ATP inhibits hydroxyl radical formation and the inflammatory response of stimulated whole blood even under circumstances of severe oxidative stress

We recently reported that Adenosine-5'-triphosphate (ATP) is able to inhibit the inflammatory reaction in stimulated whole blood. Many diseases, in which inflammatory reactions are involved, are associated with oxidative stress. In the present study, we therefore, investigated the effect of ATP on cytokine release in stimulated whole blood under conditions of oxidative stress, as simulated by pre-incubation of blood with hydrogen peroxide (H(2)O(2)). In the presence of H(2)O(2), ATP at concentrations of 100 and 300 microM inhibited Tumour Necrosis factor-alpha (TNF-alpha) release and stimulated IL-10 release in LPS-PHA stimulated whole blood. Moreover, electron spin resonance (ESR) measurements showed that ATP and its breakdown product Adenosine-5'-diphosphate (ADP) attenuated spin trap-hydroxyl radical adduct formation in the Fenton reaction. Our results demonstrate that even in circumstances of severe oxidative stress, ATP has marked anti-inflammatory properties in stimulated whole blood. Moreover, the inhibition of the hydroxyl radical ESR signal indicates a direct attenuation of oxidative stress by ATP.     

Free radical production in roots of Phaseolus vulgaris subjected to phosphate deficiency stress

We subjected bean plants (Phaseolus vulgaris L. cv. ‘Zlota Saxa’) to phosphate deficiency stress and studied free radical production in whole root extracts. Starting from the 12th day of growth a carbon-centred free radical was detected, by means of electron spin resonance (ESR) after spin trapping with 5,5-dimethyl-L-pyrroline-N-oxide (DMPO), only in phosphate-deficient plants. The simulated hyperfine coupling constants of this spectrum (a_N = 16.2 G; a_H = 23.6 G) are consistent with an aliphatic or an aromatic carbon-centred radical; this species could derive from lipid peroxidation or phenol oxidation processes, respectively. Hydrogen peroxide production was also enhanced. Production of both H₂O₂ and DMPO adduct were related to the length of growth on the phosphate-deficient medium. Roots from phosphate-deficient plants showed increased content of phenols and a redox state of ascorbate similar to the control. These results indicate that phosphate starvation imposes a mild oxidative stress.     

Oxidative stress augments toll-like receptor 8 mediated neutrophilic responses in healthy subjects

Background

Excessive oxidative stress has been reported to be generated in inflamed tissues and contribute to the pathogenesis of inflammatory lung diseases, exacerbations of which induced by viral infections are associated with toll-like receptor (TLR) activation. Among these receptors, TLR8 has been reported as a key receptor that recognizes single-strand RNA virus. However, it remains unknown whether TLR8 signaling is potentiated by oxidative stress. The aim of this study is to examine whether oxidative stress modulates TLR8 signaling in vitro.

Methods

Human peripheral blood neutrophils were obtained from healthy non-smokers and stimulated with TLR 7/8 agonist imidazoquinoline resiquimod (R848) in the presence or absence of hydrogen peroxide (H₂O₂). Neutrophilic responses including cytokine release, superoxide production and chemotaxis were examined, and the signal transduction was also analyzed.

Results

Activation of TLR8, but not TLR7, augmented IL-8 release. The R848-augmented IL-8 release was significantly potentiated by pretreatment with H₂O₂ (p < 0.01), and N-acetyl-L-cysteine reversed this potentiation. The combination of H₂O₂ and R848 significantly potentiated NF-kB phosphorylation and IkBα degradation. The H₂O₂-potentiated IL-8 release was suppressed by MG-132, a proteosome inhibitor, and by dexamethasone. The expressions of TLR8, myeloid differentiation primary response gene 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were not affected by H₂O₂.

Conclusion

TLR8-mediated neutrophilic responses were markedly potentiated by oxidative stress, and the potentiation was mediated by enhanced NF-kB activation. These results suggest that oxidative stress might potentiate the neutrophilic inflammation during viral infection.     

Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): Nonperoxidative purine and pyrimidine nucleotide depletion

Hydrogen peroxide (H₂O₂) overload may contribute to cardiac ischemia-reperfusion injury. We report utilization of a previously described cardiomyocyte model (J. Cell. Physiol., 149:347, 1991) to assess the effect of H₂O₂-induced oxidative stress on heart-muscle purine and pyrimidine nucleotides and high-energy phosphates (ATP, phosphocreatine). Oxidative stress induced by bolus H₂O₂ elicited the loss of cardiomyocyte purine and pyrimidine nucleotides, leading to eventual de-energization upon total ATP and phosphocreatine depletion. The rate and extent of ATP and phosphocreatine loss were dependent on the degree of oxidative stress within the range of 50 μM to 1.0 mM H₂O₂. At the highest H₂O₂ concentration, 5 min was sufficient to elicit appreciable cardiomyocyte highenergy phosphate loss, the extent of which could be limited by prompt elimination of H₂O₂ from the culture medium. Only H₂O₂ dismutation completely prevented ATP loss during H₂O₂-induced oxidative stress, whereas various freeradical scavengers and metal chelators afforded no significant ATP preservation. Exogenously-supplied catabolic substrates and glycolytic or tricarboxylic acidcycle intermediates did not ameliorate the observed ATP and phosphocreatine depletion, suggesting that cardiomyocyte de-energization during H₂O₂-induced oxidative stress reflected defects in substrate utilization/energy conservation. Compromise of cardiomyocyte nucleotide and phosphocreatine pools during H₂O₂-induced oxidative stress was completely dissociated from membrane peroxidative damage and maintenance of cell integrity. Cardiomyocyte de-energization in response to H₂O₂ overload may constitute a distinct nonperoxidative mode of injury by which cardiomyocyte energy balance could be chronically compromised in the post-ischemic heart. © 1993 Wiley-Liss, Inc.     

Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): Lethal peroxidative membrane injury

Oxidative stress induced by hydrogen peroxide (H₂O₂) may contribute to the pathogenesis of ischemic-reperfusion injury in the heart. For the purpose of investigating directly the injury potential of H₂O₂ on heart muscle, a cellular model of H₂O₂-induced myocardial oxidative stress was developed. This model employed primary monolayer cultures of intact, beating neonatal-rat cardiomy-ocytes and discrete concentrations of reagent H₂O₂ in defined, supplement-free culture medium. Cardiomyocytes challenged with H₂O₂ readily metabolized it such that the culture content of H₂O₂ diminished over time, but was not depleted. The consequent H₂O₂-induced oxidative stress caused lethal sarcolemmal disruption (as measured by lactate dehydrogenase release), and cardiomyocyte integrity could be preserved by catalase. During oxidative stress, a spectrum of cellular derangements developed, including membrane phospholipid peroxidation, thiol oxidation, consumption of the major chain-breaking membrane antiperoxidant (α-tocopherol), and ATP loss. No net change in the protein or phospholipid contents of cardiomyocyte membranes accompanied H₂O₂-induced oxidative stress, but an increased turnover of these membrane constituents occurred in response to H₂O₂. Development of lethal cardiomyocyte injury during H₂O₂-induced oxidative stress did not require the presence of H₂O₂ itself; a brief “pulse” exposure of the cardiomyocytes to H₂O₂ was sufficient to incite the pathogenic mechanism leading to cell disruption. Cardiomyocyte disruption was dependent upon an intracellular source of redox-active iron and the iron-dependent transformation of internalized H₂O₂ into products (e.g., the hydroxyl radical) capable of initiating lipid peroxidation, since iron chelators and hydroxyl-radical scavengers were cytoprotective. The accelerated turnover of cardiomyocyte-membrane protein and phospholipid was inhibited by antiperoxidants, suggesting that the turnover reflected molecular repair of oxidized membrane constituents. Likewise, the consumption of α-tocopherol and the oxidation of cellular thiols appeared to be epiphenomena of peroxidation. Antiperoxidant interventions coordinately abolished both H₂O₂-induced lipid peroxidation and sarcolemmal disruption, demonstrating that an intimate pathogenic relationship exists between sarcolemmal peroxidation and lethal compromise of cardiomyocyte integrity in response to H₂O₂-induced oxidative stress. Although sarcolemmal peroxidation was causally related to cardiomyocyte disruption during H₂O₂-induced oxidative stress, a nonperoxidative route of H₂O₂ cytotoxicity was also identified, which was expressed in the complete absence of cardiomyocyte-membrane peroxidation. The latter mode of H₂O₂-induced cardiomyocyte injury involved ATP loss such that membrane peroxidation and cardiomyocyte disruption on the one hand and cellular de-energization on the other could be completely dissociated. The cellular pathophysiology of H₂O₂ as a vectorial signal for cardiomyocyte necrosis that “triggers” irreversible peroxidative disruption of the sarcolemma has implications regarding potential mechanisms of oxidative injury in the postischemic heart.     

