Spectroscopic studies have been used to describe the mechanism of the decomposition of hydrogen peroxide by solutions of a dimeric Cu(II) complex of a dissymetric Schiff base, [CuSALAD]2.H2O, and imidazole or methyl substituted imidazoles, B, which form monomeric CuSALAD.B2 complexes, in aqueous ethanol solvent. Freezing point depression and vapor pressure lowering studies were carried out to confirm the dimeric nature of the [CuSALAD]2.H2O complex that had been previously reported. The stoichiometry of the [CuSALAD]2.H2O-imidazole equilibrium was extensively studied pointing to a 1:4 stoichiometry. The CuSALAD.B2 adducts exhibited certain catalytic properties that mimic those of catalase enzymes. The different imidazoles were buffered to acidic, neutral and basic pH media in order to investigate the pH effects of this reaction. Two charge transfer (CT) bands were observed near 420 and 450 nm upon addition of hydrogen peroxide to CuSALADB2 solutions, and were associated with two proposed intermediates (CuBOOH and CuBOOCu). A mechanism consistent with these results has been developed. First order dependence of the rate on CuSALAD.B2 was observed in the presence of excess CuSALAD.B2 over hydrogen peroxide, whereas second order dependence was observed with the latter in excess. The CuBOOCu intermediate was unstable in the presence of EDTA, and a first order dependence of rate of formation of intermediate on both CuSALAD.B2, and hydrogen peroxide was observed.

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Objectives: To measure the level of hydrogen peroxide penetration into the pulp chamber and to evaluate its whitening efficacy when the tooth is treated with an innovative double layer of potassium nitrate desensitizing agent and hydrogen peroxide whitening gel. Methods: Extracted human molar teeth (n=160) were collected. Roots were trimmed 3 mm apical to the cemento-enamel junction, the pulp was removed and a cavity prepared, leaving a standardized wall of 2 mm thickness, which retained 100 µL of acetate buffer solution. A standardized whitening area of 6 mm in diameter was established buccally by painting the rest of the tooth with gray nail varnish. The teeth were randomly assigned into four groups. Group 1: no treatment (glycerin gel, negative control); Group 2: double layer of 20 L 5% potassium nitrate (Relief ACP, Philips Oral Healthcare) and 100 L 25% hydrogen peroxide (Zoom Chairside Whitening Gel, Philips Oral Healthcare); Group 3: double layer of 40 L 5% potassium nitrate and 100 L 25% hydrogen peroxide; and Group 4: 100 L 25% hydrogen peroxide (positive control). All groups received three 45-minute sessions of in-office whitening with light activation at 3-day intervals. Hydrogen peroxide penetration was assessed spectrophotometrically using leucocrystal violet and horseradish peroxidase. Color measurements were assessed with VITA Easyshade at baseline, 1-day, and 1-month post-whitening. Tooth color change was measured per Commission Internationale de l'Eclairage methodology. One-way ANOVA with Tukey multiple comparisons adjustment was performed to compare group differences in overall tooth color change (Delta E*) and hydrogen peroxide penetration (type I error = 0.05). Results: Hydrogen peroxide penetration levels were not significantly different between Group 2 and Group 4; however, the levels were significantly higher than Group 1 and Group 3 (p< 0.0001). Groups 2, 3 and 4 showed no differences for overall tooth color change but differed significantly from the negative control group (Group 1) at 1-day and 1-month post-whitening. Conclusions: Hydrogen peroxide penetration was affected when pretreated by potassium nitrate desensitizer; however, the double layer technique did not adversely affect the whitening efficacy.

