Orientador: Carlos Alberto de Souza Costa / Banca: Marcelo Ferrarezi de Andrade / Banca: José Carlos Pereira / Resumo: O objetivo da presente pesquisa foi avaliar in vitro os efeitos citotóxicos de diferentes concentrações de um agente clareador de ativação dual à base de peróxido de hidrogênio, sobre células de linhagem odontoblástica MDPC-23. Para isso, 50 discos de dentina com 0,5mm de espessura, obtidos de dentes terceiros molares humanos íntegros, foram tratados com EDTA 0,5 M, pH 7,4 por 1 minuto, sendo a condutância hidráulica (Lp; æL/mm2/min/cm H2O) determinada para cada um deles. Estes discos foram distribuídos em 5 grupos de acordo com os valores de permeabilidade. Os discos de dentina adaptados a um dispositivo metálico foram autoclavados, posicionados de maneira invertida (superfície pulpar dos discos voltados para cima) em recipientes plásticos de 24 compartimentos ("wells"). Então, 5 x 104 células MDPC-23/cm2 foram semeadas em cada compartimento. Após 72 horas de incubação na temperatura de 37º C, 5% de CO2 e 95% de ar, os dispositivos com os discos foram invertidos nos compartimentos, de tal maneira que os materiais experimentais pudessem ser aplicados (2 trocas de 15 minutos cada) sobre a superfície oclusal dos discos, caracterizando os 164 seguintes grupos experimentais: G1: H2O2 7,5%; G2: H2O2 20%; G3: H2O2 35%; e G4: gel base (sem o H2O2). No grupo controle (G5) os discos não foram tratados. Após esta etapa do experimento, o metabolismo das células MDPC-23 que permaneceram aderidas à superfície pulpar dos discos de dentina foi avaliado pela técnica de MTT. A morfologia das células e dentina tratada com os agentes experimentais foram avaliadas em microscopia eletrônica de varredura. A análise estatística de Kruskal-Wallis demonstrou que as três concentrações do agente clareador à base de H2O2 causaram intenso efeito citotóxico sobre as células MDPC-23. As concentrações de 7,5%, 20% e 35% de H2O2 aplicadas sobre os discos de dentina com 0,5mm... (Resumo completo, clicar acesso eletrônico abaixo). / Abstract: The aim of this in vitro study was evaluate the transdentinal effects of an experimental bleaching agent with different concentration of hydrogen peroxide on the immortalized odontoblast-cell line MDPC-23. Fifty dentin disks, 0.5mm thick, cut from extracted non-carious permanent third human molar were conditioned for 1 minute with 0.5M EDTA, pH 7.4 and the hydraulic conductance (Lp; æL/mm2/min/cm H2O) was determined. The dentin discs were adapted to a metallic device which were sterilized and placed in wells of 24-well dishes (the pulpal surface of the discs remained in up position). Then, the cells were plated (5 x 104 cells/cm2) on the pulpal surface of the discs and incubated for 72 hours at 37oC, with 5% CO2 and 95% air. The metallic devices with the adapted dentin discs were inverted into the wells in such way that the experimental bleaching agents were applied on the oclusal surface of the discs for 30 minutes (2 changes of 15 minutes). The different concentration of H2O2 present in the bleaching agent gave rise to the following experimental groups: G1: 7.5% H2O2; G2: 20% H2O2; G3: 35% H2O2; and G4: 0% H2O2. In control group the dentin discs were not 167 submitted to treatment. The metabolism of MDPC-23 cells which remained attached to the pulpal surface of the discs was evaluated by the methyltetrazolium assay (MTT) and the cell and dentin morphology was assessed under scanning electron microscope (SEM). The statistical analysis of Kruskal-Wallis determined that the experimental bleaching agent caused an intense cytotoxic effects on the MDPC-23 cells. This experimental agent containing H2O2 at 7.5%; 20%; and 35% concentration decreased the cell metabolism by 62.65%; 62.28%; and 65.69%, respectively. The complementary statistical test of Mann-Withney demonstrated no difference between groups 1 and 2. The bleaching agent with... (Complete abstract, click electronic address below). / Mestre

