper, Melanie J. McConnell

Glioblastoma multiforme (GBM) has a very poor prognosis due to its chemo- and radiation therapy resistance. Here we investigated the ability of pharmacological concentrations of ascorbate to radio-sensitize primary cells isolated from six GBM patients, mouse astrocytoma cells and mouse astrocytes. We measured cell viability by trypan blue exclusion, generation of double stranded DNA breaks by H2AX phosphorylation using fluorescently labelled antibodies and FACS analysis, apoptosis by annexin V/propidium iodide staining, inhibition of autophagy by 3-methyladenine and cell cycle progression by propidium iodide staining of permeabilized cells. We showed that 5 mM ascorbate in combination with 6 Gy radiation killed more GBM primary cells by generating significantly more double stranded breaks than either treatment alone (p < 0.05). Combined treatment affected viability and double stranded break generation in normal astrocytes to a much smaller extent. Radiation, but not 5 mM ascorbate, caused G2/M arrest in GBM cell and ascorbate prevented radiation-induced G2/M arrest in combined treatment. Cell death in response to 5 mM ascorbate or combination treatment was not mediated by apoptosis or autophagy.In conclusion, pharmacological concentrations of ascorbate radio-sensitize GBM primary cells to a much greater extent than astrocytes; this large therapeutic ratio may be of clinical significance in radiation resistant cancers.

Highlights

► 0.5 mM ascorbate sensitized GBM primary cells from six patients to 1 Gy radiation. ► 5 mM ascorbate plus 6 Gy significantly increased the number of DSBs. ► 5 mM ascorbate did not cause G2/M arrest ► 5mN ascorbate inhibited radiation-induced G2/M arrest in combined treatment.

Fri, 03 Feb 2012 06:02

Purified human paraoxonase-1 interacts with plasma membrane lipid rafts and mediates cholesterol efflux from macrophages

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 1 February 2012

Hicham Berrougui, Soumaya Loued, Abdelouahed Khalil

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a major contribution to the antioxidant and anti-inflammatory capacities of HDLs. However, the role of PON1 in the modulation of cholesterol efflux is poorly understood. The aim of our study was to investigate the involvement of PON1 in the regulation of cholesterol efflux, especially the mechanism by which it modulates HDL-mediated cholesterol transport. The enrichment of HDL3with human PON1 enhanced, in a dose-dependent manner, cholesterol efflux from THP-1 macrophage-like cells and ABCA1-enriched J774 macrophages. Moreover, an additive effect was observed when ABCA1-enriched J774 macrophages were incubated with both PON1 and apo-AI. Interestingly, PON1 alone was able to mediate cholesterol efflux from J774 macrophages and to up-regulate ABCA1 expression on J774 macrophages. Immunofluorescence measurement showed an increase of PON1 levels in the cytoplasm of J774 macrophages over-expressing ABCA1. PON1 used an apo-AI-like mechanism to modulate cholesterol efflux from rapid and slow efflux pools derived from the lipid raft and non-raft domains of the plasma membrane, respectively. This was supported by the fact that ABCA1 protein was incrementally expressed by J774 macrophages within the first few hours of incubation with cholesterol-loaded J774 macrophages and that cyclodextrin significantly inhibited the capacity of PON1 to modulate cholesterol efflux from macrophages. This finding suggested that PON1 plays an important role in the anti-atherogenic properties of HDLs and may exert its protective function outside the lipoprotein environment.

Highlights

► PON1 stimulates the capacity of HDL to mediate cholesterol efflux ► PON1 up-regulates the ABCA1 gene and protein expression on J774 macrophages ► PON1 stimulates the association of apoA-I to ABCA1 to stimulate cholesterol efflux ► PON1 acts via an apo-AI-like mechanism to mediate cholesterol efflux.

Fri, 03 Feb 2012 06:02

A high-selenium diet induced insulin resistance in gestating rats and their offspring

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 1 February 2012

Min-Shu Zeng, Xi Li, Yan Liu, Hua Zhao, Ji-Chang Zhou, ...

While supranutrition of selenium (Se) is considered to be a promising anti-cancer strategy, recent human studies have shown an intriguing association between high body Se status and diabetic risk. This study was to determine if a prolonged high intake of dietary Se actually induced gestational diabetes in rat dams and insulin resistance in their offspring. Forty-five 67-d old female Wistar rats (n = 15/diet) were fed a Se-deficient (0.01 mg/kg), corn-soy basal diet (BD) or BD + Se (as Se-yeast) at 0.3 or 3.0 mg/kg from 5 wk before breeding to d 14 postpartum. Offspring (n = 8/diet) of the 0.3 and 3.0 mg Se/kg dams were fed with the same respective diet until age of 112 d. Compared with the 0.3 mg Se/kg diet, the 3.0 mg/kg diet induced hyperinsulinemia (P < 0.01), insulin resistance (P < 0.01), and glucose intolerance (P < 0.01) in the dams at late gestation and(or) d 14 postpartum and in the offspring at age of 112 d. Those impairments concurred with decreased (P < 0.05) mRNA and(or) protein levels of 6 insulin signal proteins in liver and muscle of dams and(or) pups. Dietary Se produced dose-dependent increases inGpx1mRNA or GPX1 activity in pancreas, liver, and erythrocytes of dams. The 3.0 mg Se/kg diet decreasedSelh(P < 0.01),Sepp1(P = 0.06), andSepw1(P < 0.01), but increasedSels(P < 0.05) mRNA levels in liver of the offspring, compared with the 0.3 mg Se/kg diet. In conclusion, supranutrition of Se as the Se-enriched yeast in rats induced gestational diabetes and insulin resistance. Expression of 6 selenoprotein genes, in particularGpx1, was linked to this metabolic disorder.

Highlights

► We studied if supranutrition of Se induced gestational diabetes in rat dams. ► Gestational diabetes was induced by feeding dams with Se-yeast at 3 mg Se/kg. ► High maternal Se exposure and high Se feeding led to insulin resistant pups. ► Tissue expressions of 6 insulin signal proteins were altered by the high Se diet.► Gpx1and 5 other selenoprotein genes were linked to the metabolic disorder.

Fri, 03 Feb 2012 06:02

Amplification of Pro-inflammatory Phenotype, Damage, and Weakness by Oxidative Stress in the Diaphragm Muscle ofmdxMice

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 31 January 2012

Jong-Hee Kim, John M. Lawler

Duchenne muscular dystrophy (DMD) is a common and devastating type of childhood-onset muscular dystrophy, attributed to an X-linked defect in the gene that encodes for dystrophin. Myopathy with DMD is most pronounced in the diaphragm muscle and fast-twitch limb muscles, and is dependent upon susceptibility to damage, inflammatory cell infiltration, and pro-inflammatory signaling (nuclear factor-kappaB: NF-B). While recent papers have re-awakened the notion that oxidative stress links inflammatory signaling with pathology in DMD in limb muscle, the importance of redox mechanisms had been clouded by inconsistent results from indirect scavenger approaches, including the diaphragm muscle. Therefore, we used a novel catalytic mimetic of superoxide dismutase and catalase (EUK-134) as a direct of oxidative stress on myopathy in the diaphragm of themdxmouse model. EUK-134 reduced 4-hydroynonenal and total hydroperoxides, markers of oxidative stress in themdxdiaphragm. EUK-134 also attenuated positive staining of macrophage and T-cells as well as activation of NF-B and p65 protein abundance. Moreover, EUK-134 ameliorated markers of muscle damage including internalized nuclei, variability of cross-sectional area, and Type IIc fibers. Finally, impairment of contractile force was partially rescued by EUK-134 in the diaphragm ofmdxmice. We conclude that oxidative stress amplifies DMD pathology in the diaphragm muscle.

► EUK-134 reduces oxidative stress in themdxdiaphragm. ► EUK-134 reduces muscle damage in themdxdiaphragm. ► EUK-134 attenuates elevation of inflammatory cell invasion in themdxdiaphragm. ► EUK-134 attenuates NF-B activity and p65 subunit protein levels inmdxdiaphragm. ► EUK-134 provides partial protection against suppressed diaphragm contractility.

Fri, 03 Feb 2012 06:02

Mechanistic overview of reactive species-induced degradation of the endothelial glycocalyx during hepatic ischemia/reperfusion injury

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 31 January 2012

Rowan F. van Golen, Thomas M. van Gulik, Michal Heger

Endothelial cells are covered by a delicate meshwork of glycoproteins known as the glycocalyx. Under normophysiological conditions the glycocalyx plays an active role in maintaining vascular homeostasis by deterring primary and secondary hemostasis and leukocyte adhesion and by regulating vascular permeability and tone. During (micro)vascular oxidative and nitrosative stress, which prevails in numerous metabolic (diabetes), vascular (atherosclerosis, hypertension), and surgical (ischemia/reperfusion injury, trauma) disease states, the glycocalyx is oxidatively and nitrosatively modified and degraded, which culminates in an exacerbation of the underlying pathology. Consequently, glycocalyx degradation due to oxidative/nitrosative stress has far reaching clinical implications. In this review the molecular mechanisms of reactive oxygen and nitrogen species-induced destruction of the endothelial glycocalyx are addressed in the context of hepatic ischemia/reperfusion injury as a model disease state. Specifically, the review focuses on (I) the mechanisms of glycocalyx degradation during hepatic ischemia/reperfusion, (II) the molecular and cellular players involved in the degradation process, and (III) its implications on hepatic pathophysiology. These topics are projected against a background on liver anatomy, glycocalyx function and structure, and the biology/biochemistry and the sources/targets of reactive oxygen and nitrogen species. The majority of the glycocalyx-related mechanisms elucidated for hepatic ischemia/reperfusion are extrapolatable to the other aforementioned disease states.

