RESUMEN
The defining trait of obligate anaerobes is that oxygen blocks their growth, yet the underlying mechanisms are unclear. A popular hypothesis was that these microorganisms failed to evolve defences to protect themselves from reactive oxygen species (ROS) such as superoxide and hydrogen peroxide, and that this failure is what prevents their expansion to oxic habitats. However, studies reveal that anaerobes actually wield most of the same defences that aerobes possess, and many of them have the capacity to tolerate substantial levels of oxygen. Therefore, to understand the structures and real-world dynamics of microbial communities, investigators have examined how anaerobes such as Bacteroides, Desulfovibrio, Pyrococcus and Clostridium spp. struggle and cope with oxygen. The hypoxic environments in which these organisms dwell - including the mammalian gut, sulfur vents and deep sediments - experience episodic oxygenation. In this Review, we explore the molecular mechanisms by which oxygen impairs anaerobes and the degree to which bacteria protect their metabolic pathways from it. The emergent view of anaerobiosis is that optimal strategies of anaerobic metabolism depend upon radical chemistry and low-potential metal centres. Such catalytic sites are intrinsically vulnerable to direct poisoning by molecular oxygen and ROS. Observations suggest that anaerobes have evolved tactics that either minimize the extent to which oxygen disrupts their metabolism or restore function shortly after the stress has dissipated.
Asunto(s)
Bacterias Anaerobias/metabolismo , Oxígeno/toxicidad , Especies Reactivas de Oxígeno/toxicidad , Anaerobiosis , Bacterias Anaerobias/crecimiento & desarrollo , Bacteroides/crecimiento & desarrollo , Bacteroides/metabolismo , Clostridium/crecimiento & desarrollo , Clostridium/metabolismo , Desulfovibrio/crecimiento & desarrollo , Desulfovibrio/metabolismo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Oxígeno/metabolismo , Pyrococcus/crecimiento & desarrollo , Pyrococcus/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Superóxidos/toxicidadRESUMEN
Copper and superoxide are used by the phagocytes to kill bacteria. Copper is a host effector encountered by uropathogenic Escherichia coli (UPEC) during urinary tract infection in a non-human primate model, and in humans. UPEC is exposed to higher levels of copper in the gut prior to entering the urinary tract. Effects of pre-exposure to copper on bacterial killing by superoxide has not been reported. We hypothesized that copper-replete E. coli is more sensitive to killing by superoxide in vitro, and in activated macrophages. We utilized wild-type UPEC strain CFT073, and its isogenic mutants lacking copper efflux systems, superoxide dismutases (SODs), regulators of a superoxide dismutase, and complemented mutants to address this question. Surprisingly, our results reveal that copper protects UPEC against killing by superoxide in vitro. This copper-dependent protection was amplified in the mutants lacking copper efflux systems. Increased levels of copper and manganese were detected in UPEC exposed to sublethal concentration of copper. Copper activated the transcription of sodA in a SoxR- and SoxS-dependent manner resulting in enhanced levels of SodA activity. Importantly, pre-exposure to copper increased the survival of UPEC within RAW264.7 and bone marrow-derived murine macrophages. Loss of SodA, but not SodB or SodC, in UPEC obliterated copper-dependent protection from superoxide in vitro, and from killing within macrophages. Collectively, our results suggest a model in which sublethal levels of copper trigger the activation of SodA and SodC through independent mechanisms that converge to promote the survival of UPEC from killing by superoxide. A major implication of our findings is that bacteria colonizing copper-rich milieus are primed for efficient detoxification of superoxide.
