Role Of Superoxide Research Articles

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets.

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions byhuman platelets,understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually,identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

Abstract 363: Intracellular redox milieu regulates Mcl-1 and decreases overall mitochondrial priming in hematopoietic cancers

Abstract Background: The dichotomy of redox signaling in cancer cell fate decisions has emerged as a topic of importance over the years. In particular, our group has highlighted the role of superoxide (O2 .-)-mediated signaling in the acquisition of drug resistance and cancer survival. Mechanistically, this involves post-translational modification of proteins involved in oncogenic signaling, such as c-Myc and NF-κB, as well as apoptosis resistance like Bcl-2 at the serine 70 phosphorylation site. Here we present evidence that O2 .--mediated signaling stabilizes anti-apoptotic protein Mcl-1, a commonly amplified gene in human cancers and is implicated in resistance to venetoclax, the Bcl-2 specific BH3 mimetic. Methods: Intracellular O2 .- levels was increased either pharmacologically or by siRNA mediated knockdown of SOD1 in MOLM14, RPMI8226, OCI-AML3 hematopoietic cell lines. Cell viability and apoptosis were assessed by MTT and Annexin V/DAPI staining. Protein expression was determined by Western blot analysis. BH3 profiling of patient-derived CLL cells was used to assess mitochondrial priming. Results: Elevated O2 .- levels increased Mcl-1 stability through Thr163 phosphorylation of Mcl-1, which in turn reduced ubiquitination-dependent degradation. Furthemore, venetoclax resistant cells possessed higher O2 .- levels and increase in intracellular O2 .- levels promoted resistance to Mcl-1 inhibitor, S63845 through increased binding between Mcl-1 and pro-apoptotic proteins NOXA and Bak. Contrarily, scavenging of O2 .- re-sensitized venetoclax-resistant leukemia cells to venetoclax. Importantly, BH3 profiling revealed that increase in intracellular O2 .- levels decreased overall mitochondrial priming in primary CLL cells by promoting sequestration of pro-apoptotic proteins that specifically target Mcl-1. Conclusion: Oncogenic activity of O2 .- is mediated by increased Mcl-1 stability through Thr163 phosphorylation, which resulted in a reduction in overall mitochondrial priming and increased binding of Mcl-1 to NOXA and Bak. Scavenging O2 .- reduces Mcl-1 levels and sensitizes resistant cells to venetoclax-induced apoptosis thereby demonstrating that manipulation of the redox microenvironment could be a potential vulnerability to be exploited in venetoclax resistant cancers Citation Format: Jolin Xiao Lai, Stephen Jun Chong, Matthew Davids, Shazib Pervaiz. Intracellular redox milieu regulates Mcl-1 and decreases overall mitochondrial priming in hematopoietic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 363.

Perivascular adipose tissue modulates the effects of flavonoids on rat aorta rings: Role of superoxide anion and β3 receptors

Several studies demonstrate the beneficial effects of dietary flavonoids on the cardiovascular system. Since perivascular adipose tissue (PVAT) plays an active role in the regulation of vascular tone in both health and diseases, the present study aimed to assess the functional interaction between PVAT and flavonoids in vitro on rat aorta rings. Several flavonoids proved to display both antispasmodic and spasmolytic activities towards noradrenaline-induced contraction of rings deprived of PVAT (-PVAT). However, on PVAT-intact (+PVAT) rings, both actions of some flavonoids were lost and/or much decreased. In rings-PVAT, the superoxide donor pyrogallol mimicked the effect of PVAT, while in rings+PVAT the antioxidant mito-tempol restored both activities of the two most representative flavonoids, namely apigenin and chrysin. The Rho-kinase inhibitor fasudil, or apigenin and chrysin concentration-dependently relaxed the vessel active tone induced by the Rho-kinase activator NaF; the presence of PVAT counteracted apigenin spasmolytic activity, though only in the absence of mito-tempol. Similar results were obtained in rings pre-contracted by phenylephrine. Finally, when β3 receptors were blocked by SR59230A, vasorelaxation caused by both flavonoids was unaffected by PVAT. These data are consistent with the hypothesis that both noradrenaline and apigenin activated adipocyte β3 receptors with the ensuing release of mitochondrial superoxide anion, which once diffused toward myocytes, counteracted flavonoid vasorelaxant activity. This phenomenon might limit the beneficial health effects of dietary flavonoids in patients affected by either obesity and/or other pathological conditions characterized by sympathetic nerve overactivity.