Highlights

► Hepatic I/R injury is characterized by the production of reactive oxygen and nitrogen species. ► The glycocalyx is a protective sugar layer that normally coats the endothelium. ► Reactive oxygen and nitrogen species degrade the glycocalyx to exacerbate hepatic I/R injury. ► Glycocalyx loss increases oxidative stress and facilitates leukocyte adhesion. ► Oxidative glycocalyx degradation yields immunogenic circulating fragments.

Tue, 31 Jan 2012 09:01

β-caryophyllene ameliorates cisplatin-induced nephrotoxicity in a cannabinoid 2 receptor-dependent manner

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 31 January 2012

Béla Horváth, Partha Mukhopadhyay, Malek Kechrid, Vivek Patel, Galin Tanashian, ...

(E)-β-caryophyllene (BCP) is a natural sequiterpene found in many essential oils of spice (best known for contributing to the spiciness of black pepper) and food plants with recognized anti-inflammatory properties. Recently it was shown that BCP is a natural agonist of endogenous cannabinoid 2 (CB2) receptors, which are expressed in immune cells and mediate anti-inflammatory effects. In this study we aimed to test the effects of BCP in a clinically relevant murine model of nephropathy (induced by the widely used antineoplastic drug cisplatin) in which the tubular injury is largely dependent on inflammation and oxidative/nitrative stress.β-caryophyllene dose-dependently ameliorated cisplatin-induced kidney dysfunction, morphological damage, and renal inflammatory response (chemokines MCP-1 and MIP-2, cytokines TNF-α and IL-1β, adhesion molecule ICAM-1, and neutrophil and macrophage infiltration). It also markedly mitigated oxidative/nitrative stress (NOX-2, NOX-4 expression, 4-HNE and 3-NT content) and cell death. The protective effects of BCP against biochemical and histological markers of nephropathy were absent in CB2knockout mice. Thus, BCP may be an excellent therapeutic agent to prevent cisplatin-induced nephrotoxicity through a CB2receptor dependent pathway. Given the excellent safety profile of BCP in humans it has tremendous therapeutic potential in multitude of diseases associated with inflammation and oxidative stress.

Highlights

► β-caryophyllene (BCP) is a constituent of many essential oils of spice/food plants ► Cisplatin (CIS) is a widely used anticancer drug which induces nephrotoxicity ► CIS nephrotoxicity involves oxidative stress and inflammatory response ► BCP attenuates the CIS-induced kidney inflammation, oxidative stress and injury ► The protective effect of BCP is mediated via cannabinoid 2 (CB2) receptors

Tue, 31 Jan 2012 09:01

Calorie Restriction Increases Cerebral Mitochondrial Respiratory Capacity in a NO-Mediated Mechanism: Impact on Neuronal Survival

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 26 January 2012

Fernanda M. Cerqueira, Fernanda M. Cunha, Francisco R.M. Laurindo, Alicia J. Kowaltowski

Calorie restriction (CR) enhances animal lifespan and prevents age-related diseases, including neurological decline. Recent evidence suggests a mechanism involved in CR-induced lifespan extension is NO-stimulated mitochondrial biogenesis. We examine here the effects of CR on brain mitochondrial content. CR increased eNOS and nNOS and the content of mitochondrial proteins (cytochrome c oxidase, citrate synthase and mitofusin) in the brain. Furthermore, we established anin vitrosystem to study the neurological effects of CR using serum extracted from animals on this diet. In cultured neurons, CR serum enhanced nNOS expression and increased nitrite levels (a NOproduct). CR serum also enhanced the levels of cytochrome c oxidase and increased citrate synthase activity and respiratory rates in neurons. CR serum effects were inhibited by L-NAME and mimicked by the NOdonor SNAP. Furthermore, both CR sera and SNAP were capable of improving neuronal survival. Overall, our results indicate that CR increases mitochondrial biogenesis in a NO-mediated manner, resulting in enhanced reserve respiratory capacity and improved survival in neurons.

Highlights

► Calorie restriction (CR) increases eNOS, nNOS and mitochondrial content in brain. ► Serum from CR animals increased nNOS and NOin neuronal cultures. ► CR serum enhances mitochondrial biogenesis and respiratory rates in neurons. ► CR serum effects are inhibited by L-NAME and mimicked by the NOdonor SNAP. ► CR serum and SNAP increase neuronal survival.

Sat, 28 Jan 2012 09:01

Assessment of antioxidant capacity for scavenging free radicalsin vitro: A rational basis and practical application

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 26 January 2012

Mizuki Takashima, Masanori Horie, Mototada Shichiri, Yoshihisa Hagihara, Yasukazu Yoshida, ...

With increasing evidence showing the involvement of oxidative stress induced by free radicals in the development of various diseases, the role of radical scavenging antioxidants has received much attention. Although many randomized controlled clinical trials do not support the beneficial effects of indiscriminate supplementation of antioxidants, more recent studies suggest that antioxidants such as vitamin E may be effective for prevention and treatment of some diseases when given to right subjects at right time. Many studies on the antioxidant capacity assessed by various available methods showed inconsistent results and the assessment of antioxidant capacity has been the subject of extensive studies and arguments. The present study was performed to elucidate basic chemistry required for the development of reliable method for assessment of antioxidant capacity for radical scavenging in vitro. In the present study, the capacity of α-tocopherol and its related compounds, ascorbic acid, and uric acid for scavenging radicals was assessed from their effects on the rate of decay of hydrophilic and lipophilc probes with different reactivity toward free radicals induced by hydrophilic and lipophilic radicals in homogeneous solution and heterogeneous micelle systems. Fluorescein, pyranine, and pyrogallol red were used as hydrophilic probe, while BODIPY andN,N-diphenyl-p-phenylenediamine were used as lipophilic probe. It was shown that the rate and amount of radical scavenging by antioxidant, termed Antioxidant Radical Absorbance Capacity (ARAC), could be assessed respectively by an appropriate combination of radical initiator and probe. This method was applied for the assessment of radical scavenging capacity of human plasma, wine, and green tea powder.

Highlights

► Method for assessment of antioxidant capacity for radical scavenging was developed. ► Reactivity and concentration of antioxidants in mixtures were measured separately. ► Probes with different reactivity and solubility were used for the assessment. ► Effect of localization of radical and antioxidant in water and oil phase was assessed. ► The method was applied for human plasma, wine and green tea powder.

Sat, 28 Jan 2012 09:01

The thermodynamics of thiol sulfenylation

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 26 January 2012

Lionel Billiet, Paul Geerlings, Joris Messens, Goedele Roos

Protein sulfenic acids are essential cysteine oxidations in cellular signaling pathways. The thermodynamics that drive protein sulfenylation are not entirely clear. Experimentally, sulfenic acid reduction potentials are hard to measure, due to their highly reactive nature. We designed a calculation method, the Reduction potentials from Electronic Energies (REE) method, to give for the first time insight in the thermodynamic aspects of protein sulfenylation. The REE method is based on the correlation between reaction path independent reaction energies and free energies of a series of analogous reactions. For human peroxiredoxin (Tpx-B), an anti-oxidant enzyme that forms a sulfenic acid on one of its active site cysteines during reactive oxygen scavenging, we found that the reduction potential depends on the composition of the active site and on the protonation state of the cysteine. Interaction with polar residues directs the RSO/RSreduction to a lower potential than the RSOH/RSH reduction. A conserved arginine that thermodynamically favors the sulfenylation reaction might be a good candidate to favor the reaction kinetics. The REE method is not limited to thiol sulfenylation, but can broadly be applied to understand protein redox biology in general.

Highlights

► Reduction potentials for sulfenylation calculated at low computational cost. ► For analogous reactions, the reaction energy correlates with the free energy. ► Polar residues thermodynamically drive the oxidation of thiolate. ► A hydrophobic environment thermodynamically drives the thiol oxidation. ► In human peroxiredoxin, Arg127 determines the sulfenylation thermodynamics

Sat, 28 Jan 2012 09:01

RNA modifications by oxidation: A novel disease mechanism?

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 26 January 2012

Henrik E. Poulsen, Elisabeth Specht, Kasper Broedbaek, Trine Henriksen, Christina Ellervik, ...