Asunto(s)
Cobre/farmacología , Infecciones por Escherichia coli/tratamiento farmacológico , Interacciones Huésped-Patógeno/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Superóxidos/toxicidad , Infecciones Urinarias/tratamiento farmacológico , Escherichia coli Uropatógena/efectos de los fármacos , Animales , Infecciones por Escherichia coli/inducido químicamente , Infecciones por Escherichia coli/microbiología , Femenino , Regulación Bacteriana de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Superóxido Dismutasa/genética , Infecciones Urinarias/inducido químicamente , Infecciones Urinarias/microbiologíaRESUMEN
Reactive oxygen species (ROS) play important roles in aging, inflammation, and cancer. Mitochondria are an important source of ROS; however, the spatiotemporal ROS events underlying oxidative cellular damage from dysfunctional mitochondria remain unresolved. To this end, we have developed and validated a chemoptogenetic approach that uses a mitochondrially targeted fluorogen-activating peptide (Mito-FAP) to deliver a photosensitizer MG-2I dye exclusively to this organelle. Light-mediated activation (660 nm) of the Mito-FAP-MG-2I complex led to a rapid loss of mitochondrial respiration, decreased electron transport chain complex activity, and mitochondrial fragmentation. Importantly, one round of singlet oxygen produced a persistent secondary wave of mitochondrial superoxide and hydrogen peroxide lasting for over 48 h after the initial insult. By following ROS intermediates, we were able to detect hydrogen peroxide in the nucleus through ratiometric analysis of the oxidation of nuclear cysteine residues. Despite mitochondrial DNA (mtDNA) damage and nuclear oxidative stress induced by dysfunctional mitochondria, there was a lack of gross nuclear DNA strand breaks and apoptosis. Targeted telomere analysis revealed fragile telomeres and telomere loss as well as 53BP1-positive telomere dysfunction-induced foci (TIFs), indicating that DNA double-strand breaks occurred exclusively in telomeres as a direct consequence of mitochondrial dysfunction. These telomere defects activated ataxia-telangiectasia mutated (ATM)-mediated DNA damage repair signaling. Furthermore, ATM inhibition exacerbated the Mito-FAP-induced mitochondrial dysfunction and sensitized cells to apoptotic cell death. This profound sensitivity of telomeres through hydrogen peroxide induced by dysregulated mitochondria reveals a crucial mechanism of telomere-mitochondria communication underlying the pathophysiological role of mitochondrial ROS in human diseases.
Asunto(s)
Mitocondrias/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Telómero/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , ADN Mitocondrial/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Potenciales de la Membrana , Enfermedades Mitocondriales/metabolismo , Estrés Oxidativo , Oxígeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/toxicidad , Transducción de Señal , Superóxidos/metabolismo , Superóxidos/toxicidad , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Clinically active drugs for the treatment of acute pain have their prescription limited due to the significant side effects they induce. An increase in reactive oxygen species (ROS) has been linked to several conditions, including inflammation and pain processing. Therefore, new or repurposed drugs with the ability of reducing ROS-triggered responses are promising candidates for analgesic drugs. Vinpocetine is a clinically used nootropic drug with antioxidant, anti-inflammatory, and analgesic properties. However, the effects of vinpocetine have not been investigated in a model with a direct relationship between ROS, inflammation, and pain. Based on that, we aimed to investigate the effects of vinpocetine in a model of superoxide anion-induced pain and inflammation using potassium superoxide (KO2) as a superoxide anion donor to trigger inflammation and pain. In the KO2 model, vinpocetine dose-dependently reduced pain-like behaviors (spontaneous pain and hyperalgesia), paw edema, and neutrophil and mononuclear cell recruitment to the paw skin (assessed by H&E staining, fluorescence, and enzymatic assays) and to the peritoneal cavity. Vinpocetine also restored tissue endogenous antioxidant ability and Nrf2 and Ho-1 mRNA expression and reduced superoxide anion production and gp91phox mRNA expression. We also observed the inhibition of IκBα degradation by vinpocetine, which demonstrates a reduction in the activation of NF-κB explaining the diminished production of IL-33, IL-1ß, and TNF-α. Collectively, our data show that vinpocetine alleviates pain and inflammation induced by KO2, which is a mouse model with a direct role of ROS in triggering pain and other inflammatory phenomena. Thus, the results suggest the repurposing of vinpocetine as an anti-inflammatory and analgesic drug.
Asunto(s)
Analgésicos/uso terapéutico , Antiinflamatorios/uso terapéutico , Reposicionamiento de Medicamentos/métodos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Nootrópicos/uso terapéutico , Superóxidos/toxicidad , Alcaloides de la Vinca/uso terapéutico , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Edema/tratamiento farmacológico , Edema/metabolismo , Hemo-Oxigenasa 1 , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Trypanosoma cruzi, the causative agent of Chagas disease (CD), contains exclusively Fe-dependent superoxide dismutases (Fe-SODs). During T. cruzi invasion to macrophages, superoxide radical (O2â¢-) is produced at the phagosomal compartment toward the internalized parasite via NOX-2 (gp91-phox) activation. In this work, T. cruzi cytosolic Fe-SODB overexpressers (pRIBOTEX-Fe-SODB) exhibited higher resistance to macrophage-dependent killing and enhanced intracellular proliferation compared with wild-type (WT) parasites. The higher infectivity of Fe-SODB overexpressers compared with WT parasites was lost in gp91-phox-/- macrophages, underscoring the role of O2â¢- in parasite killing. Herein, we studied the entrance of O2â¢- and its protonated form, perhydroxyl radical [(HO2â¢); pKa = 4.8], to T. cruzi at the phagosome compartment. At the acidic pH values of the phagosome lumen (pH 5.3 ± 0.1), high steady-state concentrations of O2â¢- and HO2⢠were estimated (â¼28 and 8 µM, respectively). Phagosomal acidification was crucial for O2â¢- permeation, because inhibition of the macrophage H+-ATPase proton pump significantly decreased O2â¢- detection in the internalized parasite. Importantly, O2â¢- detection, aconitase inactivation, and peroxynitrite generation were lower in Fe-SODB than in WT parasites exposed to external fluxes of O2â¢- or during macrophage infections. Other mechanisms of O2â¢- entrance participate at neutral pH values, because the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid decreased O2â¢- detection. Finally, parasitemia and tissue parasite burden in mice were higher in Fe-SODB-overexpressing parasites, supporting the role of the cytosolic O2â¢--catabolizing enzyme as a virulence factor for CD.