One-step preparation of Ag-incorporated BiVO4 thin films: plasmon-heterostructure effect in photocatalytic activity enhancement

Ag-incorporated BiVO4 thin film with high photocatalytic activity is a great candidate for industrial wastewater treatment, enhancing the photoactivity of BiVO4, as well as solving the recyclability and reusability drawback of powder photocatalysts. Therefore, a novel one-step process of the reactive magnetron sputtering technique was employed, which to this day was never utilized to synthesis Ag-incorporated BiVO4 thin films. The effect of Ag incorporation on the photocatalytic properties was comparatively investigated. The photoactive phases e.g. monoclinic BiVO4, Ag4V2O7, α-AgVO3 were detected by X-ray diffraction (XRD). Various morphologies, depending on the Ag content, were observed by field emission electron microscopy (FESEM) along with Ag nanoparticles on the film surface. X-ray photoelectron spectroscopy (XPS) proved the existence of Bi and V exclusively in their Bi+3 and V+5 oxidation states, while Ag was present in Ag+ and Ag0, which are correspondent with silver vanadates and Ag nanoparticles, respectively. The thin film with 24 at. % Ag exhibited superior photocatalytic performance with a 99 % photodegradation of Rhodamine-B at neutral pH under visible light, as a result of having the lowest bandgap (1.96 eV), proper distribution of Ag4V2O7 phase, the plasmonic effect of Ag nanoparticles on the film surface, and the lower recombination rate compared to the pristine sample demonstrated by photoluminescence (PL) spectra. The recyclability experiment ensured the high stability of the thin films after three consecutive cycles, and the role of superoxides (•O2−) and photogenerated holes were found to be crucial using scavengers. The results confirmed the synergistic enhancement of the photocatalytic activity through Ag-incorporated BiVO4 heterostructure and the plasmonic effect, as well as solving the low photoactivity of BiVO4 at neutral pH.

Macrophage 12(S)-HETE Enhances Angiotensin II-Induced Contraction by a BLT2 (Leukotriene B4 Type-2 Receptor) and TP (Thromboxane Receptor)-Mediated Mechanism in Murine Arteries.

12/15-LO (12/15-lipoxygenase), encoded by Alox15 gene, metabolizes arachidonic acid to 12(S)-HETE (12-hydroxyeicosatetraenoic acid). Macrophages are the major source of 12/15-LO among immune cells, and 12/15-LO plays a crucial role in development of hypertension. Global Alox15- or macrophage-deficient mice are resistant to Ang II (angiotensin II)-induced hypertension. This study tests the hypothesis that macrophage 12(S)-HETE contributes to Ang II-mediated arterial constriction and thus to development of Ang II-induced hypertension. Ang II constricted isolated abdominal aortic and mesenteric arterial rings. 12(S)-HETE (100 nmol/L) alone was without effect; however, it significantly enhanced Ang II-induced constriction. The presence of wild-type macrophages also enhanced the Ang II-induced constriction, while Alox15-/- macrophages did not. Using this model, pretreatment of aortic rings with inhibitors, receptor agonists/antagonists, or removal of the endothelium, systematically uncovered an endothelium-mediated, Ang II receptor-2-mediated and superoxide-mediated enhancing effect of 12(S)-HETE on Ang II constrictions. The role of superoxide was confirmed using aortas from p47phox-/- mice where 12(S)-HETE failed to enhance constriction to Ang II. In cultured arterial endothelial cells, 12(S)-HETE increased the production of superoxide, and 12(S)-HETE or Ang II increased the production of an isothromboxane-like metabolite. A TP (thromboxane receptor) antagonist inhibited 12(S)-HETE enhancement of Ang II constriction. Both Ang II-induced hypertension and the enhancing effect of 12(S)-HETE on Ang II contractions were eliminated by a BLT2 (leukotriene B4 receptor-2) antagonist. These results outline a mechanism where the macrophage 12/15-LO pathway enhances the action of Ang II. 12(S)-HETE, acting on the BLT2, contributes to the hypertensive action of Ang II in part by promoting endothelial synthesis of a superoxide-derived TP agonist.