A scientific and cultural revolution occurred with the sequencing of the human genome. The information provided by this accomplishment has provided tools for researchers to test new ideas in silico and on the bench. In redox biology many of the genes, transcripts, proteins, and redox active species have been well characterized. However, the vast majority have not been quantitated in an absolute manner. This is a necessary step to provide the tools for mathematical modeling and systems biology approaches for predicting changes in the cellular redox environment and the biochemical and biological consequences. Here we demonstrate techniques for the absolute quantitation of human catalase, glutathione peroxidase, peroxiredoxin, thioredoxin, and superoxide dismutase within cells. These techniques can be parsed into two groups: detection of activity and detection of total amount of species. Methods for the absolute quantitation of active catalase, peroxiredoxins, and superoxide dismutase have been developed by utilizing specific characteristics of each enzyme. Catalase generates oxygen in the presence of hydrogen peroxide that can easily be detected with a Clark electrode (oxygen monitor); the data are fit to a single-exponential to determine the observed pseudo-first-order rate constant. From this the effective number of fully active catalase enzymes in the sample can be determined. Peroxiredoxin in the disulfide state can be reduced by thioredoxin; thioredoxin from E. coli loses fluorescence upon oxidation. The loss of fluorescence over time is mathematically fit to a single-exponential to determine the observed pseudo first-order rate constant from which the number of active enzymes can be determined. Using an inhibition assay to detect superoxide dismutase activity along with the rate constants at which superoxide reacts with the dismutase and the competing superoxide-reacting-indicator-molecule, the concentration of active superoxide dismutase can be determined. To detect the total amount of protein of an enzyme in a biological sample, an immunoassay was first implemented. This method utilized Bio-Plex® beads from Bio-Rad; however, it was problematic because the antibodies applied did not perform satisfactorily not allowing sufficient signal-to-noise to be deployed. Quantitative mass spectrometry was then implemented to detect total catalase, glutathione peroxidase 1, peroxiredoxin 2, and thioredoxin 1 in human red blood cells. With the absolute concentration of these enzymes and proteins along with data for oxygen consumption rates and peroxisomal hydrogen peroxide concentration for several cell lines, we hypothesize that a reasonable model of hydrogen peroxide and superoxide flux can be constructed. Quantitative data such as these provide the foundation for the new redox biology of the 21st century. Presented here is a roadmap for the obligatory first steps to dissect quantitatively the cellular and tissue metabolic pathways and redox networks that are the basis of all of biology.

Development of an in vitro model for the early stages of cardiovascular disease is a current necessity. Cardiovascular disease is the leading cause of death in the United States and throughout the world. Oxidative stress and reactive oxygen species have been implicated in cardiovascular disease development. An in vitro model of these processes will improve our understanding of cardiovascular disease development and allow for the development of additional treatments. Atherosclerosis is an inflammatory disease and increased levels of H2O2 are associated with inflammation. The model focuses on H2O2-induced oxidative stress under static and shear conditions. Previous studies have documented increased O2.- and increased cytotoxicity in smooth muscle cells exposed to H2O2. Under static culture, endothelial cells exposed to H2O2, exhibited increased O2.- over basal levels via NOS and NAPDH oxidase pathways. Increased O2.- was attenuated by MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. Endothelial cell cytotoxicity was increased with H2O2 exposure. The increase in cytotoxicity was diminished upon exposure to Tiron or L-NAME. Under shear conditions (8.2 dynes/cm2), endothelial cells exposed to H2O2 exhibited increased O2.- compared to control via an L-NAME (specific inhibitor NOS) and Apocynin (NADPH oxidase inhibitor) inhibitable mechanism. This suggests NOS and NADPH oxidase as sources of increased O2.- under H2O2-induced oxidative stress. The increased O2.- was attenuated with MnSOD adenoviral-mediated upregulation and endothelial cell exposure to Tiron (an O2.-scavenger). Endothelial cell attachment under shear with exposure to H2O2 was improved with MnSOD adenoviral-mediated upregulation as observed by decreased loss of the endothelial cell monolayer compared with H2O2 exposed endothelial cells. Endothelial cells exposed to H2O2 exhibit increased O2.-, suggesting that H2O2-induced oxidative stress may be a reasonable model for atherosclerosis. NOS and NADPH oxidase co-inhibition under shear and static culture demonstrated that NOS and NADPH oxidase inhibition is non-additive under static culture, yet additive under shear. Co-inhibition results suggest a complex relationship between the two enzymes that requires additional experimentation to deconvolve.