Asunto(s)
Citosol/enzimología , Macrófagos/metabolismo , Superóxido Dismutasa/metabolismo , Superóxidos/toxicidad , Trypanosoma cruzi/enzimología , Animales , Enfermedad de Chagas/parasitología , Regulación Enzimológica de la Expresión Génica , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Consumo de Oxígeno , Ácido Peroxinitroso/metabolismo , Fagosomas , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/patogenicidad , VirulenciaRESUMEN
Fifty-seven compounds were purified from the stems of Tinospora sinensis, including three new compounds characterized as a lignan (1), a pyrrole alkaloid (11), and a benzenoid (17), respectively. Their structures were elucidated and established by various spectroscopic and spectrometric analytical methods. Among the isolates, fifteen compounds were examined for their anti-inflammatory potential in vitro. The results showed that several compounds displayed moderate inhibition of N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release.
Asunto(s)
Alcaloides/farmacología , Lignanos/farmacología , Elastasa Pancreática/metabolismo , Pirroles/farmacología , Alcaloides/química , Citocalasina B/antagonistas & inhibidores , Citocalasina B/toxicidad , Humanos , Lignanos/química , Estructura Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Elastasa Pancreática/biosíntesis , Elastasa Pancreática/efectos de los fármacos , Tallos de la Planta/química , Pirroles/química , Superóxidos/antagonistas & inhibidores , Superóxidos/toxicidad , Tinospora/químicaRESUMEN
Oxidative stress (OS) is characterized by an unbalance between increased levels of reactive oxygen species (ROS) and/or impaired antioxidant protection. In this context, the composition of seminal plasma (SP) plays a key role in protecting sperm against OS. However, reproductive biotechnologies applied to dogs recommend the removal of SP. Thus, antioxidant therapy may be an important alternative when applying biotechniques such as semen cryopreservation in this specie. However, in order to be efficient, the choice of the ideal antioxidant in each condition is essential since each ROS is preferably neutralized by different antioxidant systems. Therefore, this study aims to evaluate the susceptibility of canine spermatozoa to different oxidative challenges (superoxide anion [O2-], hydrogen peroxide [H2O2], hydroxyl radical [OH-] and malondialdehyde [MDA]) in the present or absence of SP. We used ejaculates of eight dogs and submitted to induce oxidative challenges (with or without SP). After incubations, samples were evaluated for the susceptibility to lipid peroxidation, motility, mitochondrial activity and function, DNA integrity, plasma membrane and acrosome integrity. Sperm with SP had mitochondrial function preserved against ROS. However, in the absence of SP, H2O2 reduced mitochondrial membrane potential. In addition, regardless on SP, H2O2 was deleterious to sperm kinetics and plasma/acrosomal membranes. Incubation with OH- reduced mitochondrial activity and increased DNA fragmentation also independent on the absence of presence of SP. Furthermore, samples with SP were more resistant to lipid peroxidation (i.e., decreased concentration of TBARS). In conclusion, H2O2 and OH- appears to be the most deleterious ROS to dog sperm and SP protects the spermatozoa against mitochondrial injuries and lipid peroxidation.