The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50–70% of specific mRNA molecules are reported oxidized, whereas other RNA molecules show virtually no oxidation. The iron-storage disease hemochromatosis exhibits the most prominent general increase in RNA oxidation ever observed. Oxidation of RNA primarily leads to strand breaks and to oxidative base modifications. Oxidized mRNA is recognized by the ribosomes, but the oxidation results in ribosomal stalling and dysfunction, followed by decreased levels of functional protein as well as the production of truncated proteins that do not undergo proper folding and may result in protein aggregation within the cell. Ribosomal dysfunction may also signal apoptosis by p53-independent pathways. There are very few reports on interventions that reduce RNA oxidation, one interesting observation being a reduction in RNA oxidation by ingestion of raw olive oil. High urinary excretion of 8-oxo-guanosine, a biomarker for RNA oxidation, is highly predictive of death in newly diagnosed type 2 diabetics; this demonstrates the clinical relevance of RNA oxidation. Taken collectively the available data suggest that RNA oxidation is a contributing factor in several diseases such as diabetes, hemochromatosis, heart failure, and β-cell destruction. The mechanism involves free iron and hydrogen peroxide from mitochondrial dysfunction that together lead to RNA oxidation that in turn gives rise to truncated proteins that may cause aggregation. Thus RNA oxidation may well be an important novel contributing mechanism for several diseases.

Highlights

► RNA oxidation is more abundant than DNA oxidation. ► High levels of oxidized RNA are found in degenerative brain diseases. ► General RNA oxidation is measured by 8-oxoGuo excretion into urine. ► General RNA oxidation predicts death in type 2 diabetes. ► RNA may be a novel and important mechanism of diseases such as diabetes, degenerative brain diseases, and hemochromatosis.

Sat, 28 Jan 2012 09:01

Editorial Board

Publication year: 2012
Source: Free Radical Biology and Medicine, Volume 52, Issue 4, 15 February 2012, Pages IFC

[No author name available]

Fri, 27 Jan 2012 05:01

Announcements and Calendar

Publication year: 2012
Source: Free Radical Biology and Medicine, Volume 52, Issue 4, 15 February 2012, Pages 818

[No author name available]

Fri, 27 Jan 2012 05:01

Editorial Board

Publication year: 2012
Source: Free Radical Biology and Medicine, Volume 52, Issue 3, 1 February 2012, Pages IFC

[No author name available]

Fri, 27 Jan 2012 05:01

Announcements and Calendar

Publication year: 2012
Source: Free Radical Biology and Medicine, Volume 52, Issue 3, 1 February 2012, Pages 724

[No author name available]

Fri, 27 Jan 2012 05:01

NovelS-acyl-glutathione derivatives prevent amyloid oxidative stress and cholinergic dysfunction in Alzheimer's disease models

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 27 January 2012

Mariagioia Zampagni, Daniel Wright, Roberta Cascella, Giampiero D'Adamio, Fiorella Casamenti, ...

Oxidative stress-mediated neuronal death may be initiated by a decrease in glutathione (GSH), whose levels are reduced in mitochondrial and synaptosomal fractions of specific CNS regions in Alzheimer's disease (AD) patients. Currently, the use of GSH as a therapeutic agent is limited by its unfavourable pharmacokinetic properties. In this study, we designed the synthesis of newS-acyl-glutathione (acyl-SG) thioesters of fatty acidsvia N-acyl benzotriazole-intermediate production, and investigated their potential for targeted delivery of the parent GSH and free fatty acid to amyloid-exposed fibroblasts from familial AD patients and human SH-SY5Y neuroblastoma cells. Cell culture supplementation with acyl-SG derivatives triggers a significant decrease in lipid peroxidation and mitochondrial dysfunction in a fatty acid unsaturation degree-dependent fashion. Acyl-SG thioesters also protect cholinergic neurons against Aβ-induced damage and reduce glial reaction in rat brains.Collectively, these findings suggest that acyl-SG thioesters could prove useful as a tool for controlling AD-induced cerebral deterioration.

Schematic diagram depicting the dual antioxidant feature enclosed in a single molecule of acyl-SG thioester. The lipophilic nature of the novel pro-drugs allowed them to easily cross the plasma membrane and be trapped intracellularly by thioesterase-catalyzed hydrolysis, resulting in the release of the parent free reduced GSH, which acts as a scavenger, and PUFA, which acts as carrier and itself has inherent antioxidant properties.

Highlights

► We report a novel and efficient synthesis of a series of new acyl-SG thioesters. ► Acyl-SGs act as pro-drug increasing the intracellular GSH. ► Polyunsaturated acyl-SGs possess inherent antioxidant properties. ► Acyl-SGs reduce lipid peroxidation and mitochondrial dysfunction in Aβ-treated cells. ► Acyl-SGs protect against Aβ-induced neurodegeneration and glial reaction in rat brain.

Fri, 27 Jan 2012 05:01

Oxidative stress, endogenous antioxidants, alcohol, and hepatitis C: Pathogenic interactions and therapeutic considerations

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 25 January 2012

Jinah Choi

Hepatitis C virus (HCV) is a bloodborne pathogen that was identified as an etiologic agent of Non-A, Non-B hepatitis in 1989. HCV is estimated to have infected at least 170 million people worldwide. The majority of patients infected with HCV does not clear the virus and becomes chronically infected, and chronic HCV infection increases the risk for hepatic steatosis, cirrhosis, and hepatocellular carcinoma. HCV induces oxidative/nitrosative stress from multiple sources including the inducible nitric oxide synthase, mitochondrial electron transport chain, hepatocyte NAD(P)H oxidases, and inflammation while decreasing glutathione. The cumulative oxidative burden is likely to promote both hepatic and extrahepatic conditions precipitated by HCV through a combination of local and more distal effects of reactive species, and clinical, animal, andin vitrostudies strongly point to a role of oxidative/nitrosative stress in the HCV-induced pathogenesis. Oxidative stress and hepatopathogenesis induced by HCV are exacerbated by even low doses of alcohol. Alcohol and reactive species may have other effects on hepatitis C patients such as modulation of the host immune system, viral replication, and positive selection of HCV sequence variants that contributes to antiviral resistance. This review summarizes current understanding of redox interactions of HCV, outlining key experimental findings, directions for future research, and potential applications to therapy.

Highlights

► HCV induces oxidative/nitrosative stress from multiple sources ► Pathogenesis involves local and more distal effects of reactive species ► Effects of alcohol on the HCV-induced oxidative stress and pathogenesis ► Redox modulation of host immune response, HCV replication, and viral genome

Fri, 27 Jan 2012 05:01

Damage to mtDNA in liver injuries of patients with extra-hepatic cholestasis: The protective effects of mitochondrial transcription factor A

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 25 January 2012

Shang-Cheng Xu, Yong-Biao Chen, Heng Lin, Hui-Feng Pi, Ni-Xian Zhang, ...

Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of chronic liver cholestasis. Mitochondrial DNA (mtDNA) is highly susceptible to oxidative stress and mtDNA damage leads to mitochondrial dysfunction. This study aimed to investigate the mtDNA alterations that occurred during liver injuries in patients with extra-hepatic cholestasis. Along with an increase in malondialdehyde (MDA) levels and a decrease in ATP levels, extrahepatic cholestatic patients presented a significant increase in mitochondrial 8-hydroxy-deoxyguanosine (8-OHdG) levels and decreases in mtDNA copy number, mtDNA transcripts levels and mtDNA nucleoid structure. In L02 cells, glycochenodeoxycholic acid (GCDCA) induced similar damage to the mtDNA and mitochondria. In line with the mtDNA alterations, the mRNA and protein levels of mitochondrial transcription factor A (TFAM) were significantly decreased both in cholestatic patients and in GCDCA-treated L02 cells. Moreover, over-expression of TFAM could efficiently attenuate the mtDNA damage induced by GCDCA in L02 cells. However, without the C-tail,ΔC-TFAM appeared less effective against the hepatotoxicity of GCDCA than the wild-type TFAM. Overall, our study demonstrates that mtDNA damage is involved in liver damage in extrahepatic cholestatic patients. The mtDNA damage is attributable to the loss of TFAM. TFAM has mtDNA-protective effects against the hepatotoxicity of bile acid during cholestasis.

Highlights

► Increased mtDNA damage was found in extrahepatic cholestasis patients. ► The mtDNA damage is attributable to the loss of TFAM. ► TFAM has mtDNA-protective effects against the hepatotoxicity of bile acid during cholestasis. ► Without the C-tail, TFAM is less effective against the hepatotoxicity of GCDCA.

Fri, 27 Jan 2012 05:01

Systemic oxidative stress is implicated in the pathogenesis of brain edema in rats with chronic liver failure

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 24 January 2012

Cristina R. Bosoi, Xiaoling Yang, Jimmy Huynh, Christian Parent-Robitaille, Wenlei Jiang, ...