Modulatory effects of low-dose hydrogen peroxide on the function of human plasmacytoid dendritic cells

Under normal conditions, plasmacytoid dendritic cells (pDCs) are located in peripheral lymphoid organs or circulate in the blood, from where they can migrate to sites of infection or inflammation. In inflamed tissues, pDCs can be exposed to elevated levels of reactive oxygen species produced by inflammatory cells and we presume that oxidative stress could affect the cellular responses of pDCs to microenvironmental stimuli. To explore this possibility, human pDCs isolated from peripheral blood of healthy donors were treated with H₂O₂ and R837 (a Toll-like receptor 7 ligand), separately and in combination. Our results demonstrate that treatment with a low concentration (0.01 μM) of H₂O₂ resulted in only slight changes in the expression of CD40, CD80, CD86, and CD83; however, low-dose H₂O₂ markedly decreased the expression of HLA-DQ on pDCs. Exposure to H₂O₂ did not trigger the release of IL-6, TNF-α, IL-8, or IFN-α from pDCs. Although addition of H₂O₂ did not modify the capacity of pDCs to activate allogeneic IL-17- or IFN-γ-producing T cells, it significantly increased the ability of pDCs to stimulate IL-4-secreting T cells. Exposure of pDCs to H₂O₂ before cocultivation with naïve autologous T cells significantly lowered IL-10 production by T cells, but did not affect IL-17 release. It was also observed that H₂O₂-exposed pDCs provided stronger stimuli for Th2 than for Th1 differentiation upon autologous activation, compared to untreated pDCs, possibly because of elevated surface expression of OX40-L. Most importantly, when pDCs were stimulated with R837 in the presence of H₂O₂, decreased phenotypic activation, decreased chemokine and cytokine release, and impaired allo- and autostimulatory functions of pDCs were detected, indicating that pDCs exposed to oxidative stress in vivo may have an anti-inflammatory or tolerogenic role in regulating adaptive immune responses.     

Inhibition of phagocytic activity of ARPE-19 cells by free radical mediated oxidative stress