O processo de clarificação do caldo de cana-de-açúcar tem sido alvo de vários trabalhos de pesquisa, no intuito de melhorar a qualidade do açúcar obtido, tanto do ponto de vista de novas tecnologias em equipamentos e processos, quanto a respeito do estudo das propriedades físico-químicas da sacarose durante sua decomposição na clarificação. Os POA (Processos Oxidativos Avançados) têm sido aplicados, em especial, ozonização do caldo, tal qual este projeto propõe estudar uma alternativa ao processo convencional de sulfitação do caldo para a obtenção do açúcar cristal branco, através da utilização do peróxido de hidrogênio como agente de redução de cor ICUMSA do caldo e o impacto na degradação da sacarose em compostos não-cristalizáveis, reduzindo o rendimento industrial. Não há relatos na literatura sobre condições ideais de uso do peróxido de hidrogênio, bem como quais alterações essa tecnologia pode ocasionar no caldo. Por isso, buscaram-se elucidar quais são as melhores condições de trabalho e quais fatores influenciam na sua ação, bem como quais são os seus efeitos sobre o caldo tratado. As melhores condições para o uso do peróxido de hidrogênio são: pH entre 3,0 e 7,0, temperatura entre 40 a 70ºC, peróxido de hidrogênio maior que 600 ppm e dextrana menor que 750 ppm. Pode-se verificar que a maturidade da cana-de-açúcar no corte pode influenciar na ação do peróxido de hidrogênio, uma vez que quanto maior o grau de maturação da cana-de-açúcar, maior quantidade de compostos fenólicos e maior a cor inicial do caldo. A cinética de degradação da cor ICUMSA não apresentou distribuição regular, oscilando em pequenos intervalos de tempo, devido provavelmente à pequena quantidade de peróxido de hidrogênio utilizada nos ensaios. Não houve diminuição visual da cor do caldo quando utilizado doses até 5.000 ppm de H2O2. Com relação à turbidez, não foi possível identificar a influência da peroxidação nos valores. Houve degradação de sacarose quando foi feito o tratamento combinando temperatura elevada (62ºC) com pH ácido (3,8). A rede neural artificial (RNA) mostrou um bom ajuste na maioria dos casos apresentados e indicou a variável temperatura como a que apresentou maior influência na diminuição da absorbância à 420 nm. A segunda variável com maior influência foi o Brix do caldo de cana-de-açúcar. A espectrometria de massa mostrou que a peroxidação, nas condições reacionais avaliadas, não foi capaz de reduzir significativamente a cor do caldo, sugerindo que haja uma promoção de sedimentação de algumas impurezas do caldo, o que faz com que haja uma diminuição visual da cor do mesmo, não ocorrendo aparentemente reação química no caldo, quando utilizamos doses de 50.000 ppm. Assim o peróxido de hidrogênio não funcionou como um agente clarificante, nas condições estudadas. / The process of sugarcane juice clarification has been the subject of several research papers in order to improve the quality of sugar obtained both from the point of view of new technologies in equipment and processes , as concerning the study of physico- chemical properties of sucrose during decomposition in clarification . The AOP \'s (Advanced Oxidation Process ) have been applied in particular ozonation of the juice as such this design proposed to study an alternative to conventional process sulphiting of the juice to obtain sugar white crystal through the use of hydrogen peroxide as reduction ICUMSA color of juice and the impact on the degradation of sucrose into non- crystallizable compounds by reducing industrial productivity agent. There are no reports in the literature on optimal conditions of use of hydrogen peroxide as well as the technology changes which may result in the juice. Therefore , we sought to elucidate what are the best working conditions and factors which influence in its action, and what are its effects on the treated juice. The best conditions for the use of hydrogen peroxide are: pH lower than 7.0 or higher than 3.0, temperature greater than 40 °C and below 70 °C, hydrogen peroxide greater than 600 ppm and lower than 750 ppm dextran. We observed that the maturity of the sugarcane cutting can influence the action of hydrogen peroxide, since the more mature sugarcane, a greater number of phenolic compounds are produced and the higher the initial color of the juice. The kinetics of ICUMSA color degradation showed no regular distribution, oscillating at short time intervals, probably due to the small amount of hydrogen peroxide used in the tests. There was no visual color decrease of the juice. Regarding turbidity, it was not possible to identify the influence of peroxidation values . There was sucrose degradation when the treatment was made by combining high temperature (62°C) at acid pH (3.8). The artificial neural network (ANN) showed a good fit in most cases presented and indicated the variable temperature with the highest influence on the absorbance decrease at 420 nm. The second variable with the greatest influence was the Brix of sugarcane juice. Mass spectrometry showed that peroxidation in the reaction conditions evaluated was not able to significantly reduce the sugarcane juice color, suggesting a promotion of sedimentation of some impurities in the juice, hindering a reduction of its visual color, and apparently, there was no chemical reaction in the juice, using rate of hydrogen peroxide of 50,000 ppm. Thus hydrogen peroxide did not work as a clarifying agent, in the studied conditions