Asunto(s)
Peróxido de Hidrógeno/toxicidad , Radical Hidroxilo/toxicidad , Semen/fisiología , Espermatozoides/efectos de los fármacos , Superóxidos/toxicidad , Animales , Perros , Masculino , Estrés Oxidativo , Especies Reactivas de Oxígeno , Análisis de Semen/veterinaria , Motilidad Espermática/efectos de los fármacosRESUMEN
High-dose ascorbic acid (AsA) treatment, known as pharmacological AsA, has been shown to exert carcinostatic effects in many types of cancer cells and in vivo tumour models. Although pharmacological AsA has potential as a complementary and alternative medicine for anticancer treatment, its effects on human tongue carcinoma have not yet been elucidated. In this study, we investigated the effect of AsA treatment on human tongue carcinoma HSC-4 cells compared with non-tumourigenic tongue epithelial dysplastic oral keratinocyte (DOK) cells. Our results show that treatment with 1 and 3 mM of AsA for 60 min preferentially inhibits the growth of human tongue carcinoma HSC-4 over DOK cells. Furthermore, AsA-induced effects were accompanied by increased intracellular oxidative stress and were repressed by treatment with a hydrogen peroxide (H2O2) scavenger catalase and a superoxide anion radical (O2-) scavenger, tempol. Time-lapse observation and thymidine analog EdU incorporation revealed that AsA treatment induces not only cell death but also suppression of DNA synthesis and cell growth. Moreover, the growth arrest was accompanied by abnormal cellular morphologies whereby cells extended dendrite-like pseudopodia. Taken together, our results demonstrate that AsA treatment can induce carcinostatic effects through induction of cell death, growth arrest, and morphological changes mediated by H2O2 and O2- generation. These findings suggest that high-dose AsA treatment represents an effective treatment for tongue cancer as well as for other types of cancer cells.
Asunto(s)
Ácido Ascórbico/administración & dosificación , Carcinoma/tratamiento farmacológico , Depuradores de Radicales Libres/metabolismo , Neoplasias de la Lengua/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Ácido Ascórbico/efectos adversos , Carcinoma/metabolismo , Carcinoma/patología , Catalasa/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Peróxido de Hidrógeno/toxicidad , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Superóxidos/toxicidad , Neoplasias de la Lengua/metabolismo , Neoplasias de la Lengua/patologíaRESUMEN
The aim of this study was to investigate the photolysis mechanism of ketoprofen (KET) under simulated sunlight. The results demonstrated that the photolysis of KET aligned well with pseudo first-order kinetics. Radical scavenging experiments and dissolved oxygen experiments revealed that the superoxide anion radical (O2Ë-) played a primary role in the photolytic process in pure water. Bicarbonate slightly increased the photodegradation of KET through generating carbonate radicals, while DOM inhibited the photolysis via both attenuating light and competing radicals. Moreover, Zhujiang river water inhibited KET phototransformation. Potential KET degradation pathways were proposed based on the identification of products using LC/MS/MS and GC/MS techniques. The theoretical prediction of reaction sites was derived from Frontier Electron Densities (FEDs), which primarily involved the KET decarboxylation reaction. The ecotoxicity of the treated solutions was evaluated by employing Daphnia magna and V. fischeri as biological indicators. Ecotoxicity was also hypothetically predicted through the "ecological structure-activity relationship" (ECOSAR) program, which revealed that toxic products might be generated during the photolysis process.
Asunto(s)
Cetoprofeno/toxicidad , Modelos Teóricos , Fotólisis , Luz Solar , Superóxidos/toxicidad , Contaminantes Químicos del Agua/toxicidad , Aliivibrio fischeri/efectos de los fármacos , Animales , Chlorophyta/efectos de los fármacos , Daphnia/efectos de los fármacos , Peces/crecimiento & desarrollo , Cetoprofeno/análisis , Cetoprofeno/efectos de la radiación , Cinética , Valor Predictivo de las Pruebas , Superóxidos/análisis , Espectrometría de Masas en Tándem , Pruebas de Toxicidad Aguda , Pruebas de Toxicidad Crónica , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/efectos de la radiaciónRESUMEN
Landomycin E (LE) is an angucycline antibiotic produced by Streptomyces globisporus. Previously, we have shown a broad anticancer activity of LE which is, in contrast to the structurally related and clinically used anthracycline doxorubicin (Dx), only mildly affected by multidrug resistance-mediated drug efflux. In the present study, cellular and molecular mechanisms underlying the anticancer activity of landomycin E towards Jurkat T-cell leukemia cells were dissected focusing on the involvement of radical oxygen species (ROS). LE-induced apoptosis distinctly differed in several aspects from the one induced by Dx. Rapid generation of both extracellular and cell-derived hydrogen peroxide already at one hour drug exposure was observed in case of LE but not found before 24h for Dx. In contrast, Dx but not LE induced production of superoxide radicals. Mitochondrial damage, as revealed by JC-1 staining, was weakly enhanced already at 3h LE treatment and increased significantly with time. Accordingly, activation of the intrinsic apoptosis pathway initiator caspase-9 was not detectable before 12h exposure. In contrast, cleavage of the down-stream caspase substrate PARP-1 was clearly induced already at the three hour time point. Out of all caspases tested, only activation of effector caspase-7 was induced at this early time points paralleling the LE-induced oxidative burst. Accordingly, this massive cleavage of caspase-7 at early time points was inhibitable by the radical scavenger N-acetylcysteine (NAC). Additionally, only simultaneous inhibition of multiple caspases reduced LE-induced apoptosis. Specific scavengers of both H2O2 and OH⢠effectively decreased LE-induced ROS production, but only partially inhibited LE-induced apoptosis. In contrast, NAC efficiently blocked both parameters. Summarizing, rapid H2O2 generation and a complex caspase activation pattern contribute to the antileukemic effects of LE. As superoxide generation is considered as the main cardiotoxic mechanism of Dx, LE might represent a better tolerable drug candidate for further (pre)clinical development.