Oxidative stress is a main factor responsible for key changes leading to the onset of age-related macular degeneration (ARMD) that occur in the retinal pigment epithelium (RPE), which is involved in phagocytosis of photoreceptor outer segments (POS). In this study, hydrogen peroxide (H₂O₂), H₂O₂ and iron ions (Fe) or rose Bengal (RB) in the presence of NADH and Fe were used to model free radical mediated oxidative stress to test if free radicals and singlet oxygen have different efficiency to inhibit phagocytosis of ARPE-19 cells. Free radical mediated oxidative stress was confirmed by HPLC-EC(Hg) measurements of cholesterol hydroperoxides in treated cells. Electron paramagnetic resonance (EPR) spin trapping was employed to detect superoxide anion. Cell survival was analyzed by the MTT assay. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS and non-specific phagocytosis of fluorescent beads were measured by flow cytometry. HPLC analysis of cells photosensitized with RB in the presence of NADH and Fe indicated substantial increase in formation of free radical-dependent 7α/7β-hydroperoxides. EPR spin trapping confirmed the photogeneration of superoxide anion in samples enriched with RB, NADH and Fe. For all three protocols sub-lethal oxidative stress induced significant inhibition of the specific phagocytosis of POS. In contrast, non-specific phagocytosis was inhibited only by H₂O₂ or H₂O₂ and Fe treatment. Inhibition of phagocytosis was transient and recoverable by 24?h. These results suggest that free radicals may exert similar to singlet oxygen efficiency in inhibiting phagocytosis of RPE cells, and that the effect depends on the location where initial reactive species are formed.     

Sonic hedgehog promotes neurite outgrowth of cortical neurons under oxidative stress: Involving of mitochondria and energy metabolism

Oxidative stress has been demonstrated to be involved in the etiology of several neurobiological disorders. Sonic hedgehog (Shh), a secreted glycoprotein factor, has been implicated in promoting several aspects of brain remodeling process. Mitochondria may play an important role in controlling fundamental processes in neuroplasticity. However, little evidence is available about the effect and the potential mechanism of Shh on neurite outgrowth in primary cortical neurons under oxidative stress. Here, we revealed that Shh treatment significantly increased the viability of cortical neurons in a dose-dependent manner, which was damaged by hydrogen peroxide (H₂O₂). Shh alleviated the apoptosis rate of H₂O₂-induced neurons. Shh also increased neuritogenesis injuried by H₂O₂ in primary cortical neurons. Moreover, Shh reduced the generation of reactive oxygen species (ROS), increased the activities of SOD and and decreased the productions of MDA. In addition, Shh protected mitochondrial functions, elevated the cellular ATP levels and amelioratesd the impairment of mitochondrial complex II activities of cortical neurons induced by H₂O₂. In conclusion, all these results suggest that Shh acts as a prosurvival factor playing an essential role to neurite outgrowth of cortical neuron under H₂O₂ -induced oxidative stress, possibly through counteracting ROS release and preventing mitochondrial dysfunction and ATP as well as mitochondrial complex II activities against oxidative stress.     

Heat-shock response protects peripheral blood mononuclear cells (PBMCs) from hydrogen peroxide-induced mitochondrial disturbance

The present study was designed to investigate ex vivo the protective mechanisms of heat-shock response against H₂O₂-induced oxidative stress in peripheral blood mononuclear cells (PBMCs) of rats. Twenty-four hours later, heat-shock treatment was executed in vivo; rat PBMCs were collected and treated with H₂O₂. The accumulation of reactive oxygen species and the mitochondrial membrane potential were evaluated by intracellular fluorescent dHE and JC-1 dye staining, respectively, and expression of HSP72 and cytochrome c was detected by Western blot analysis. Cellular apoptosis was assayed by TUNEL staining and double staining of Annexin V and PI. The results showed that H₂O₂-induced oxidative stress leads to intracellular superoxide accumulation and collapse of the mitochondrial membrane potential in rat PBMCs. Moreover, cellular apoptosis was detected after H₂O₂ treatment, and the release of mitochondrial cytochrome c from mitochondria to cytosol was significantly enhanced. Heat-shock pretreatment decreases the accumulation of intracellular superoxide in PBMCs during H₂O₂-induced oxidative stress. Moreover, heat-shock treatment prevents the collapse of the mitochondrial membrane potential and cytochrome c release from mitochondria during H₂O₂-induced oxidative stress. In conclusion, mitochondria are critical organelles of the protective effects of heat-shock treatment. Cellular apoptosis during H₂O₂-induced oxidative stress is decreased by heat-shock treatment through a decrease in superoxide induction and preservation of the mitochondrial membrane potential.     