Neste estudo avaliamos a ocorrência de fagocitose por parte de alguns tipos celulares presentes na hemolinfa do carrapato bovino B. microplus e a produção de espécies reativas de oxigênio durante a resposta imune. As técnicas empregadas para avaliação da produção de espécies reativas de oxigênio foram luminescência amplificada por luminol, oxidação de fenol vermelho, microscopia de fluorescência e fluorimetria com o corante dihidrorrodamina 123 (DHR). Observamos um aumento da luminescência amplificada por luminol quando hemócitos foram incubados na presença de bactérias Micrococcus luteus ou zimosam ou PMA. Esta luminescência foi inibida por superóxido dismutase (SOD) e por catalase (CAT), enzimas antioxidantes que removem superóxido e peróxido de hidrogênio, respectivamente. LPS não elicitou aumento da luminescência dos hemócitos em relação ao controle. Através da oxidação de fenol vermelho em reação inibida por CAT, verificamos aumento nos níveis de H2O2 produzido pelos hemócitos quando estimulados com PMA e Micrococcus luteus, enquanto não houve aumento quando o estímulo foi LPS, corroborando os resultados da luminescência. Usando microscopia de fluorescência para avaliar a produção de ERO pelos hemócitos, encontramos que cerca de 25% dos hemócitos fluorescem com maior intensidade quando estimulados com zimosam, sendo esta fluorescência inibida por CAT. Através de fluorimetria usando DHR observamos um aumento na intensidade de fluorescência dos hemócitos estimulados com PMA em reação inibida por cisteína, substância redutora que remove peróxido de hidrogênio e peroxinitrito. Nosso conjunto de resultados permitem concluir que os hemócitos do carrapato bovino produzem espécies reativas de oxigênio durante a resposta imune, semelhantemente ao que ocorre em vertebrados e em invertebrados como moluscos, crustáceos e insetos. Este é o primeiro trabalho mostrando produção de ERO pelos hemócitos de aracnídeos. / The phagocytic activity and the reactive oxygen species (ROS) production during immune response of some hemocytes of the cattle tick Boophilus microplus were evaluated in this study. The ROS production was evaluated by luminol amplified luminescence, phenol red oxidation, dyhydrorhodamine (DHR) fluorescence microscopy and fluorimetry. The luminol-amplified luminescence increased when hemocytes were incubated with bacteria (Micrococcus luteus) or zymosam or phorbol 12-miristate 13 acetate (PMA). The superoxide dismutase (SOD) and catalase (CAT), antioxidant enzymes that removes superoxide and hydrogen peroxide, respectively, inhibited this luminescence. Lipopolysaccharide (LPS) did not elicit luminescence of hemocytes in relation to controls. The catalase-inhibittable phenol red oxidation assay also showed an increase in the level of hydrogen peroxide produced by hemocytes stimulated with PMA or Micrococcus luteus. LPS did not stimulate the hemocytes, similarly to the observed by luminescence assay's. We also evaluated ROS production by fluorescence microscopy and we found approximately 25% more fluorescent hemocytes when zymosam was used. This fluorescence was inhibited by catalase. In DHR fluorimetry assay we observed an increase in the intensity of fluorescence in PMA stimulated hemocytes. This fluorescence was inhibited by cystein, a reducing agent that removes hydrogen peroxide and peroxinitrite. We conclude that hemocytes of the tick, like other invertebrate such as mollusks, crustaceans and insects and vertebrate, produce reactive oxygen species during the immune response. This is the first report of reactive oxygen species production by arachnid hemocytes.

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.

No description available.

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.