Orientador: Alessandra Bühler Borges / Coorientadora: Rayssa Ferreira Zanatta / Banca: Cesar Rogério Pucci / Banca: Daniele Mara da Silva Ávila Nogueira / Resumo: O objetivo deste estudo foi avaliar a degradação do peróxido de hidrogênio em altas concentrações (35-40%), durante uma simulação de sessão de clareamento de consultório, utilizando produtos comercialmente disponíveis com dois protocolos de aplicação. Foram medidas também a eficácia clareadora dos géis, a alteração de pH dos mesmos, bem como o potencial de alteração da microdureza e rugosidade superficial do esmalte. Para tal, espécimes de dentes bovinos em forma de disco com 4 mm de diâmetro e 2 mm de espessura (1 mm de esmalte e 1 mm de dentina) foram preparados para tal 105 espécimes, embutidos em resina acrílica, polidos e divididos nos seguintes grupos, de acordo com o tipo de gel clareador: Pola Office Plus (POP - SDI PH-37,5%), Opalescence Boost (OPB - Ultradent PH-40%) e Whiteness HP (WHP - FGM PH-35%). Cada grupo foi dividido em dois subgrupos conforme o protocolo de aplicação (n = 15): com e sem troca do gel clareador durante a sessão de clareamento. A alteração de cor das amostras foi medida por espectrofotometria de acordo com o sistema CIE L*a*b*, antes e após a sessão de clareamento. A superfície do esmalte foi analisada com microdurômetro e perfilômetro, para mensuração da microdureza Knoop (KHN) e rugosidade superficial (Ra). Outros 30 espécimes adicionais em dentes bovinos foram confeccionados para mensuração da concentração do peróxido de hidrogênio e pH. A concentração e pH dos géis foram medidos por meio da titulação com permanganato de potássio (KMnO4) e ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the degradation of hydrogen peroxide at high concentrations (35-40%) during a clinic whitening session simulation using commercially available products with two application protocols. The bleaching efficiency of the gels, pH change, as well as the alteration potential of the enamel microhardness and surface roughness (Ra) were measured. For this, disc-shaped bovine tooth specimens of 4 mm diameter and 2 mm thickness (1 mm enamel and 1 mm dentin) were prepared 105 specimens, embedded in acrylic resin, polished, and divided into the following groups according to the type of bleaching gel: Pola Office Plus (POP - SDI PH-37,5%), Opalescence Boost (OPB - Ultradent PH-40%), and Whiteness HP (WHP - FGM PH-35%). Each group was divided into two subgroups, according to the application protocol (n = 15): with and without exchange of the gel during the whitening session. The specimens' color change was measured by spectrophotometry according to the CIE system L*a*b*, before and after the bleaching session. The enamel surface was analyzed with a microhardner and profilometer, to measure the Knoop microhardness and surface roughness (Ra). Another 30 specimens in bovine teeth were made to measure the hydrogen peroxide concentration and pH. The concentration and pH of the gels were measured by titration with potassium permanganate (KMnO4) and by a pH meter and specific electrode, respectively, before and after said application times thereof. The degradation of the gels was calculated as a function of the initial concentration. The results were submitted to the ANOVA test and Tukey's multiple comparison test (5%). All gels presented similar bleaching efficacy (ΔE and ΔE00) comparing SA and MA. There were no significant differences between KHN and Ra means comparing SA and MA for the same gel. Although peroxide degradation significantly increased with time, the final gels concen.. / Mestre

The clinical potential of pharmacological ascorbate (P-AscH-; IV delivery achieving mM concentrations in blood) as an adjuvant in cancer therapy is being re-evaluated. At mM concentrations, P-AscH- is thought to exhibit anti-cancer activity via generation of a flux of H2O2 in tumors, which leads to oxidative distress. Here, we use cell culture models of pancreatic cancer, MIA PaCa-2, PANC-1, and 339 cells, to examine the effects of P-AscH- on DNA damage, and downstream consequences, including changes in bioenergetics. We have found that the high flux of H2O2 produced by P-AscH- induces both nuclear and mitochondrial DNA damage. In response to this DNA damage, we observed that poly (ADP-ribose) polymerase-1 (PARP-1) is hyperactivated, as determined by increased formation of poly (ADP-ribose) polymer. Using our unique absolute quantitation, we found that the P-AscH--mediated the overactivation of PARP-1, which results in consumption of NAD+, and subsequently depletion of ATP (potential energy crisis) leading to mitotic cell death. Time-course studies with MIA PaCa-2 cells showed that the level of NAD+ and ATP were reduced by 80% immediately after a 1-h exposure to P-AscH- (4 mM; 14 pmol cell-1); both species returned to near basal levels within 24 h. In parallel with these metabolic and energetic restorations, the lesions in nuclear DNA were removed within 3 h; however, even after 24 h, lesions in mitochondrial DNA were only partially repaired. We have also found that the Chk1 pathway has a major role in the maintenance of genomic integrity following treatment with P-AscH-. Hence, combinations of P-AscH- and Chk1 inhibitors could have the potential to improve outcomes of cancer treatment. Hyperactivation of PARP-1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 Analyzer, we observed no changes in OCR or ECAR/PPR following treatment with P-AscH-. OCR and ECAR/PPR together indicate the rate of production of intracellular ATP; therefore, the rate of production is unchanged after challenge with P-AscH-. Thus, the severe decrease in ATP is due solely to increased demand. Genetic deletion and pharmacological inhibition of PARP-1 preserved both NAD+ and ATP; however, the toxicity of P-AscH- remained. These data indicate that loss of NAD+ and ATP are secondary factors in the toxicity of P-AscH-, and damage to DNA is the primary factor. These preclinical findings can guide the best use of P-AscH- as an adjuvant in cancer therapy.