Chronic liver failure leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy (HE); however, a correlation between blood ammonia levels and HE severity remains controversial. It is believed oxidative stress plays a role in modulating the effects of hyperammonemia. This study aimed to determine the relationship between chronic hyperammonemia, oxidative stress, and brain edema (BE) in two rat models of HE: portacaval anastomosis (PCA) and bile-duct ligation (BDL). Ammonia and reactive oxygen species (ROS) levels, BE, oxidant and antioxidant enzyme activities, as well as lipid peroxidation were assessed both systemically and centrally in these two different animal models. Then, the effects of allopurinol (xanthine oxidase inhibitor, 100 mg/kg for 10 days) on ROS and BE and the temporal resolution of ammonia, ROS, and BE were evaluated only in BDL rats. Similar arterial and cerebrospinal fluid ammonia levels were found in PCA and BDL rats, both significantly higher compared to their respective sham-operated controls (p < 0.05). BE was detected in BDL rats (p < 0.05) but not in PCA rats. Evidence of oxidative stress was found systemically but not centrally in BDL rats: increased levels of ROS, increased activity of xanthine oxidase (oxidant enzyme), enhanced oxidative modifications on lipids and proteins, as well as decreased antioxidant defense. In PCA rats, a preserved oxidant/antioxidant balance was demonstrated. Treatment with allopurinol in BDL rats attenuated both ROS and BE, suggesting systemic oxidative stress is implicated in the pathogenesis of BE. Analysis of ROS and ammonia temporal resolution in the plasma of BDL rats suggests systemic oxidative stress might be an important “first hit,” which, followed by increases in ammonia, leads to BE in chronic liver failure. In conclusion, chronic hyperammonemia and oxidative stress in combination lead to the onset of BE in rats with chronic liver failure.

Highlights

► Hyperammonemia does not lead independently to brain edema in chronic liver failure. ► Systemic oxidative stress plays a role in brain edema in chronic liver failure. ► Systemic oxidative stress induces brain edema synergistically with hyperammonemia. ► Systemic oxidative stress is an important “first hit” in chronic liver failure.

Tue, 24 Jan 2012 08:01

Nitric oxide in adaptation to altitude

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 20 January 2012

Cynthia M. Beall, Daniel Laskowski, Serpil C. Erzurum

This review summarizes published information on levels of nitric oxide gas (NO) in the lungs and NO-derived liquid phase molecules in the acclimatization of visitors newly arrived at altitudes of 2500 m or more and adaptation of populations whose ancestors arrived thousands of years ago. Studies of acutely exposed visitors to high altitude focus on the first 24–48 hours with just a few extending to days or weeks. Among healthy visitors, NO levels in the lung, plasma and/or red blood cells fell within three hours, but then returned toward baseline or slightly higher by 48 hours, and increased above baseline by 5 days. Among visitors ill with high-altitude pulmonary edema at the time of the study or in the past, NO levels were lower than their healthy counterparts. As for highland populations, Tibetans had NO levels in the lung, plasma and red blood cells that were at least double and in some cases orders of magnitude greater than other populations regardless of altitude. Red blood cell associated nitrogen oxides were more than two hundred times higher. Other highland populations had generally higher levels although not to the degree showed by Tibetans. Overall, responses of those acclimatized and those presumed to be adapted are in the same direction although the Tibetans have much larger responses. Missing are long-term data on lowlanders at altitude showing how similar they become to the Tibetan phenotype. Also missing are data on Tibetans at low altitude to see the extent to which their phenotype is a response to the immediate environment or expressed constitutively. The mechanisms causing the visitors’ and the Tibetans’ high levels of NO and NO-derived molecules at altitude remain unknown. Limited data suggest processes including hypoxic upregulation of NO synthase gene expression, hemoglobin-NO reactions and genetic variation. Gains in understanding will require integrating appropriate methods and measurement techniques with indicators of adaptive function under hypoxic stress.

This figure shows nitric oxide (NO) and NO-derived products in the lungs and circulation that influence gas transfer in the lungs and oxygen delivery and utilization in the tissues. Increase of NO and NO-derived reaction products is part of the integrated set of human responses to high altitude that is beneficial for survival under high-altitude hypoxia.

Highlights

► Published data identify NO as integral to human adaptation to altitudes above 2500 m. ► After a transitory early decline, most nitrogen oxides exceed low-altitude baseline. ► Lack of increase in NO or nitrogen oxides associates with acute mountain sickness. ► Tibetans have high levels of NO that are associated with greater oxygen delivery.

Fri, 20 Jan 2012 06:01

Identification of RhitH as a novel transcriptional repressor of human Mpv17-like protein with a mitigating effect on mitochondrial dysfunction, and its transcriptional regulation by FOXD3 and GABP

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 20 January 2012

Reiko Iida, Misuzu Ueki, Toshihiro Yasuda

Mpv17-like protein (M-LP) is a protein that has been suggested to be involved in metabolism of reactive oxygen species. To elucidate the molecular basis of M-LP expression, we recently searched for regulatory elements of M-LP and identified a novel mouse KRAB-containing protein, Rhit, (regulator ofheat-inducedtranscription) as a repressor of the transcriptional regulation of M-LP. In this study, we identified zinc finger protein 205 (ZNF205) as a candidate human Rhit (RhitH), and subsequently confirmed its participation in transcriptional regulation of human M-LP (M-LPH). To clarify the function of RhitH and M-LPH, we searched forcis-regulatory elements in the promoter region of RhitH, and identified two transcription factors: forkhead box D3 (FOXD3) as a negative regulatory element, and GA-binding protein (GABP), one of the key regulators of the mitochondrial electron transport system, as a positive regulatory element. Additionally, it was demonstrated that knockdown of RhitH or overexpression of M-LPH reduces the generation of intracellular H2O2and loss of mitochondrial membrane potential caused by an inhibitor of the respiratory chain, antimycin A. These results suggested that M-LPH functions to protect cells from oxidative stress and/or initiation of the mitochondrial apoptotic cascade under stressed conditions.

Highlights

►RhitH is involved in transcriptional regulation of M-LPH.►FOXD3 and GABP are involved in transcriptional regulation of RhitH.►M-LPH reduces intracellular H2O2and loss of mitochondrial membrane potential.►Our data suggest that M-LPH has a mitigating effect on mitochondrial dysfunction.

Fri, 20 Jan 2012 06:01

Mitochondrial permeability transition pore opening induces the initial process of renal calcium crystallization

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 18 January 2012

Kazuhiro Niimi, Takahiro Yasui, Masahito Hirose, Shuzo Hamamoto, Yasunori Itoh, ...

Renal tubular cell injury induced by oxidative stress via mitochondrial collapse is thought to be the initial process of renal calcium crystallization. Mitochondrial collapse is generally caused by mitochondrial permeability transition pore (mPTP) opening, which can be blocked by cyclosporine A (CsA). Definitive evidence for the involvement of mPTP opening in the initial process of renal calcium crystallization, however, is lacking. In this study, we examined the physiological role of mPTP opening in renal calcium crystallization in vitro and in vivo. In the in vitro study, cultured renal tubular cells were exposed to calcium oxalate monohydrate (COM) crystals and treated with CsA (2 μM). COM crystals induced depolarization of the mitochondrial membrane potential and generated oxidative stress as evaluated by Cu-Zn SOD and 4-HNE. Furthermore, the expression of cytochromecand cleaved caspase 3 was increased and these effects were prevented by CsA. In the in vivo study, Sprague–Dawley rats were administered 1% ethylene glycol (EG) to generate a rat kidney stone model and then treated with CsA (2.5, 5.0, and 10.0 mg/kg/day) for 14 days. EG administration induced renal calcium crystallization, which was prevented by CsA. Mitochondrial collapse was demonstrated by transmission electron microscopy, and oxidative stress was evaluated by measuring Cu-Zn SOD, MDA, and 8-OHdG generated by EG administration, all of which were prevented by CsA. Collectively, our results provide compelling evidence for a role of mPTP opening and its associated mitochondrial collapse, oxidative stress, and activation of the apoptotic pathway in the initial process of renal calcium crystallization.

Highlights

► The physiological role of mitochondrial permeability transition pore (mPTP) opening in renal calcium crystallization was examined. ► Cyclosporine A decreases oxidative stress by blocking mPTP opening in the kidney. ► Decreasing renal oxidative stress prevents renal calcium crystallization. ► Blocking mPTP opening prevents renal calcium crystallization.

Wed, 18 Jan 2012 10:01

Geranylgeranylacetone protects mice against morphine-induced hyperlocomotion, rewarding effect, and withdrawal syndrome

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 18 January 2012

Fu-Cheng Luo, Lei Qi, Tao Lv, Sheng-Dong Wang, Hua Liu, ...

There are few efficacious interventions to combat morphine dependence. Thioredoxin-1 (Trx-1) and heat shock protein 70 (Hsp70) are emerging as important modulators of neuronal function. They have been shown to be involved in cellular protective mechanisms against a variety of toxic stressors. This study was designed to investigate the effects of geranylgeranylacetone (GGA), a pharmacological inducer of Trx-1 and Hsp70, on morphine-induced hyperlocomotion, rewarding effect, and withdrawal syndrome. Trx-1 and Hsp70 expression was increased in the frontal cortex, hippocampus, ventral tegmental area, and nucleus accumbens of mice after GGA treatment. GGA administration reduced morphine-induced motor activity and inhibited conditioned place preference. GGA markedly attenuated the morphine–naloxone-induced withdrawal signs, including jumping, rearing, and forepaw tremor. Furthermore, the activation of cAMP-responsive element-binding protein and the expression of ΔFosB and cyclin-dependent kinase 5 were decreased in the nucleus accumbens by GGA treatment after morphine withdrawal. In the nucleus accumbens, GGA enhanced morphine-induced expression of Trx-1 and Hsp70 after morphine withdrawal. These results suggest that strengthening the expression of Trx-1 and Hsp70 in the brain by using noncytotoxic pharmacological inducers may provide a novel therapeutic strategy for morphine dependence. GGA could be a safe and novel therapeutic agent for morphine dependence.