The Role of Superoxide in Sex Differences in Angiotensin II‐induced Hypertensive and Natriuretic Responses in Mice with Proximal Tubule‐Specific Deletion of Mitochondrial Protein Sirtuin 3 in The Kidney

Sirtuin 3 (SIRT3), aNAD-dependent deacetylase, is a major mitochondrial protein in the matrix that is encoded by the SIRT3 gene. The key functions of SIRT3 in the mitochondria include anti-oxidative, anti-aging, anti-inflammation, and blood pressure-regulating effects by decreasing reactive oxygen species/superoxide production. However, the role of SIRT3 via superoxide in the proximal tubules (PT) of the kidney in angiotensin II-induced hypertensive or natriuretic responses remains poorly understood. To test our hypothesis, five groups of adult male and female wild-type (WT) and PT-SIRT3-/- mice were treated without (Control) or with Ang II (~0.65 mg/kg/day, i.p.) treated with 2% NaCl diet or tempol, a membrane-permeable radical scavenger and superoxide dismutase-mimetic (50 mg/kg/day, p.o.) for 2 weeks. Under basal conditions, systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) were significantly lower in adult male and female PT-SIRT3-/- mice than male and female wild-type mice (~13 ± 3 mmHg, P<0.01). Although there were no sex differences in male and female WT mice, SBP and MAP were significantly lower in female than male PT-SIRT3-/- mice (P<0.01). Urinary excretion of electrolytes including sodium (Na+), potassium (K+), chloride (Cl-), and lithium (Li+) were also significantly lower in female PT-SIRT3-/- mice (P<0.01). In response to Ang II infusion and 2% high salt treatment, SBP was increased similarly to 136 ± 7 mmHg in male and 135 ± 7 mmHg in female PT-SIRT3-/- mice (P<0.01). Urinary Na+, K+, Cl-, and Li+ excretion were all significantly increased in male and female PT-SIRT3-/- mice without significant sex differences in these responses to Ang II-induced hypertension. There were also no sex differences found in response to Ang II infusion alone. Tempol treatment alone had no significant effects on blood pressure and urinary electrolyte excretory responses compared with their basal levels. Interestingly, however, tempol treatment of Ang II-infused and 2% NaCl-fed male and female PT-SIRT3-/- mice revealed significant sex differences in these responses (P<0.01). SBP and MAP increased further in male (P<0.01), but not in female PT-SIRT3-/- mice (n.s.), whereas the natriuretic response was significantly greater in female PT-SIRT3-/- mice (P<0.01). Taken together, we concluded that SIRT3 in the mitochondria of the proximal tubules plays an important role in the development of ANG II-dependent hypertension, and that superoxide may be implicated in sex differences in Ang II-induced hypertensive and natriuretic responses in PT-SIRT3-/- mice.

The Roles of Superoxide on At-Level Spinal Cord Injury Pain in Rats.