Asunto(s)
Aminoglicósidos/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Células Jurkat/metabolismo , Leucemia/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Acetilcisteína/administración & dosificación , Apoptosis/efectos de los fármacos , Caspasa 7/metabolismo , Caspasa 9/metabolismo , Doxorrubicina/administración & dosificación , Humanos , Peróxido de Hidrógeno/toxicidad , Células Jurkat/efectos de los fármacos , Células Jurkat/patología , Leucemia/metabolismo , Leucemia/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Streptomyces/química , Superóxidos/toxicidadRESUMEN
Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target.
Asunto(s)
Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidad , Proteínas Fúngicas/química , Estrés Oxidativo , Peroxirredoxinas/química , Animales , Aspergilosis/microbiología , Cristalografía por Rayos X , Femenino , Eliminación de Gen , Cinética , Ratones , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxidación-Reducción , Peroxidasa/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Superóxidos/toxicidad , VirulenciaRESUMEN
BACKGROUND: Misfolded α-synuclein (α-Syn) aggregates participate in the pathogenesis of synucleinopathies, such as Parkinson's disease. Whereas much is known about how the various domains within full-length α-Syn (FL-α-Syn) contribute to the formation of α-Syn aggregates and therefore to their neurotoxicity, little is known about whether the individual peptides that can be generated from α-syn, possibly as intermediate metabolites during degradation of misfolded α-Syn aggregates, are neurotoxic themselves. METHODS: A series of synthesized α-Syn peptides, corresponding to the locus in FL-α-Syn containing alanine 30, substitution of which with a proline causes a familial form of Parkinson's disease, were examined for their capacity of inducing release of microglial superoxide. The neurotoxicity of these peptides was measured according to their influence on the ability of neuroglial cultures deficient in gp91 (phox) , the catalytic unit of NADPH oxidase (Nox2), or wild-type cultures to take up (3)H-labeled dopamine and on the number of tyrosine hydroxylase-staining-positive neurons. Western blots and confocal images were utilized to analyze membrane translocation of p47 (phox) and p67 (phox) , phosphorylation of p47 (phox) and Erk1/2 kinase, and binding of α-Syn peptides to gp91 (phox) . Activation of brain microglia in mice injected with α-Syn peptides was demonstrated by immunostaining for major histocompatibility complex (MHC)-II along with qPCR for Iba-1 and MHC-II. RESULTS: We report α-Syn (29-40) as a specific peptide capable of activating microglial Nox2 to produce superoxide and cause dopaminergic neuronal damage. Administered to mice, this peptide also activated brain microglia to increase expression of MHC-II and Iba-1 and stimulated oxidation reaction. Exploring the underlying mechanisms showed that α-Syn (29-40) peptide triggered Nox2 to generate extracellular superoxide and its metabolite H2O2 by binding to the catalytic unit gp91 (phox) of Nox2; diffusing into cytosol, H2O2 activated Erk1/2 kinase to phosphorylate p47 (phox) and p67 (phox) and further activated Nox2, establishing a positive feedback loop to amplify the Nox2-mediated response. CONCLUSIONS: Collectively, our study suggests novel information regarding how α-Syn causes neuronal injury, possibly including mechanisms involving abnormal metabolites of α-Syn aggregates.