Highlights

► Geranylgeranylacetone attenuated morphine dependence. ► Geranylgeranylacetone induced thioredoxin-1 and heat shock protein 70 expression. ► Geranylgeranylacetone suppressed protein expression associated with drug addiction.

Wed, 18 Jan 2012 10:01

Identification of a redox-sensitive switch within the JAK2 catalytic domain

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 14 January 2012

John K. Smith, Chetan N. Patil, Srikanth Patlolla, Barak W. Gunter, George W. Booz, ...

Four cysteine residues (Cys866, Cys917, Cys1094 and Cys1105) have direct roles in cooperatively regulating Janus kinase 2 (JAK2) catalytic activity. Additional site-directed mutagenesis experiments now provide evidence that two of these residues (Cys866 and Cys917) act together as a redox-sensitive switch, allowing JAK2's catalytic activity to be directly regulated by the redox state of the cell. We created several variants of the truncated JAK2 (GST/(N∆661)rJAK2) which incorporated cysteine-to-serine or cysteine-to-alanine mutations. The catalytic activities of these mutant enzymes were evaluated byin vitroautokinase assays and byin situautophosphorylation and transphosphorylation assays. Cysteine-to-alanine mutagenesis revealed that the mechanistic role of Cys866 and Cys917 is functionally distinct from that of Cys1094 and Cys1105. Most notable is the observation that the robust activity of the CC866,917AA mutant is unaltered by pre-treatment with dithiothreitol oro-iodosobenzoate, unlike all other JAK2 variants previously examined. This work provides the first direct evidence for a cysteine-based redox-sensitive switch that regulates JAK2 catalytic activity. The presence of this redox-sensitive switch predicts that reactive oxygen species can impair the cell's response to JAK-coupled cytokines under conditions of oxidative stress, which we confirm in a murine pancreatic β-islet cell line.

Highlights

► Cys866 and Cys917 function as a redox sensor switch in JAK2 ► Converting these cysteines to alanines creates a redox-refractive enzyme ► This switch is conserved as Cys892 and Cys944 in JAK1 ► Provides a biochemical basis for direct redox-regulation of JAK activity ► Explains H2O2inhibition of JAK2-dependent STAT5 phosphorylation in βTC-6 cells

Sun, 15 Jan 2012 09:01

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

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 14 January 2012

Rosa Bretón-Romero, Cecilia González de Orduña, Natalia Romero, Francisco J. Sánchez, Cristina de Álvaro, ...

ObjectivesLaminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function, and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species (ROS). LSS activates several endothelial signaling responses including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways.Methods and resultsUsing the cone/plate model we found that LSS (12 dyn/cm) rapidly (5–60 min) promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H2O2). Physiological concentrations of H2O2(flux of 0.1 nM/min and 15 μM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decrease LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H2O2- and LSS-induced eNOS phosphorylation and reducedNO levels.ConclusionsWe propose a model in which LSS promotes the formation of signaling levels of H2O2, which in turn activate p38α MAPK and then stimulate eNOS, leading to increasedNO generation and protection of endothelial function.

Highlights

► Laminar shear stress (LSS) generates signaling levels of ROS in endothelial cells ► Low levels of hydrogen peroxide activate p38 MAPK and eNOS ► LSS-dependent activation of p38 MAPK is upstream of eNOS activation and NO synthesis ► LSS-dependent activation of p38 MAPK requires peroxide generation by NOX4

Sat, 14 Jan 2012 09:01

Combined Effects between Singlet Oxygen and Hydroxyl Radical in Photodynamic Therapy with Photostable Bacteriochlorins: Evidence from Intracellular Fluorescence and Increased Photodynamic Efficacy In Vitro

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 13 January 2012

Janusz M. Dąbrowski, Luis G. Arnaut, Mariette M. Pereira, Krystyna Urbańska, Sérgio Simões, ...

Sulfonamides of halogenated bacteriochlorins bearing Cl or F substituents in theorthopositions of the phenyl rings have adequate properties for photodynamic therapy, including strong absorptions in the near infrared (λmax ≈ 750 nm, ε ≈ 10 M cm), controlled photodecomposition, large cellular uptake, intracellular localization in the endoplasmic reticulum, low cytotoxicity and high phototoxicity against A549 and S91 cells. The role of type I and type II photochemical processes is assessed by singlet oxygen luminescence and intracellular hydroxyl radical detection. Phototoxicity of halogenated sulfonamide bacteriochlorins does not correlate with singlet oxygen quantum yields and must be mediated both by electron transfer (superoxide ion, hydroxyl radicals) and by energy transfer (singlet oxygen). The photodynamic efficacy is enhanced when cellular death is induced both by singlet oxygen and hydroxyl radicals.

Highlights

► NIR absorbing sulfonamide bacteriochlorins as photostable PDT sensitizers. ► High intracellular uptake and localization in ER explain improved photodynamic effect. ► Combined effects between singlet oxygen and hydroxyl radical make them efficient PDT agents. ► Pro-oxidant and anti-oxidant effects of ascorbate in bacteriochlorin-PDT are discussed.

Sat, 14 Jan 2012 09:01

Dual contradictory roles of cAMP signaling pathways on hydroxyl radical production in rat striatum

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 12 January 2012

Shuichi Hara, Masamune Kobayashi, Fumi Kuriiwa, Toshiji Mukai, Hajime Mizukami

Studies have suggested that cAMP signaling pathways may be associated with the production of reactive oxygen species. In the present study, we examined how modifications in cAMP signaling affected the production of hydroxyl radicals in rat striatum using microdialysis to measure extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA), which is a hydroxyl radical adduct of salicylate. Up to 50 nmol of the cell-permeable cAMP mimetic, 8-bromo-cAMP (8-Br-cAMP), increased 2,3-DHBA in a dose-dependent manner (there was no additional increase in 2,3-DHBA at 100 nmol). Another cAMP mimetic, dibutyryl cAMP (db-cAMP), caused a non-significant increase in 2,3-DHBA at 50 nmol and a significant decrease at 100 nmol. Up to 20 nmol of forskolin, which is a direct activator of adenylyl cyclase, increased 2,3-DHBA similar to the effect of 8-Br-cAMP; however, forskolin resulted in a much greater increase in 2,3-DHBA. A potent inhibitor of protein kinase A (PKA), H89 (500 μM), potentiated the 8-Br-cAMP- and forskolin-induced increases in 2,3-DHBA and antagonized the inhibitory effect of 100 nmol db-cAMP. Interestingly, the administration of 100 nmol 8-bromo-cGMP alone or in combination with H89 had no significant effect on 2,3-DHBA levels. Doses of 100 nmol of a preferential PKA activator (6-phenyl-cAMP) or a preferential PKA inhibitor (8-bromoadenosine-3’,5’-cyclic monophosphorothionate, Rp-isomer; Rp-8-Br-cAMPS), which also inhibits the cAMP-mediated activation of Epac, suppressed or enhanced formation of 2,3-DHBA, respectively. Up to 100 nmol of 8-(4-chlorophenylthio)-2’-O-methyladenosine-cAMP, which is a selective activator of Epac (the exchange protein directly activated by cAMP), dose-dependently stimulated the formation of 2,3-DHBA. These findings suggest that cAMP signaling plays contradictory roles (stimulation and inhibition) in the production of hydroxyl radicals in rat striatum by differential actions of Epac and PKA. These roles might contribute to the production of hydroxyl radicals concomitantly with cAMP in carbon monoxide poisoning, because the formation of 2,3-DHBA was potentiated by the PKA inhibitor H89 and suppressed by Rp-8-Br-cAMPS, which inhibits PKA and Epac.

Highlights

► Dual contradictory roles of cAMP signaling pathways on hydroxyl radical production. ► Stimulation of hydroxyl radical production by activating Epac ► Suppression of hydroxyl radical production by activating PKA.

Fri, 13 Jan 2012 09:01

Dual contradictory roles of cAMP signaling pathways on hydroxyl radical production in rat striatum

Highlights

CFTR is the primary known apical glutathione transporter involved in cigarette smoke-induced adaptive responses in the lung

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 12 January 2012

Neal S. Gould, Elysia Min, Richard J. Martin, Brian J. Day

One of the most abundant antioxidants in the lung is glutathione (GSH), a low-molecular-weight thiol, which functions to attenuate both oxidative stress and inflammation. GSH is concentrated in the epithelial lining fluid (ELF) of the lung and can be elevated in response to the increased oxidant burden from cigarette smoke (CS). However, the transporter(s) responsible for the increase in ELF GSH with cigarette smoke is not known. Three candidate apical GSH transporters in the lung are CFTR, BCRP, and MRP2, but their potential roles in ELF GSH transport in response to CS have not been investigated. In vitro, the inhibition of CFTR, BCRP, or MRP2 resulted in decreased GSH efflux in response to cigarette smoke extract. In vivo, mice deficient in CFTR, BCRP, or MRP2 were exposed to either air or acute CS. CFTR-deficient mice had reduced basal and CS-induced GSH in the ELF, whereas BCRP or MRP2 deficiency had no effect on ELF GSH basal or CS-exposed levels. Furthermore, BCRP or MRP2 deficiency had little effect on lung tissue GSH. These data indicate that CFTR is predominantly involved in maintaining basal ELF GSH and increasing ELF GSH in response to CS.