Background: In the present study, we examined superoxide-mediated excitatory nociceptive transmission on at-level neuropathic pain following spinal thoracic 10 contusion injury (SCI) in male Sprague Dawley rats. Methods: Mechanical sensitivity at body trunk, neuronal firing activity, and expression of superoxide marker/ionotropic glutamate receptors (iGluRs)/CamKII were measured in the T7/8 dorsal horn, respectively. Results: Topical treatment of superoxide donor t-BOOH (0.4 mg/kg) increased neuronal firing rates and pCamKII expression in the naïve group, whereas superoxide scavenger Tempol (1 mg/kg) and non-specific ROS scavenger PBN (3 mg/kg) decreased firing rates in the SCI group (* p < 0.05). SCI showed increases of iGluRs-mediated neuronal firing rates and pCamKII expression (* p < 0.05); however, t-BOOH treatment did not show significant changes in the naïve group. The mechanical sensitivity at the body trunk in the SCI group (6.2 ± 0.5) was attenuated by CamKII inhibitor KN-93 (50 μg, 3.9 ± 0.4) or Tempol (1 mg, 4 ± 0.4) treatment (* p < 0.05). In addition, the level of superoxide marker Dhet showed significant increase in SCI rats compared to the sham group (11.7 ± 1.7 vs. 6.6 ± 1.5, * p < 0.05). Conclusions: Superoxide and the pCamKII pathway contribute to chronic at-level neuropathic pain without involvement of iGluRs following SCI.

Investigation on photocatalytic degradation of crystal violet dye using bismuth ferrite nanoparticles

Gadolinium doped bismuth ferrite (Gd:BFO) nanoparticles were synthesized using an auto combustion method. The structural analysis of the prepared Gd: BFO nanoparticles showed that all samples exhibit a rhombohedral structure. The optical analysis shows the doping of Gd ions reduces the band gap value of BFO from 2.21 eV to 2.17 eV. The presence of Fe-O stretching vibrations is confirmed by Fourier transform infrared (FTIR) spectroscopic analysis. The magnetic hysteresis (M-H) analysis shows a weak ferromagnetic nature for the prepared BFO samples and the value of saturation magnetization (Ms) increases upon an increase in the Gd doping concentration. The dielectric analysis shows that the doping of Gd ions in BFO has a notable control in the dielectric loss of the prepared BFO nanoparticles. It was observed the Gd (2 wt%) doped BFO nanoparticles exhibit 84.5% degradation toward crystal violet (CV) dye under visible light irradiation. The formation of trap levels due to Gd 4f state and the role of superoxide anion () and OHo radicals toward the degradation of CV dye were analyzed.

Trichosanthes dioica Roxb. prevents hepatic inflammation and fibrosis in CCl4-induced ovariectomized rats

Summary Plant-derived natural products have shown beneficial effects in many diseases associated with liver. The present study aimed to appraise the effects of Trichosanthes dioica (TD) peel extract on oxidative stress, inflammation as well as fibrosis exhibited by the ovariectomized rats in which hepatic damage had been induced by the administration of carbon tetrachloride (CCl4). The CCl4 administration in the ovariectomized rats elevated the plasma ALT, AST, and ALP level in comparison with the control while TD peel extract showed significant reduction of those activities. Besides, oxidative stress and lipid peroxidation also increased in CCl4-treated rats. TD peel extract significantly reduced the oxidative stress marker's concentration and improved the catalase and SOD activities in CCl4-administered ovariectomized rats. These results suggest that TD peel extract is capable of protecting the liver from CCl4induced damage through preventing oxidative stress, lipid peroxidation, inflammation, fibrosis and boosting the diminished cellular antioxidant defense.

The efficacy of Co3O4 loaded WO3 sheets for the enhanced photocatalytic removal of 2,4,6-trichlorophenol in natural sunlight exposure

Owing to the promising photocatalytic performance, the sheet-like WO3 was modified by depositing nanostructured Co3O4 at the surface. The appearance of the varying dual absorption edges in the optical analysis exposed the composite nature of the synthesized materials. The structural analysis revealed the deposition of Co3O4 particles at the surface without altering the lattice of WO3 however, during the processing the cracking of disc was also evidenced. The FESEM and HRTEM analysis corroborated the uniform surface dispersion of Co3O4 nanoparticles. The co-existence of 2+ and 3+ oxidation states of Co in the deposited Co3O4 was examined by XPS analysis. The efficient trapping of excitons by Co3O4 surface entities was witnessed in the emission measurements whereas the same was authenticated by the photo-electrochemical chronopotentiometry. The Co3O4 loaded sheets exhibited substantially enhanced activity for the removal of 2,4,6-trichlorophenol as compared to pure WO3 in the complete spectrum and visible region of natural sunlight exposure. The progress of the degradation process was monitored by HPLC whereas the degradation products were identified by GC–MS. The measurement and identification of the ion released during the photocatalytic process facilitated the estimation of the probable route and role of reactive oxygen species involved in the removal process. Although, the careful analysis of the findings from the analytical tools revealed the major involvement of hydroxyl radicals however, the role of superoxide anions was also exposed. An alternative mechanism of the generation of the superoxide radical involving the 2+ and 3+ oxidation states of Co was also proposed. The effect of the pH and the added concentration of H2O2 on the ease of removal process was also investigated.