Asunto(s)
Neuronas Dopaminérgicas/efectos de los fármacos , Neuroglía/efectos de los fármacos , Superóxidos/metabolismo , Superóxidos/toxicidad , alfa-Sinucleína/farmacología , Animales , Animales Recién Nacidos , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Embrión de Mamíferos , Antígenos de Histocompatibilidad Clase II/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Neuroglía/metabolismo , Fragmentos de Péptidos/farmacología , Transporte de Proteínas/efectos de los fármacos , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Tirosina 3-Monooxigenasa/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
Extensive clinical investigations, in hand with biochemical and biophysical research, have associated brain iron accumulation with the pathogenesis of the amyotrophic lateral sclerosis (ALS) disease. The origin of iron is still not identified, but it is proposed that it forms redox active complexes that can participate in the Fenton reaction generating the toxic hydroxyl radical. In this paper, the state of iron in the neural tissues isolated from SOD1(G93A) transgenic rats was investigated using low temperature EPR spectroscopy and is compared with that of nontransgenic (NTg) littermates. The results showed that iron in neural tissues is present as high- and low-spin, heme and non-heme iron. It appears that the SOD1(G93A) rat neural tissues were most likely exposed in vivo to higher amounts of reactive oxygen species when compared to the corresponding NTg tissues, as they showed increased oxidized [3Fe-4S](1+) cluster content relative to [4Fe-4S](1+). Also, the activity of cytochrome c oxidase (CcO) was found to be reduced in these tissues, which may be associated with the observed uncoupling of heme a3 Fe and CuB in the O2-reduction site of the enzyme. Furthermore, the SOD1(G93A) rat spinal cords and brainstems contained more manganese, presumably from MnSOD, than those of NTg rats. The addition of potassium superoxide to all neural tissues ex vivo, led to the [4Fe-4S]â[3Fe-4S] cluster conversion and concurrent release of Fe. These results suggest that the superoxide anion may be the cause of the observed oxidative damage to SOD1(G93A) rat neural tissues and that the iron-sulfur clusters may be the source of poorly liganded redox active iron implicated in ALS pathogenesis. Low temperature EPR spectroscopy appears to be a valuable tool in assessing the role of metals in neurodegenerative diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Hierro/metabolismo , Estrés Oxidativo/genética , Superóxido Dismutasa-1/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Espectroscopía de Resonancia por Spin del Electrón , Complejo IV de Transporte de Electrones/metabolismo , Hemo/química , Hemo/genética , Humanos , Hierro/química , Oxidación-Reducción , Ratas , Ratas Transgénicas , Azufre/química , Superóxido Dismutasa-1/metabolismo , Superóxidos/toxicidadRESUMEN
We evaluated the effect of pyrrolidine dithiocarbamate (PDTC) in superoxide anion-induced inflammatory pain. Male Swiss mice were treated with PDTC and stimulated with an intraplantar or intraperitoneal injection of potassium superoxide, a superoxide anion donor. Subcutaneous PDTC treatment attenuated mechanical hyperalgesia, thermal hyperalgesia, paw oedema and leukocyte recruitment (neutrophils and macrophages). Intraplantar injection of superoxide anion activated NF-κB and increased cytokine production (IL-1ß, TNF-α and IL-10) and oxidative stress (nitrite and lipid peroxidation levels) at the primary inflammatory foci and in the spinal cord (L4-L6). PDTC treatment inhibited superoxide anion-induced NF-κB activation, cytokine production and oxidative stress in the paw and spinal cord. Furthermore, intrathecal administration of PDTC successfully inhibited superoxide anion-induced mechanical hyperalgesia, thermal hyperalgesia and inflammatory response in peripheral foci (paw). These results suggest that peripheral stimulus with superoxide anion activates the local and spinal cord oxidative- and NF-κB-dependent inflammatory nociceptive mechanisms. PDTC targets these events, therefore, inhibiting superoxide anion-induced inflammatory pain in mice.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , FN-kappa B/metabolismo , Estrés Oxidativo/efectos de los fármacos , Dolor/metabolismo , Pirrolidinas/administración & dosificación , Médula Espinal/metabolismo , Tiocarbamatos/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Edema/metabolismo , Edema/prevención & control , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/prevención & control , Masculino , Ratones , FN-kappa B/antagonistas & inhibidores , Estrés Oxidativo/fisiología , Dolor/inducido químicamente , Dolor/prevención & control , Médula Espinal/efectos de los fármacos , Superóxidos/toxicidadRESUMEN
A 16 h exposure of U937 cells to 2.5 µM arsenite promotes superoxide (O2(·-)) formation and inhibition of the activity of aconitase, a O2(·-) sensitive enzyme. Both responses were abolished by the complex I inhibitor rotenone, or by the respiration-deficient phenotype. Interestingly, a similar suppressive effect was mediated by a short term pre-exposure to a low concentration of L-ascorbic acid (AA), previously shown to be actively taken up by the cells and by their mitochondria. The mitochondrial origin of O2(·-) was confirmed by fluorescence microscopy studies, whereas different approaches failed to detect a contribution of NADPH oxidase. Under similar conditions, arsenite induced autophagy as well as a decline in mitochondrial membrane potential resulting in delayed (48 h) apoptosis. Importantly, all these events turned out to be sensitive to treatments associated with prevention of O2(·-) formation, including AA, and were only partially blunted by inhibitors of autophagy. As a final note, the toxic effects mediated by O2(·-)were entirely dependent on its conversion to H2O2. AA-sensitive mitochondrial O2(·-) formation is therefore involved in autophagy and apoptosis induced by arsenite in U937 cells, although part of the lethal response appears mediated by an autophagy-independent mechanism.