Highlights

► Cigarette smoke (CS) exposure induces an increase in lung epithelial lining fluid (ELF) GSH levels. ► CFTR mediates 50% of the ELF GSH under both basal and CS exposure. ► Loss of GSH-transporting proteins BCRP and MRP2 had no effect on ELF GSH levels.

Thu, 12 Jan 2012 05:01

Molecular mechanism of indomethacin-induced gastropathy

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 11 January 2012

Sudhir K. Yadav, Biplab Adhikary, Saswati Chand, Biswanath Maity, Sandip K. Bandyopadhyay, ...

The probable cross talk among large numbers of inflammatory and angiogenic parameters in indomethacin (IND)-induced gastropathy and the associated signaling mechanism were studied in a mouse model. A single dose of IND (18 mg/kg, po) produced robust gastric ulceration in mice without any mortality, which peaked on the third day, but started healing from the fifth day onward. The ulceration was associated with increased myeloperoxidase activity and expression of proinflammatory (TNF-α, adhesion molecules, COX-2) and antiangiogenic (endostatin) parameters. The levels of proangiogenic factors such as COX-1, prostaglandin E, VEGF, and von Willebrand factor VIII were downregulated by IND. Our results revealed that although the maximal and minimal levels of these parameters were attained sequentially at different time points, TNF-α upregulation was the primary event to initiate and induce gastric ulceration. IND also activated NF-κB and all the MAP kinases, but only the inhibitors of TNF-α, NF-κB, and JNK MAP kinase could abrogate the IND-induced damages. Further TNF-α inhibition also reduced the IND-mediated activation of NF-κB and JNK MAP kinase. All this evidence strongly suggests that mitigation of TNF-α may offer a potential solution to IND-mediated gastropathy.

Highlights

► This article presents the formulation of a reliable mouse model of acute gastric ulceration without mortality. ► We identified key factors involved in indomethacin-induced gastropathy. ► We established a primary role for TNF-α in initiating and sustaining inflammation. ► TNF-α is a potential target for drug development against indomethacin-induced gastropathy.

Thu, 12 Jan 2012 05:01

Inactivation of thiol-dependent enzymes by hypothiocyanous acid: Role of sulfenyl thiocyanate and sulfenic acid intermediates

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 8 January 2012

Tessa J. Barrett, David I. Pattison, Stephen E. Leonard, Kate S. Carroll, Michael J. Davies, ...

Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage via targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (< 5-fold molar excess). Experiments with DAz-2, a cell permeable, chemical trap for sulfenic acids, demonstrates that these intermediates are formed on many cellular proteins including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells, and highlights the potential of this oxidant to perturb redox signaling processes.

Highlights

► HOSCN inactivates thiol dependent enzymes in mammalian cells. ► HOSCN forms sulfur oxy acids on isolated proteins and in cellular systems. ► Reversible enzyme inactivation involves the formation of sulfenyl thiocyanate and sulfenic acid intermediates. ► Sulfinic and sulfonic acid formation results in irreversible enzyme inactivation.

Mon, 09 Jan 2012 07:01

Inactivation of thiol-dependent enzymes by hypothiocyanous acid: Role of sulfenyl thiocyanate and sulfenic acid intermediates

Highlights

Cathepsin D and L reduce the toxicity of advanced glycation end products

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 4 January 2012

Stefanie Grimm, Melanie Horlacher, Betül Catalgol, Annika Hoehn, Thomas Reinheckel, ...

Advanced glycation end product-modified proteins are known for accumulating during aging and in several pathological conditions such as diabetes, renal failure and neurodegenerative disorders. There is little information about the intracellular fate of endocytosed advanced glycation end products (AGEs) and their influence on proteolytic systems. However, it is known that the lysosomal system is impaired during aging. Therefore, undegraded material may accumulate and play a considerable role in the development of diverse diseases. To investigate if AGEs can be degraded and to test whether they accumulate due to impaired lysosomal proteases we studied the effect of advanced glycation end products on the endosomal-lysosomal system.Five different types of advanced glycation end products were generated by bovine serum albumin-incubation with glyoxal, methylglyoxal, glucose, fructose and ribose. The first experiments reveal the uptake of advanced glycation endproducts by the macrophage cell line RAW 264.7. Further investigations demonstrate an increase of cathepsin D and L activity and an increase of mature cathepsin D and L. Increased activities are accompanied by the presence of more lysosomes, measured by staining with LysoTracker blue. To specify the role of cathepsin D and L we used knock out cells to test the role of both cathepsins on the toxicity of advanced glycation end products. In summary we concluded that both cathepsins are required for a reduction of advanced glycation end product-induced cytotoxicity.

Highlights

► increase protein amounts of mature cathepsin D and L after AGE treatment in macrophages ► increase of cathepsin D and L activity after AGE treatment in macrophages ► enhanced accumulation of AGE deposits in cathepsin D and L knock out cells ► reduced viability and enhanced reactive oxygen species production in cathepsin D and L deficient cells

Wed, 04 Jan 2012 09:01

Dietary-regulation of catabolic disposal of 4-hydroxynonenal analogs in rat liver

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 4 January 2012

Qingling Li, Kristyen Tomcik, Shenghui Zhang, Michelle A. Puchowicz, Guo-Fang Zhang

Our previous work in perfused rat livers has demonstrated that 4-hydroxynonenal (HNE) is catabolized predominantlyviabeta oxidation. Therefore, we hypothesized that perturbations of beta oxidation, such as diet-altered fatty acid oxidation activity, could lead to changes in HNE levels. To test our hypothesis, we (i) developed a simple and sensitive GC/MS method combined with mass isotopomer analysis to measure HNE and HNE analogs, 4-oxononenal (ONE) and 1,4-dihydroxynonene (DHN), and (ii) investigated the effects of four diets (standard, low fat, ketogenic, and high fat mix diets) on HNE, ONE, and DHN concentrations in rat livers. Our results showed that livers from rats fed ketogenic diet or high fat mix diet had high ω-6 polyunsaturated fatty acid concentrations and markers of oxidative stress. However, high concentrations of HNE (1.6 ± 0.5 nmol/g) and ONE (0.9 ± 0.2 nmol/g) were only found in livers from rats fed the high fat mix diet. Livers from rats fed the ketogenic diet had low HNE (0.8 ± 0.1 nmol/g) and ONE (0.4 ± 0.07 nmol/g), similar to rats fed the standard diet. A possible explanation is that the predominant pathway of HNE catabolism (i.e. beta oxidation) is activated in the liver by the ketogenic diet. This is consistent with a 10 fold decrease in malonyl-CoA in livers from rats fed a ketogenic diet compared to a standard diet. The accelerated catabolism of HNE lowers HNE and HNE analog concentrations in livers from rats fed the ketogenic diet. On the other hand, rats fed the high fat mix diet had high rates of lipid synthesis and low rates of fatty acid oxidation, resulting in the slowing down of the catabolic disposal of HNE and HNE analogs. Thus, decreased HNE catabolism by a high fat mix diet induces high concentrations of HNE and HNE analogs. The results of the present work suggested a potential causal relationship to metabolic syndrome induced by western diets (i.e. high fat mix), as well as the effects of the ketogenic diet on the catabolism of lipid peroxidation products in liver.

Highlights

► Diet-altered 4-hydroxynonenal analogs in the rat livers were assayed by GC-MS. ► Ketogenic diets accelerated the catabolic disposal of 4-hydroxynoenal analogs. ► High fat mix diets decreased the catabolic disposal of 4-hydroxynoenal analogs. ► 4-Hydroxynonenal analogs accumulated in rat livers from high fat mix diet groups.

Wed, 04 Jan 2012 09:01

Editorial Board

Publication year: 2012
Source: Free Radical Biology and Medicine, Volume 52, Issue 2, 15 January 2012, Pages IFC

[No author name available]

Tue, 03 Jan 2012 08:01

Announcements and Calendar

Publication year: 2012
Source: Free Radical Biology and Medicine, Volume 52, Issue 2, 15 January 2012, Pages 537

[No author name available]

Tue, 03 Jan 2012 08:01

Sub-cytotoxic mercury chloride inhibits gap junction intercellular communication by a redox- and phosphorylation-mediated mechanism

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 2 January 2012

Claudia Piccoli, Annamaria D'Aprile, Rosella Scrima, Luigi Ambrosi, Roberto Zefferino

Gap junctions play a central role in coordinating intercellular signal-transduction pathways to control tissue homeostasis. Deregulation of gap junctional intercellular communication is a common phenotype of cancer cells and supports its involvement in the carcinogenesis process. Many carcinogens, like environmental heavy-metal chemical pollutants, are known to activate various signal transduction mechanisms and modulate GJIC. They act as tumour promoters on pre-existing "initiated" cells, rather than as genotoxic initiators, albeit their mode of action is often unknown.In this study we investigated the effect of Hg(II) (HgCl2) on GJIC in cultured human keratinocytes. It is shown that sub-cytotoxic concentration of HgCl2as low as 10 nM causes inhibition of the GJIC, assessed by dye transfer assay, in spite of enhanced expression of connexins. In addition, HgCl2-treated keratinocytes exhibited decrease of free thiols and accumulation of mitochondria-derived reactive oxygen species albeit no effect on the respiratory chain activity was observed . Treatment of HgCl2-exposed keratinocytes with the PKC inhibitor calphostin C and with all-trans retinoic acid resulted in rescue of the mitochondrial ROS over-production and full recovery of the GJIC. Similar results were obtained with the PKA activator db-cAMP.Overall, the presented results support a cross-talk between the altered intracellular redox tone and PKA- and PKC-mediated signalling in HgCl2-challenged keratinocytes. These events, although not cytotoxic, lead to inhibition of GJIC and possibly to carcinogenic priming.