Objectives: Evaluate and assess the effect of an innovative double layer, single application desensitizing/whitening technique on Nitrate penetration and total color change. Methods: Specimens were prepared from extracted caries free human molars (n=160). Teeth were randomly assigned into four groups: 100μl 25% hydrogen peroxide “Philips Zoom chairside” as control group (CTRL), double layer treatment of 20μl 5% potassium nitrate “Relief ACP, Philips oral care” and 100μl 25% hydrogen peroxide (DL20), double layer treatment of 40μl 5% potassium nitrate and 100μl 25% hydrogen peroxide (DL40), and one layer treatment of 40 μl 5% potassium nitrate (PN40). Spectrophotometric color measurements (Vita EasyShade) were done at base line (T0), one day (T1), and one month (T2) following the treatment. Nitrate penetration was measured using a nitrate/nitrite assay kit. Group comparisons of tooth color difference measurements, and nitrate penetration readings were made using the Kruskal-Wallis test. Adjustment was made for pairwise treatment comparisons using the Tukey method in conjunction with an overall 0.05 level of significance. Results: 160 teeth were used. Color difference (ΔE) results at (T1) and (T2) showed no significant difference among the CTRL, DL20, DL40 groups. But there was a statistical significant difference between those groups and the single layer (PN40) group (p

Three experiments were conducted to evaluate low quality forages treatment by anhydrous ammonia (NH3) and(or) hydrogen peroxide (H2O2). In experiment I, a control and three treatments of barley straw were compared: NH3, NH3 after rehydration of the straw with water (NH3+H2O), and NH3 after rehydration with a H2O2 solution (NH3+H2O2). Forages were fed, with a supplement, at two levels of intake (ad libitum and 75% of ad lib.). Treatments were administered in a split-plot in a Latin square design to 8 ewes. Total collections and rumen digesta measurements were made. Ammoniation, rehydration, and H2O2 increased primarily dry matter intake (DMI) but also digestibility of different nutrients. A similar trend was observed in rumen fermentation characteristics. Dry matter (DM) digestibility was slightly raised by limiting DMI. Digestible DMI (DDMI) was correlated with both the increase of forage CP content and the decrease of forage neutral detergent fiber. Water intake and output were highly correlated with fiber. Intake and digestibility better than acid detergent lignin insoluble ash (ADLIA). Both markers were adequate in determining hemicellulose digestibility. In experiment II, ten solutions were prepared to rehydrate wheat straw. Six solutions were adjusted (with NaOH) to a pH of 9, 11, or 13 and contained 2% H2O2. Four solutions had a pH of 7 or 11 and contained no H2O2. Half of the straws were treated with NH3. In situ DM disappearance (DMD) of the different straws was measured at different times in 10 Holstein cows and three periods (Incomplete block design). The evolution of DMD was slow and almost linear. The positive effect of ammoniation on DMD was consistently apparent at all pH levels though depressed at pH 11. The effect of H2O2 was minor, but was complementary with ammoniation. In experiment III, mature baltic rush (Juncus balticus Willd.) was either treated or not with NH3. Eight wethers had ad libitum access to the nonsupplemented forages in a cross-over design. total collections and rumen digesta measurements were made. DMI and DDMI were not affected by treatment, but DM digestibility was decreased by ammoniation.