Asunto(s)
Ácido Ascórbico/administración & dosificación , Autofagia/efectos de los fármacos , Mitocondrias/metabolismo , Superóxidos/metabolismo , Aconitato Hidratasa/antagonistas & inhibidores , Aconitato Hidratasa/metabolismo , Apoptosis/efectos de los fármacos , Arsenitos/toxicidad , Ácido Ascórbico/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/patología , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Rotenona/administración & dosificación , Superóxidos/toxicidad , Células U937RESUMEN
OBJECTIVE: This study aimed at evaluating the activity of curcumin in superoxide anion-induced pain-like behavior and leukocyte recruitment in mice. TREATMENT: Administration of curcumin 10 mg/kg subcutaneously 1 h before stimulus. METHODS: KO2 was used as superoxide anion donor. Overt pain-like behaviors were determined by the number of abdominal writhings, paw flinches and time spent licking the paw. Mechanical and thermal hyperalgesia were determined using an electronic anesthesiometer and hot plate, respectively. Cytokine concentration and NF-κB activity were determined by ELISA, antioxidant effect by nitrobluetretrazolium assay and ABTS radical scavenging ability. Myeloperoxidase activity was measured by colorimetric assay. The Nrf2, heme oxygenase-1 (HO-1) and gp91phox mRNA expression was determined by quantitative PCR. Data were analyzed by ANOVA followed by Tukey's post hoc and considered significant when p<0.05. RESULTS: Curcumin inhibited superoxide anion-induced overt pain-like behaviors as well as mechanical and thermal hyperalgesia. Curcumin also inhibited superoxide anion-induced leukocyte recruitment in the peritoneal cavity and in the paw skin inhibited myeloperoxidase activity, oxidative stress, IL-1ß and TNF-α production and NF-κB activation as well as enhanced IL-10 production, and HO-1 and Nrf2 mRNA expression. CONCLUSION: Curcumin inhibits superoxide anion-induced inflammatory pain-like behaviors and leukocyte recruitment by targeting inflammatory molecules and oxidative stress; and inducing antioxidant and anti-inflammatory pathways.
Asunto(s)
Antioxidantes/farmacología , Curcumina/farmacología , Leucocitos/efectos de los fármacos , Factor 2 Relacionado con NF-E2/biosíntesis , FN-kappa B/antagonistas & inhibidores , Oxidantes/toxicidad , Dolor/psicología , Superóxidos/antagonistas & inhibidores , Superóxidos/toxicidad , Animales , Antioxidantes/administración & dosificación , Curcumina/administración & dosificación , Citocinas/biosíntesis , Hemo-Oxigenasa 1/biosíntesis , Hiperalgesia/inducido químicamente , Hiperalgesia/prevención & control , Inyecciones Subcutáneas , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , NADPH Oxidasa 2 , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/genética , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/efectos de los fármacos , Óxidos/química , Dolor/inducido químicamente , Compuestos de Potasio/química , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Our previous studies suggest that reactive oxygen species (ROS) scavengers have analgesic effect on neuropathic pain through spinal mechanisms in the rat. The studies suggest that superoxide in spinal cord is one of important mediators of persistent pain. To test the hypothesis that increase of superoxide-derived intermediates leads to central sensitization and pain, the effects of an intrathecal injection of chemical ROS donors releasing either OH(â), OCl(-), or H2O2 were examined on pain behaviors. Following treatment with t-BOOH (OH(â) donor), dorsal horn neuron responses to mechanical stimuli in normal rats and the changes of neuronal excitability were explored on substantia gelatinosa (SG) neurons using whole-cell patch clamping recordings. Intrathecal administration of t-BOOH or NaOCl (OCl(-) donor), but not H2O2, significantly decreased mechanical thresholds of hind paws. The responses of wide dynamic range neurons to mechanical stimuli increased after a local application of t-BOOH. The t-BOOH increased the frequency and the amplitude of excitatory postsynaptic potentials, depolarized membrane potential in SG neurons, and increased the frequency of action potentials evoked by depolarizing current pulses. These results suggest that elevated ROS, especially OH(â), in the spinal cord sensitized dorsal horn neurons and produced hyperalgesia in normal rats.