Highlights

► Hg(II) induces redox unbalance and inhibits GJIC at sub-cytotoxic concentrations. ► Hg(II)-mediated reactive oxygen species production derives from mitochondria ► Antioxidants prevent the Hg(II)-mediated inhibition of GJIC ► PKC-inhibitors or PKA-activators revert Hg(II)-mediated inhibition of GJIC and reactive oxygen species release

Mon, 02 Jan 2012 07:01

Overexpression of GRP94 in breast cancer cells resistant to oxidative stress promotes high levels of cancer cell proliferation and migration: Implications for tumor recurrence

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 2 January 2012

Nicolas Dejeans, Christophe Glorieux, Samuel Guenin, Raphael Beck, Brice Sid, ...

Targeting the altered redox status of cancer cells is emerging as an interesting approach to potentiate chemotherapy. However, to maximize the effectiveness of this strategy and define the correct chemotherapeutic associations, it is important to understand the biological consequences of chronically exposing cancer cells to reactive oxygen species (ROS). Using an H2O2generating system, we selected a ROS-resistant MCF-7 breast cancer cell line, namely Resox cells. By exploring different survival pathways that are usually induced during oxidative stress, we identified a constitutive overexpression of the endoplasmic reticulum chaperone, GRP94, in these cells, whereas levels of its cytoplasmic homolog HSP90, or GRP78, were not modified. This overexpression was not mediated by constitutive unfolded protein response (UPR) activation. The increase in GRP94 is tightly linked to an increase in cell proliferation and migration capacities, as shown by GRP94 silencing experiments. Interestingly, we also observed that GRP94 silencing inhibits migration and proliferation of the highly aggressive MDA-MB-231 cells. By immunohistochemistry, we showed that GRP94 expression was higher in recurrent human breast cancers than in their paired primary neoplasias. Similarly to the situation in the Resox cells, this increase was not associated with an increase in UPR activation in recurrent tumors. In conclusion, this study suggests that GRP94 overexpression may be a hallmark of aggressiveness and recurrence in breast cancers.

Highlights

► GRP94 promotes proliferation and migration of Reactive Oxidant Species (ROS)-resistant breast cancer cells.GRP94 favors cell proliferation under reducing conditions.GRP94 protein level is higher in recurrent breast cancers than in primary neoplasias.Our data point out a major role of GRP94 in cancer aggressiveness.

Graphical abstract

Mon, 02 Jan 2012 07:01

Free Radical Generation Induces Epithelial-to-Mesenchymal Transition in Lung Epitheliumviaa TGF-β1-dependent Mechanism

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 2 January 2012

Marta R. Gorowiec, Lee A. Borthwick, Sean M. Parker, John A. Kirby, Gabriele C. Saretzki, ...

Fibrotic remodelling of lung parenchymal and airway compartments is the major contributor to life-threatening organ dysfunction in chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and Chronic Obstructive Pulmonary Disease (COPD). Since transforming growth factor-β1 (TGF-β1) is believed to play a key role in disease pathogenesis and markers of oxidative stress are also commonly detected in bronchoalveolar lavage (BAL) from such patients we sought to investigate whether both factors might be interrelated.Here we investigated the hypothesis that oxidative stress to the lung epithelium promotes fibrotic repair by driving epithelial-to-mesenchymal transition (EMT)viathe augmentation of TGF-β1.We show that in response to 400 μM hydrogen peroxide (H2O2) A549 cells, used a model for alveolar epithelium, and human primary bronchial epithelial cells (PBECs) undergo EMT displaying morphology changes, decreased expression of epithelial markers (E-cadherin and ZO-1), increased expression of mesenchymal markers (vimentin and α-smooth muscle actin) as well as increased secretion of extracelluar matrix components. The same oxidative stress also promotes expression of TGF-β1. Inhibition of TGF-β1 signalling as well as treatment with antioxidants such as phenyl tert-butylnitrone (PBN) and superoxide dismutase 3 (SOD3) prevent the oxidative stress driven EMT-like changes described above. Interventions also inhibited EMT-like changes.This study identifies a link between oxidative stress, TGF-β1 and EMT in lung epithelium and highlights the potential for antioxidant therapies to limit EMT and its potential contribution to chronic lung disease.

Highlights

► Exposure of lung epithelium to H2O2results in epithelial-to-mesenchymal transition. ► H2O2-induced EMT changes in lung epithelial cells are mediated via TGF-β. ► Antioxidants such as PBN and SOD3 prevent formation of EMT in response to H2O2.

Mon, 02 Jan 2012 07:01

Skin mild hypoxia enhances killing of UVB-damaged keratinocytes through reactive oxygen species-mediated apoptosis requiring Noxa and Bim

Publication year: 2012
Source: Free Radical Biology and Medicine, Available online 2 January 2012

Kris Nys, Hannelore Maes, Graciela Andrei, Robert Snoeck, Maria Garmyn, ...

The naturally occurring skin hypoxia has emerged as a crucial host factor of the epidermal micro-environment. We wanted to systematically investigate how reduced oxygen availability of the epidermis modulates the response of keratinocytes and melanocytes to noxious ultraviolet B radiation (UVB).We report that the exposure of normal human keratinocytes (NHKs) or melanocytes (NHEMs) to mild hypoxia drastically impacts cell death responses following UVB irradiation. The hypoxic micro-environment favors survival and reduces apoptosis of UVB-irradiated NHEMs and their malignant counterparts (melanoma cells). In contrast, NHKs, but not the transformed keratinocytes, under hypoxic conditions display increased levels of reactive oxygen species (ROS) and are significantly sensitized to UVB-mediated apoptosis as compared to NHKs treated under normoxic conditions. Prolonged exposure of UVB-treated NHKs to hypoxia triggers a sustained and reactive oxygen species-dependent activation of the stress kinases p38and JNKs, which in turn, engage the activation of Noxa and Bim pro-apoptotic proteins. Combined silencing of Noxa and Bim significantly inhibits UVB-mediated apoptosis in hypoxic conditions, demonstrating that hypoxia results in an amplification of the intrinsic apoptotic pathway.Physiologically occurring skin hypoxia, by facilitating the specific removal of UVB-damaged keratinocytes, may represent a decisive host factor impeding important steps of the photocarcinogenesis process.

Highlights

► Mild hypoxia(1,5%O2) sensitizes NHKs to UVB-induced apoptosis while it protects NHEMs ► Mild hypoxia increases reactive oxygen species specifically in NHKs but not in NHEMs ► UVB-induced activation of p38/JNK is more sustained under mild hypoxia in NHKs ► JNK contributes to mitochondrial localization of Bim while Noxa is p38-dependent ► Mild hypoxia-mediated sensitization to UVB-induced apoptosis is lost in cancer cells.

Mon, 02 Jan 2012 07:01

15d-PGJ2 modulates manganese-induced activation of NF-kB, Nrf2 and PI3K pathways in astrocytes

Publication year: 2011
Source: Free Radical Biology and Medicine, Available online 29 December 2011

Eunsook Lee, Zhaobao Yin, Marta Sidoryk-Węgrzynowicz, Haiyan Jiang, Michael Aschner

Excessive exposure to manganese (Mn) increases levels of oxidative stressors and proinflammatory mediators, such as cycloxygenase-2 (COX-2) and prostaglandin E2 (PGE2). Mn also activates NF-κB, an important mediator of inflammation. The signaling molecule 15-deoxy-12,14-PGJ2(15 d-PGJ2) is an anti-inflammatory prostaglandin (PG). Here, we tested the hypothesis that 15 d-PGJ2modulates Mn-induced activation of astrocytic intracellular signaling, including nuclear factor-kappaB (NF-κB) and nuclear factor erythroid 2-related factor (Nrf2), a master regulator of antioxidant transcriptional responses. The results establish that 15 d-PGJ2suppresses Mn-induced NF-κB activation by interacting with several signaling pathways. The PI3K/Akt pathway, which is upstream of NF-κB plays a role in this activation, since (i) pretreatment with 15 d-PGJ2(10 μM for 1 h) significantly (p < 0.01) inhibited Mn (500 μM)-induced PI3K/Akt activation, and (ii) inhibition of the PI3K/Akt pathway with LY29004 significantly (p < 0.05) decreased NFκB activation. 15 d-PGJ2also significantly (p < 0.05) attenuated Mn-induced astrocytic NFκB activation by inhibiting the Mn-induced phosphorylation of IκB kinase (IKK) and subsequent IKB-α degradation. Because Mn-induced oxidative stress is also associated with Nrf2 activation, additional studies addressed the ability of 15 d-PGJ2to modulate the Nrf2 pathway. 15 d-PGJ2significantly (p < 0.01) increased Nrf2 expression in whole cell lysates. Consistent with its pro-oxidant properties, Mn also increased Nrf2 expression. Nevertheless, co-treatment of whole cell lysates with both Mn and 15 d-PGJ2partially suppressed (p < 0.01) the 15 d-PGJ2-induced increase in astrocytic Nrf2 protein expression. Mn treatment also decreased (p < 0.001) expression of DJ-1, a Parkinson's disease (PD)-associated protein and a stabilizer of Nrf2, and 15 d-PGJ2attenuated Mn-induced astrocytic inhibition of DJ-1 expression. Collectively, these results demonstrate that 15 d-PGJ2exerts a protective effect in astrocytes against Mn-induced inflammation and oxidative stress by modulating the activation of NFκB and Nrf2 signaling pathways.