Abstract 2648: Hypoxia-induced NADPH oxidase 5 (NOX5) promotes tumor growth of head and neck squamous cell carcinoma through HIF-2á signaling

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) has an increased content of reactive oxygen species (ROS). However, clinical trials of antioxidant resulted in no benefit towards HNSCC anti-cancer treatment. NADPH oxidase (NOX) family is found to be the major generator of ROS which might contribute to the failure of antioxidant therapy. For the past 10 years, NOX family have been found with a range of functions in different cancers. Of which, NOX5 is the least well-understood one with limited details of signaling roles. Purpose of the study: To investigate the role of NOX5 in HNSCC. Experimental procedures: Online databases were used to analyze the expression of NOX5 in HNSCC patients. Immunohistochemistry was performed to detect the NOX5 expression level and its co-expression with hypoxia-inducible factors (HIFs) in paraffin-embedded tumor tissues surgically resected from HNSCC patients in Hong Kong. Subcutaneous xenograft model was established using NOX5-silenced HNSCC cells to evaluate the tumor growth potential in vivo. Sulforhodamine B assay and colony formation assay were used to measure the proliferation potential of NOX5-silenced HNSCC cells in vitro. Hypoxia chamber was applied to mimic the hypoxic condition of tumor tissue in vitro. Flow cytometry with Dihydroethidium staining was performed to measure the production of superoxide when silencing or overexpressing NOX5. Superoxide scavenger TEMPOL was applied to test the signaling role of superoxide generated by NOX5. Quantitative PCR and western blot were used to measure the mRNA and protein expression level of NOX5 and HIFs in HNSCC cell lines. Results: Relative mRNA expression of NOX5 was found significantly increased in HNSCC tumor tissues comparing to normal tissues both in Oncomine and TCGA datasets. Interestingly, NOX5 expression was found higher in central areas of tumor nests with the co-expression of HIF-1α and HIF-2α in our HNSCC patients’ samples. Further, NOX5 was upregulated in HNSCC cell lines under hypoxic condition (1% oxygen). Knocking-down of NOX5 impaired HNSCC cell growth both in vivo and in vitro, and this reduction was more significant under hypoxic condition in vitro. Loss of NOX5 resulted in superoxide reduction and HIF-2α impairment but not HIF-1α. Overexpressed NOX5 induced superoxide production and HIF-2α expression, and this induction was attenuated by TEMPOL. Conclusions: Hypoxia-induced NOX5 promotes HNSCC tumor growth by regulating HIF-2α via generating superoxide. NOX5 might serve as a novel therapeutic target for treatment of HNSCC. Citation Format: Siqi Chen, Wei Gao, Jimmy Yu-Wai Chan, Thian-Sze Wong. Hypoxia-induced NADPH oxidase 5 (NOX5) promotes tumor growth of head and neck squamous cell carcinoma through HIF-2á signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2648.