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress-induced mitochondrial dysfunction

Exogenous oxidative stress induces cell death, but the upstream molecular mechanisms involved of the process remain relatively unknown. We determined the instant dynamic reactions of intracellular reactive oxygen species (ROS, including hydrogen peroxide (H₂O₂), superoxide radical (O₂⁻), and nitric oxide (NO)) in cells exposed to exogenous oxidative stress by using a confocal laser scanning microscope. Stimulation with extracellular H₂O₂ significantly increased the production of intracellular H₂O₂, O₂⁻, and NO (P < 0.01) through certain mechanisms. Increased levels of intracellular ROS resulted in mitochondrial dysfunction, involving the impairment of mitochondrial activity and the depolarization of mitochondrial membrane potential. Mitochondrial dysfunction significantly inhibited the proliferation of human hepatoblastoma G2 (HepG2) cells and resulted in mitochondrial cytochrome c (cyt c) release. The results indicate that upstream ROS signals play a potential role in exogenous oxidative stress-induced cell death through mitochondrial dysfunction and cyt c release.     

Effects of Free Radicals on the Fluidity of Myocardial Membranes

Free radicals, including superoxide anions (O₂^?_?), hydroxyl radical (HO'), and hypohalite radical (OCl'), as well as oxidants such as hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl), have been indicated in the pathogenesis of myocardial ischemic and reperfusion injury. In this report, we compared the integrity of the myocardial membrane when exposed to these free radicals/oxidants. Isolated rat heart membrane preparations were exposed to chemically generated free radicals with or without their respective scavengers. Membrane fluidity was monitored by fluorescence polarization using the diphenylhexatriene probe, as well as by electron spin resonance (ESR) spectroscopy using 2,2,6,6-tetramethyl piperidine-n-oxyl as the spin labeling agent. HO', H₂O₂, and OCl' + HOCl increased the fluorescence polarization (FP) and microvis-cosity significantly by 1.7-fold, 1.8-fold, and 1.7-fold, respectively, as compared to an only 1.2– fold increase in FP by O₂^?_? O₂^?_? did not alter the fatty acid profiles of the membrane phospholipids. However, HO' and H₂O₂ reduced the arachidonic acid contents in phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). These radicals also stimulated the lipid peroxidation by several-fold, while that by O₂^?_? was only insignificant. These results suggest that HO' and H₂O₂ decreased the membrane fluidity and induced lipid peroxidation by releasing the arachidonic acid from PC, PE. and PI.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Abstract

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H₂O₂) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H₂O₂-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H₂O₂-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H₂O₂, and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca^{2 +}) chelator, overexpression of Homer 1a had no significant effects on H₂O₂-induced oxidative stress. These results suggest that Homer 1a has protective effects against H₂O₂-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca^{2 +} homeostasis.     

Different behavior of agmatine in liver mitochondria: Inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 μM or 100 μM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H₂O₂ generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H₂O₂ and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca²⁺. Instead, 1 mM agmatine generates larger amounts of H₂O₂ than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca²⁺-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Adrenaline stimulates H2O2 generation in liver via NADPH oxidase

It is known that adrenaline promotes hydroxyl radical generation in isolated rat hepatocytes. The aim of this work was to investigate a potential role of NADPH oxidase (Nox) isoforms for an oxidative stress signal in response to adrenaline in hepatocytes. Enriched plasma membranes from isolated rat liver cells were prepared for this purpose. These membranes showed catalytic activity of Nox isoforms, probably Nox 2 based on its complete inhibition with specific antibodies. NADPH was oxidized to convert O₂ into superoxide radical, later transformed into H₂O₂. This enzymatic activity requires previous activation with either 3 mM Mn²⁺ or guanosine 5′-0-(3-thiotriphosphate) (GTPγS) plus adrenaline. Experimental conditions for activation and catalytic steps were set up: ATP was not required; S_0.5 for NADPH was 44 μM; S_0.5 for FAD was 8 μM; NADH up to 1 mM was not substrate, and diphenyleneiodonium was inhibitory. Activation with GTPγS plus adrenaline was dose- and Ca²⁺-dependent and proceeded through α₁-adrenergic receptors (AR), whereas β-AR stimulation resulted in inhibition of Nox activity. These results lead us to propose H₂O₂ as additional transduction signal for adrenaline response in hepatic cells.     

TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout

Acute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H₂O₂), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1^+/+) or TRPA1-deficient (Trpa1^−/−) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H₂O₂ generation, interleukin-1β release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H₂O₂ in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H₂O₂-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H₂O₂ mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H₂O₂ production as potential targets for treatment of acute gout attacks.     

Hypothermia protects H9c2 cardiomyocytes from H2O2 induced apoptosis

The purpose of our study was to investigate underlying basic mechanisms of hypothermia-induced cardioprotection during oxidative stress in a cardiomyocyte cell culture model. For hypothermic treatment we cooled H9c2 cardiomyocytes to 20 °C, maintained 20 min at 20 °C during which short-term oxidative damage was inflicted with 2 mM H₂O_2, followed by rewarming to 37 °C. Later on, we analyzed lactate dehydrogenase (LDH), caspase-3 cleavage, reactive oxygen species (ROS), mitochondrial activity, intracellular ATP production, cytoprotective signal molecules as well as DNA damage. Hypothermia decreased H₂O₂ damage in cardiomyocytes as demonstrated in a lower LDH release, less caspase-3 cleavage and less M30 CytoDeath staining. After rewarming H₂O₂ damaged cells demonstrated a significantly higher reduction rate of intracellular ROS compared to normothermic H₂O₂ damaged cardiomyocytes_. This was in line with a significantly greater mitochondrial dehydrogenase activity and higher intracellular ATP content in cooled and rewarmed cells. Moreover, hypothermia preserved cell viability by up-regulation of the anti-apoptotic protein Bcl-2 and a reduction of p53 phosphorylation. DNA damage, proven by PARP-1 cleavage and H2AX phosphorylation, was significantly reduced by hypothermia. In conclusion, we could demonstrate that hypothermia protects cardiomyocytes during oxidative stress by preventing apoptosis via inhibiting mitochondrial dysfunction and DNA damage.     

The Horseradish Peroxidase Catalysed Oxidation of Deoxyribose Sugars

The ability of horseradish peroxidase (E.C. 1.11.1.7. Donor: H₂O₂ oxidoreductase) to catalytically oxidize 2-deoxyribose sugars to a free radical species was investigated. The ESR spin-trapping technique was used to denionstrate that free radical species were formed. Results with the spin trap 3.5-dibronio-4-nitrosoben-zene sulphonic acid showed that horseradish peroxidase can catalyse the oxidation of 2-deoxyribose to produce an ESR spectrum characteristic of a nitroxide radical spectrum. This spectrum was shown to be a composite of spin adducts resulting from two carbon-centered species, one spin adduct being characterized by the hyperfine coupling constants a^N = 13.6Ganda^H_β = 11.0G, and the other by a^N = 13.4G and a^H = 5.8 G. When 2-deoxyribose-5-phosphate was used as the substrate, the spectrum produced was found to be primarily one species characterized by the hyperfine coupling constants a^N = 13.4G and a^H= 5.2. All the radical species produced were carbon-centered spin adducts with a β hydrogen, suggesting that oxidation occurred at the C(2) or C(5) moiety of the sugar. Interestingly, it was found that under the same experimental conditions, horseradish peroxidase apparently did not catalyze the oxidation of either 3-deoxyribose or D-ribose to a free radical since no spin adducts were found in these cases.