Objectives: This investigation adapted the LIVE/DEAD� Baclight[TM] bacterial viability stain for the quantitative determination of bacterial cell viability of the aerotolerant anaerobes Porphyromonas gingivalis ATCC 33277 and Prevotella intermedia ATCC 25611. The Live/Dead stain was used to determine the influence of haem availability on the resistance of P. gingivalis and P. intermedia to the reactive oxygen species (ROS) superoxide anion and hydrogen peroxide and compare the sensitivities between the haem-requiring periodontal bacteria to ROS. Neutrophils use oxidative and non-oxidative killing mechanisms. During phagocytosis, neutrophils kill bacteria via a respiratory burst, producing ROS. P. gingivalis and P. intermedia are oxygen-tolerant gram-negative bacteria found in the gingival crevice. These bacteria express superoxide dismutase (SOD) activity, which extends some protection against superoxide radicals. Methods: Initially, experiments were performed to validate the reliability and accuracy of the fluorogenic Live/Dead stain using Escherichia coli ATCC 10798 (K-12), followed by experiments using P. gingivalis. The Live/Dead stain distinguishes viable:non-viable proportions of bacteria using mixtures of green (SYTO 9) and red (propidium iodide) fluorescent nucleic acid stains respectively. Bacterial cell viability was assessed with fluorescence microscopy and subsequently quantitative measurement using a fluorescence microplate reader (BMG Fluorostar plus Optima). P. gingivalis and P. intermedia colonies were subcultured from frozen cultures, in Tryptic soy broth (TSB) (Difco) and incubated anaerobically for approximately five days. They were further subcultured in pre-reduced TSB, supplemented with menadione 0.5[mu]g/ml (TSB-M) and either 5 [mu]g/ml haemin (Haem 5), 50 [mu]g/ml haemin (Haem 50) or without supplemental haemin (Haem 0). Cultures were grown anaerobically at 37�C to early stationary phase (approximately 48 hours). For experimental purposes, bacteria were harvested, washed and resuspended in 10 mM Tris-buffered saline (pH 7.5) containing peptone (TBS-P) (0.1 mg/ml), with a final adjustment to OD₅₄₀ [approximately equals] 2.0 (which corresponds to 1 x 10⁹ bacteria/ml). Bacterial suspensions were diluted ([approximately equals] 10⁸/ml) into TBS-P containing the fluorogenic viability stain (BacLight, Molecular Probes). Either pyrogallol (0.02 - 2 mM) or hydrogen peroxide (0.01 - 100 mM) was added (except to control tubes); tubes were vortexed for ten seconds and incubated at 37�C. Viability was monitored fluorimetrically for three hours. Results: For both P. gingivalis and P. intermedia, a pyrogallol concentration of 0.2 mM resulted in 80 to 90% cell death; and a hydrogen peroxide concentration of 10 mM killed approximately 80 to 90% of cells. Irrespective of the haem status, no significant difference was determined between the overall maximum rate of killing of P. gingivalis and P. intermedia, in their response to either superoxide or hydrogen peroxide; with the exception that the P. intermedia Haem 0 group was significantly less susceptible to hydrogen peroxide than the P. gingivalis Haem 0 group. For the majority of the experiments, there was no significant difference between final bacterial cell viability in the Haem 0 and Haem 5 cells for both species, after 3 hours exposure to various concentrations of ROS. However, the Haem 50 cells showed a significant increased susceptibility (albeit, a small difference) to both hydrogen peroxide and superoxide. Conclusions: The Live/Dead bacterial viability stain provided a valuable method to monitor "real-time" killing, avoiding the difficulties associated with culture-based methods for assessing viability. Haem availability had no clear influence on the resistance to ROS of either P. gingivalis or P. intermedia Haem 0 and Haem 5 cells. The Haem 50 cells showed a very slight increase in susceptibility to hydrogen peroxide and superoxide. Although P. intermedia may be isolated in significant numbers from healthy gingivae, as well as from periodontally diseased sites, it was no more resistant to ROS than was P. gingivalis, which is associated with periodontal lesions and difficult to cultivate from relatively healthy (more oxygenated) sites. This suggests that resistance to ROS does not contribute to the ecological distinction between these two species. The finding that haem availability did not influence sensitivity implies that these bacteria do not accumulate haem for the purpose of protection from ROS.