“Redox Imaging” to Distinguish Cells with Different Proliferative Indexes: Superoxide, Hydroperoxides, and Their Ratio as Potential Biomarkers

The present study was directed to the development of EPR methodology for distinguishing cells with different proliferative activities, using “redox imaging.” Three nitroxide radicals were used as redox sensors: (a) mito-TEMPO—cell-penetrating and localized mainly in the mitochondria; (b) methoxy-TEMPO—cell-penetrating and randomly distributed between the cytoplasm and the intracellular organelles; and (c) carboxy-PROXYL—nonpenetrating in living cells and evenly distributed in the extracellular environment. The experiments were conducted on eleven cell lines with different proliferative activities and oxidative capacities, confirmed by conventional analytical tests. The data suggest that cancer cells and noncancer cells are characterized by a completely different redox status. This can be analyzed by EPR spectroscopy using mito-TEMPO and methoxy-TEMPO, but not carboxy-PROXYL. The correlation analysis shows that the EPR signal intensity of mito-TEMPO in cell suspensions is closely related to the superoxide level. The described methodology allows the detection of overproduction of superoxide in living cells and their identification based on the intracellular redox status. The experimental data provide evidences about the role of superoxide and hydroperoxides in cell proliferation and malignancy.

The Effect of Nutrient Medium on Chemical Composition of Corrosive-Active Exometabolites of the Bacterium Bacillus subtilis Affecting a Zinc Surface

We have hypothesized that products of oxygen biotransformation by a microbial electron transport chain, for example, superoxide anion and the product of its disproportionation, hydrogen peroxide, play a key role in aerobic biocorrosion. The goal of the present work was to evaluate the role of superoxide anion and its derivatives in initiation of bacterial corrosion and its dependence on the composition of nutrient medium.

Mn-doped g-C3N4 composite to activate peroxymonosulfate for acetaminophen degradation: The role of superoxide anion and singlet oxygen

Peroxymonosulfate (PMS) is an alternative to hydrogen peroxide in advanced oxidation processes. Herein, we present a simple one-pot synthetic approach for the Mn-doped graphite phase carbon nitride (g-C3N4) materials and make it as a catalyst to activate PMS for acetaminophen (ACT) degradation. Fabricated Mn-g-C3N4 composites (MnCN) were characterized by SEM, EDS, XRD, XPS, and FTIR. Results showed that Mn was uniformly dispersed in the structure of g-C3N4 mostly in the form of Mn-N coordination. The effects of pH, contents of doped Mn, catalyst dosage, and reusability of catalyst and corresponding kinetic study were conducted to determine the catalytic performance of MnCN. High reaction efficiency was obtained in a wide pH range of 3.2–9. 100% removal of ACT in 15 min under optimized conditions with initial pH 6.5, 0.8 g/L of PMS and 200 mg/L of catalyst of 0.5-MnCN. Additionally, the reaction mechanism of the PMS/MnCN system was investigated using the electron spin resonance technique, quenching studies as well as different organics with electron-donating or electron-withdrawing groups. No PMS decomposition was detected without substrates. No inhibition effect by ethanol or tert-Butyl alcohol and complete inhibition effect by benzoquinone and partly by furfuryl alcohol, as well as ESR spectrum of TEMP-1O2 were observed, suggesting that the activation of PMS proceeded in a new way, which is different from traditional sulfate radical-based advanced oxidation processes. A possible mechanism was proposed for ACT removal. PMS was first bounded to Mn-N sites to generate the superoxide anion. Then singlet oxygen produced by superoxide anion would react with ACT by replacing the electron-donating group of acylamino.

Comparative toxicity studies on bromochloroacetate, dibromoacetate, and bromodichloroacetate in J774A.1 macrophages: Roles of superoxide anion and protein carbonyl compounds.

The brominated and mixed bromo-chloro-haloacetates, such as dibromoacetate (DBA), bromochloroacetate (BCA), and bromodichloroacetate (BDCA), are by-products of water chlorination and are found at lower levels than the fully chlorinated acetates in the drinking water. The toxicities of the compounds were assessed in J774A.1 cells and were found to induce concentration-dependent increases in cell death and superoxide anion and protein carbonyl compounds production. Compared to the previously tested concentrations of dichoroacetate (DCA) and trichloroacetate (TCA) in the same cell line, the tested haloacetates induced similar effects on cellular viability and superoxide anion production but at DBA and BCA concentrations that were approximately 40-160 times lower than those of DCA and TCA, and at BDCA concentrations that were 4-16 times lower than those of DCA and TCA. Also, production of super oxide anion, protein carbonyl compounds, and induction of phagocytic activation are suggested to play a role in their toxicity.