It can be readily seen that 2-deoxyribose and 2-deoxyribose-5-phosphate can be oxidized by HRP/H₂O₂ to form a free radical species that can be detected with the ESR spin-trapping technique. There are two probable sites for the formation of a CH type radical on the 2-deoxyribose sugar, these being the C(2) and the C(5) carbons. The fact that there is a species produced from 2-deoxy-ribose, but not 2-deoxy-ribose-5-phosphate, suggests that there is an involvement of the C(5) carbon in the species with the 1 1.0G β hydrogen. In the spectra formed from 2-deoxy-ribose, there is a big difference in the hyperfine splitting of the β hydrogens, suggesting that the radicals are formed at different carbon centers, while the addition of a phosphate group to the C(5) carbon seems to inhibit radical formation at one site. In related work, the chemiluminescence of monosaccharides in the presence of horseradish peroxidase was proposed to be the consequence of carbon-centered free radical formation (10).     

Redox treatment ameliorates diabetes mellitus-induced skin flap necrosis via inhibiting apoptosis and promoting neoangiogenesis

Intractable wound healing is the habitual problem of diabetes mellitus. High blood glucose limits wound healing by interrupting inflammatory responses and inhibiting neoangiogenesis. Oxidative stress is commonly thought to be a major pathogenic cause of diabetic complications. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, EDV) is a free radical scavenger which suppress oxidative stress. This study investigates whether EDV can reduce oxidative stress in wound healing HaCaT/human dermal fibroblasts cells (HDFs) in vitro and in vivo animal model. Cell viability and wound healing assays, FACS flow cytometry, and Hoechst 33342 staining were performed to confirm apoptosis and cytotoxicity in H₂O₂ and EDV-treated HaCaT and HDFs. A streptozotocin-induced hyperglycemic animal model was made in adult C57BL6 mice. Full-thickness skin flap was made on dorsomedial back and re-sutured to evaluate the wound healing process. EDV was delivered slowly in the skin flap with degradable fibrin glue. The flap was monitored and analyzed on postoperative days 1, 3, and 5. CD31/DAPI staining was done to detect newly formed blood vessels. The expression levels of NF-κB, bcl-2, NOX3, and STAT3 proteins in C57BL6 mouse tissues were also examined. The wound healing process in hyper- and normoglycemic mice showed a difference in protein expression, especially in oxidative stress management and angiogenesis. Exogenous H₂O₂ reduced cell viability in a proportion to the concentration via apoptosis. EDV protected HaCaT cells and HDFs from H₂O₂ induced reactive oxygen species cell damage and apoptosis. In the mouse model, EDV with fibrin resulted in less necrotic areas and increased angiogenesis on postoperative day 5, compared to sham-treated mice. Our results indicate that EDV could protect H₂O₂-induced cellular injury via inhibiting early apoptosis and inflammation and also increasing angiogenesis. EDV might be valuable in the treatment of diabetic wounds that oxidative stress has been implicated.     

Aminoacetone Induces Loss of Ferritin Ferroxidase and Iron Uptake Activities

The effects of inorganic phosphate (P_i), the main intracellular membrane permeable anion capable of altering mitochondrial pH gradients (ΔpH), were measured on mitochondrial H₂O₂ release. As expected, P_i decreased ΔpH and increased the electric membrane potential (ΔΨ). Mitochondrial H₂O₂ release was stimulated by P_i and also by its structural analogue arsenate. However, acetate, another membrane-permeable anion, did not stimulate mitochondrial H₂O₂ release. The stimulatory effect promoted by P_i was prevented by CCCP, which decreases transport of P_i across the inner mitochondrial membrane, indicating that P_i must be in the mitochondrial matrix to stimulate H₂O₂ release. In conclusion, we found that P_i and arsenate stimulate mitochondrial reactive oxygen release, an effect that may contribute towards oxidative stress under conditions such as ischemia/reperfusion, in which high-energy phosphate bonds are hydrolyzed.