Highlights

► 15 d-PGJ2protects astrocytes from Mn-induced inflammation by modulating NFκB. ► 15 d-PGJ2protects astrocytes from Mn-induced oxidative stress by modulating Nrf2. ► Mn decreases DJ-1 mRNA and protein expression. ► 15 d-PGJ2attenuates Mn-induced astrocytic inhibition of DJ-1 expression.

Sat, 31 Dec 2011 04:12

Trimer hydroxylated quinone (IIIHyQ) derived from apocynin targets cysteine residues of p47preventing the activation of human vascular NADPH oxidase

Publication year: 2011
Source: Free Radical Biology and Medicine, Available online 29 December 2011

Mauricio Mora-Pale, Seok Joon-Kwon, Robert J. Linhardt, Jonathan S. Dordick

Enzymatic derived oligophenols from apocynin can be effective inhibitors of human vascular NADPH oxidase. An isolated IIIHyQ has been shown to inhibit endothelial NADPH oxidase with an IC50~ 30 nM.In vitrostudies demonstrated that IIIHyQ is capable on disrupting the interaction between p47and p22, thereby blocking the activation of the Nox2 isoform. Herein, we report the role of key cysteine residues in p47as targets for the IIIHyQ. Incubation of p47with IIIHyQ results in a decrease of ~ 80% of the protein free cysteine residues; similar results were observed using 1,2- and 1,4-naphthoquinoes, while apocynin was unreactive. Mutants of p47, where each Cys was individually replaced by Ala (at residues 111, 196 and 378) and Gly (at residue 98), were generated to evaluate their individual importance in IIIHyQ-mediated inhibition of p47interaction with p22. Specific Michael addition on Cys196, within the N-SH3 domain, by the IIIHyQ is critical for disrupting the p47-p22interaction. When a C196A mutation was tested, the IIIHyQ was unable to disrupt the p47-p22interaction. However, the IIIHyQ was effective at disrupting this interaction with the other mutants, displaying IC50values (4.9, 21.0, and 2.3 μM for the C111A, C378A, and C98G mutants, respectively) comparable to that of wild type p47.

Highlights

► Inhibition of human vascular NADPH oxidase by a IIIHyQ derived from apocynin ► IIIHyQ blocks the p22-p47interaction preventing the activation of NADPH oxidase ► IIIHyQ targets cysteine residues of p47as a potential mechanism of inhibition ► Cys196 within the N-SH3 domain plays a critical role as a target of the IIIHyQ ► Toxicity of the IIIHyQ is low and comparable with the apocynin toxicity

Sat, 31 Dec 2011 04:12

Editorial Board

Publication year: 2012
Source: Free Radical Biology and Medicine, Volume 52, Issue 1, 1 January 2012, Pages IFC

[No author name available]

Wed, 28 Dec 2011 08:12

Reviewer Thank You

Publication year: 2012
Source: Free Radical Biology and Medicine, Volume 52, Issue 1, 1 January 2012, Pages v-ix

[No author name available]

Wed, 28 Dec 2011 08:12

Announcements and Calendar

Publication year: 2012
Source: Free Radical Biology and Medicine, Volume 52, Issue 1, 1 January 2012, Pages 237

[No author name available]

Wed, 28 Dec 2011 08:12

Effect of Antioxidant Gene Therapy on Retinal Neurons and Oxidative Stress in a Model of Retinal Ischemia/Reperfusion

Publication year: 2011
Source: Free Radical Biology and Medicine, Available online 24 December 2011

Yu Liu, Luosheng Tang, Baihua Chen

Retinal ischemia/reperfusion (I/R) results in neuronal death and generation of reactive oxygen species. The aim of this study is to investigate the neuroprotective effect of manganese superoxide dismutase (SOD2) on retinal ganglion cells (RGC) in I/R-induced retinal injury model. One eye of each Wistar rat was pretreated with recombinant adeno-associated virus (AAV) containing theSOD2gene (AAV-SOD2) or recombinant AAV containing GFP gene (AAV-GFP) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure for 1 hour, and reperfusion was established immediately afterward. The number of RGC and inner plexiform layer (IPL) thickness were measured by fluorogold retrograde labeling and hematoxylin and eosin staining at 6 h, 24 h, 72 h and 5 d after injury. Superoxide anion, the number of RGC, IPL thickness, malondialdehyde(MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG) , MnSOD (manganese superoxide dismutase) activity and nitrotyrosine were measured by fluorescence staining, immunohistochemistry and enzyme-linked immunosorbent analysis at 5 days after I/R injury. Severe RGC loss, reduced IPL thickness and MnSOD activity, increased superoxide ion, MDA, 8-OHdG, nitrotyrosine productions were observed after I/R injury. Administration of AAV-SOD2 significantly reduced levels of superoxide ion, MDA, 8-OHdG and nitrotyrosine, and prevented the damage of RGC and IPL. Delivery of the antioxidant gene inhibited I/R-induced RGC and IPL damage by reducing oxidative stress and nitrative stress, suggesting that MnSOD may be relevant for the neuroprotection of inner retina from I/R-related diseases.

Highlights

► The number of RGC and IPL thickness decreased significantly after retinal ischemia with time-dependent manner. ► Manganese superoxide dismutase attenuated the I/R-induced damage of RGC and IPL by enhancing the antioxidation ability and reducing the reactive oxygen, nitrogen species and DNA damage. ► Manganese superoxide dismutase may be relevant for the neuroprotection of ischemic retinal diseases.

Sat, 24 Dec 2011 04:12

4-Ketopinoresinol, a Novel Naturally-occurring ARE Activator, Induces the Nrf2/HO-1 Axis and Protects against Oxidative Stress-induced Cell Injury via Activation of PI3K/AKT Signaling

Publication year: 2011
Source: Free Radical Biology and Medicine, Available online 24 December 2011

Huang-Hui Chen, Yu-Tsen Chen, Yen-Wen Huang, Hui-Ju Tsai, Ching-Chuan Kuo

The Nrf2/ARE pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins, and has been considered as a potential target for cancer chemoprevention by eliminating harmful reactive oxygen species (ROS) or reactive intermediates generated from carcinogens. The objectives of this study were to identify novel Nrf2/ARE activators and to investigate the mechanistic signaling pathway involved in the activation of Nrf2-mediated cytoprotective effects against oxidative-induced cell injury. A stable ARE-driven luciferase reporter cell line was established to screen the potentially cytoprotective compound. 4-Ketopinoresinol (4-KPR), the (α-γ) double cyclized type of lignan obtained from adlay (Coix lachrymal-jobiL. var.ma-yuenStapf), more effectively activates ARE-driven luciferase activity than the classical ARE activator,tert-butylhydroquinone. 4-KPR treatment resulted in a transient increase in AKT phosphorylation and subsequent phosphorylation and nuclear translocation of Nrf2, along with increased expression of ARE-dependent cytoprotective genes, such as heme oxygenase-1 (HO-1), aldo-keto reductases, and glutathione synthetic enzyme. 4-KPR suppresses oxidative stress-induced DNA damage and cell death via up-regulation of HO-1. Inhibition of PI3K/AKT signal by chemical inhibitors or RNA interference not only suppressed 4-KPR-induced Nrf2/HO-1 activation, but also eliminated cytoprotective effect against oxidative damage. These observations in an ARE-regulated gene system suggest that 4-KPR is a novel Nrf2/ARE-mediated transcription activator, activates the Nrf2/HO-1 axis, and protects against oxidative stress-induced cell injury via activation of PI3K/AKT signaling.

Highlights

► 4-Ketopinoresinol (4-KPR), the (α-γ) double cyclized type of lignan, is a novel Nrf2/ARE-mediated transcription activator. ► 4-KPR suppresses oxidative stress-induced DNA damage and cell death via Nrf2/HO-1 axis. ► 4-KPR induces Nrf2/HO-1–mediated cytoprotective effect against oxidative damage via PI3K/AKT signaling.

Sat, 24 Dec 2011 04:12

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