29 - Oxygen Inhibits Nitrite Reduction to Nitric Oxide by the Molybdenum-Dependent mARC-2 Enzyme

The mitochondrial amidoxime reducing component (mARC) enzymes are molybdenum (Mo) -dependent oxidoreductases that function as part of a three-enzyme metabolon with cytochrome b5 (CYB5) and cytochrome b5 reductase (CYB5R). Previous studies in our lab have demonstrated that nitrite reduction to nitric oxide by the mARC-1 metabolon is regulated by oxygen via superoxide. However, the effect of oxygen on mARC-2, the mARC paralog expressed in human vascular tissue, has not been reported. Therefore, this study was conducted to determine the effect of oxygen on nitrite reduction to nitric oxide by mARC-2 and determine if superoxide is involved. We used purified recombinant mARC-2, CYB5, and CYBR enzymes and kinetic nitric oxide measurements to measure the effect of anoxia, hypoxia, and normoxia on nitrite reduction. We found that the NADH-dependent CYB5/CYB5R/mARC-2 metabolon is more sensitive to oxygen than the mARC-1 metabolon. However, the role of superoxide is less pronounced. This is in contrast with previous studies on the mARC-1 metabolon. Our data suggests that mARC-2 oxygen inhibition proceeds via a superoxide-independent mechanism, possibly direct and irreversible oxidation of the Mo active site.

Roles of superoxide anion and hydrogen peroxide during replication of two unrelated plant RNA viruses in Nicotiana benthamiana

ABSTRACTReactive oxygen species (ROS), including superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O2− production to inhibit pathogen infection during plant innate immunity. RBOH-derived O2− can be immediately converted into H2O2 by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O2− scavenger but insensitive to an H2O2 scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O2− and H2O2 for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.

Deoxynivalenol induced mouse skin tumor initiation: Elucidation of molecular mechanisms in human HaCaT keratinocytes.

Among food contaminants, mycotoxins are toxic to both human and animal health. Our prior studies suggest that Deoxynivalenol (DON), a mycotoxin, behaves as a tumor promoter by inducing edema, hyperplasia, ODC activity and activation of MAPK's in mouse skin. In this study, topical application of DON, 336 and 672 nmol significantly enhanced ROS levels, DNA damage and apoptosis with concomitant downregulation of Ki-67, cyclin D, cyclin E, cyclin A and cyclin-dependent kinases (CDK4 and CDK2) thereby resulting in tumor initiation in mouse skin. Further, the elucidation of molecular mechanisms of tumor initiation by DON (0.42-3.37 nmol/ml) in HaCaT keratinocytes, revealed (i) enhanced ROS generation with cell cycle phase arrest in G0/G1 phase, (ii) increase in levels of 8-OxoG (6-24 hr) and γH2AX protein, (iii) significant enhancement in oxidative stress marker enzymes LPO, GSH, GR with concomitant decrease in antioxidant enzymes catalase, GPx, GST, SOD and mitochondrial membrane potential after DON (1.68 nmol) treatment, (iv) suppression of Nrf2 translocation to nucleus, enhanced phosphorylation with subsequent activation ERK1/2, p38 and JNK MAPK's following DON (1.68 nmol) treatment, (v) overexpression of c-jun, c-fos proteins, upregulation of Bax along with downregulation of Bcl-2 proteins, (vi) increase in cytochrome-c, caspase-9, caspase-3 and poly ADP ribose polymerase levels leads to apoptosis. Pretreatment of superoxide dismutase, mannitol and ethanol to HaCaT cells resulted in significant reduction in ROS levels and apoptosis indicating the role of superoxide and hydroxyl radicals in DON induced apoptosis as an early event and skin tumor initiation as a late event.

Abstract 155: Lipopolysaccharide Selectively Decreases Circulating Endothelial Progenitor Cells in Female Mice via Superoxide Production

Background: Gender differences exist in cardiovascular disease such as coronary artery disease. Differences also exist between males and females in regard to oxidative stress, which is considered an important mechanism in the development of cardiovascular disease such as atherosclerosis. Endothelial progenitor cells (EPCs) play a critical role in maintaining the structural and functional integrity of vasculature. The number and function of EPCs are closely associated with the outcome for patients with cardiovascular diseases. The objective of the present study was to determine if the effect of oxidative stress on EPCs could differ between males and females. Methods: Male and female wild-type C57BL/6 mice were injected with low dose lipopolysaccharide (LPS) for three days to induce oxidative stress with PBS as control. To evaluate the role of superoxide in mediating the effect of LPS, the mice were treated with superoxide dismutase (SOD) prior to LPS challenge. The blood cells were collected and prepared to determine the populations of EPCs and the levels of intracellular reactive oxygen species (ROS), cytokines, and apoptosis using flow cytometry analysis. Results: The number of circulating EPCs was significantly higher in females than males at baseline. There were no differences in the basal levels of apoptosis and intracellular ROS between males and females. LPS treatment significantly increased levels of the serum inflammatory cytokines including IL-1β and TNF-α in both males and females, suggesting that LPS-induced oxidative stress mouse model was successful. Treatment with LPS significantly decreased the number of circulating EPCs in females, not in males. Treatment with SOD effectively prevented LPS-induced reduction of circulating EPC population in female mice with no effect on EPC population in males. Conclusion: The basal level of circulating EPCs was significantly higher in females than in males independent of intracellular ROS formation and apoptosis. LPS selectively decreased the circulating EPC population in females via superoxide production.

Role of Superoxide Anion in the Oxygen Reduction Reaction in Non-Aqueous Electrolytes with a Proton or Lithium Source

The non-aqueous Li-air (O2) battery has generated a great deal of research interest because it potentially possesses far greater gravimetric energy storage density than today’s rechargeable battery technologies.1-4 Presently non-aqueous Li-O2 batteries suffer from irreversible reactions, including those due to impurities such as residual protons and proton-containing compounds that can react with reduced oxygen species. To eliminate or alleviate the undesired reactions, it is crucial to have a fundamental understanding of the reactive oxygen species involved in the oxygen reduction reaction (ORR). In this talk we will present the results of a series of electrochemical studies of the ORR on a gold electrode in non-aqueous dimethyl sulfoxide (DMSO) electrolytes containing a proton or lithium source with TBAClO4 as supporting ions. The superoxide anion (O2 -), the one-electron reduction product of O2, is identified in situ spectroscopically in both systems. When the DMSO contains phenol as the proton source, O2 -, not HO2, appears as the first stable intermediate during the ORR upon cathodic scans, with H2O2 co-appearing at high overpotentials.5 When instead LiClO4 is added to DMSO, again O2 - is the first reaction intermediate to be detected, whereas surface LiO2 does not appear until the potential is much more negative of the Li2O2 formation potential. The stability of O2 - vs. the other reduced O2 species is rationalized based on first-principles theoretical modelling and calculations. Our studies show that the ORR mechanism depends intimately on the electrode potential and electrolyte composition, and contribute new mechanistic understanding for this reaction that is of fundamental importance to electrochemical energy storage and conversion technologies. 1. P.G. Bruce, S.A. Freunberger, L.J. Hardwick, J.-M. Tarascon, Nat. Mater. 2012, 11, 19. 2. J. Lu, L. Li, J.-B. Park, Y.-K. Sun, F. Wu, K. Amine, Chem. Rev. 2014, 114, 5611. 3. A.C. Luntz, B.D. McCloskey, Chem. Rev. 2014 , 114, 11721. 4. R. Black, B. Adams, L.F. Nazar, Adv. Energy Mater. 2012, 2, 801 5. Z. Peng, Y. Chen, P.G. Bruce, Y. Xu, Angew. Chem. Int. Ed. 2015, 54, 8165.