Asunto(s)
Hiperalgesia/inducido químicamente , Oxidantes/toxicidad , Células del Asta Posterior/efectos de los fármacos , Especies Reactivas de Oxígeno/farmacología , Superóxidos/toxicidad , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Radical Hidroxilo , Inyecciones Espinales , Masculino , Técnicas de Placa-Clamp , Estimulación Física , Ratas , Ratas Sprague-Dawley , Hipoclorito de Sodio/toxicidad , Sustancia Gelatinosa/efectos de los fármacosRESUMEN
We have previously shown that mitochondria-targeted vitamin E (Mito-Vit-E), a mtROS specific antioxidant, improves cardiac performance and attenuates inflammation in a pneumonia-related sepsis model. In this study, we applied the same approaches to decipher the signaling pathway(s) of mtROS-dependent cardiac inflammation after sepsis. Sepsis was induced in Sprague Dawley rats by intratracheal injection of S. pneumoniae. Mito-Vit-E, vitamin E or vehicle was administered 30 minutes later. In myocardium 24 hours post-inoculation, Mito-Vit-E, but not vitamin E, significantly protected mtDNA integrity and decreased mtDNA damage. Mito-Vit-E alleviated sepsis-induced reduction in mitochondria-localized DNA repair enzymes including DNA polymerase γ, AP endonuclease, 8-oxoguanine glycosylase, and uracil-DNA glycosylase. Mito-Vit-E dramatically improved metabolism and membrane integrity in mitochondria, suppressed leakage of mtDNA into the cytoplasm, inhibited up-regulation of Toll-like receptor 9 (TLR9) pathway factors MYD88 and RAGE, and limited RAGE interaction with its ligand TFAM in septic hearts. Mito-Vit-E also deactivated NF-κB and caspase 1, reduced expression of the essential inflammasome component ASC, and decreased inflammatory cytokine IL-1ß. In vitro, both Mito-Vit-E and TLR9 inhibitor OND-I suppressed LPS-induced up-regulation in MYD88, RAGE, ASC, active caspase 1, and IL-1ß in cardiomyocytes. Since free mtDNA escaped from damaged mitochondria function as a type of DAMPs to stimulate inflammation through TLR9, these data together suggest that sepsis-induced cardiac inflammation is mediated, at least partially, through mtDNA-TLR9-RAGE. At last, Mito-Vit-E reduced the circulation of myocardial injury marker troponin-I, diminished apoptosis and amended morphology in septic hearts, suggesting that mitochondria-targeted antioxidants are a potential cardioprotective approach for sepsis.
Asunto(s)
ADN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Sepsis/etiología , Streptococcus pneumoniae/patogenicidad , Animales , Antioxidantes/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Interleucina-1beta/análisis , Lipopolisacáridos/toxicidad , Masculino , Mitocondrias/efectos de los fármacos , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/toxicidad , Sepsis/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismo , Superóxidos/toxicidad , Receptor Toll-Like 9/antagonistas & inhibidores , Receptor Toll-Like 9/metabolismo , Troponina/análisis , Vitamina E/farmacologíaRESUMEN
Cobalt (Co) is a required divalent metal used in the production of metal alloys, batteries, and pigments and is a component of vitamin B12. Excessive uptake of Co is neurotoxic causing temporary or permanent hearing loss; however, its ototoxic effects on the sensory hair cells, neurons, and support cells in the cochlea are poorly understood. Accordingly, we treated postnatal day 3 rat cochlear organotypic cultures with various doses and durations of CoCl2 and quantified the damage to the hair cells, peripheral auditory nerve fibers, and spiral ganglion neurons (SGN). Five-day treatment with 250 µM CoCl2 caused extensive damage to hair cells and neurons which increased with dose and treatment duration. CoCl2 caused greater damage to outer hair cells than inner hair cells; damage was greatest in the base of the cochlea and decreased towards the base. CoCl2 increased expression of superoxide radical in hair cells and SGNs and SGN loss was characterized by nuclear condensation and fragmentation, morphological features of apoptosis. CoCl2 treatment increased the expression of caspase-3 indicative of caspase-mediated programmed cell death. These results identify hair cells and spiral ganglion neurons as the main targets of Co ototoxicity in vitro and implicate the superoxide radical as a trigger of caspase-mediated ototoxicity.
Asunto(s)
Cobalto/toxicidad , Cóclea/efectos de los fármacos , Cóclea/fisiopatología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasa 3/metabolismo , Cóclea/patología , Relación Dosis-Respuesta a Droga , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/patología , Neuronas Aferentes/fisiología , Técnicas de Cultivo de Órganos , Fotomicrografía , Ratas Sprague-Dawley , Superóxidos/toxicidad , Factores de TiempoRESUMEN
Chemotherapy-induced cognitive impairment (CICI) is a quality of life-altering consequence of chemotherapy experienced by a large percentage of cancer survivors. Approximately half of FDA-approved anti-cancer drugs are known to produce ROS. Doxorubicin (Dox), a prototypical ROS-generating chemotherapeutic agent, generates superoxide (O2(-)â¢) via redox cycling. Our group previously demonstrated that Dox, which does not cross the BBB, induced oxidative damage to plasma proteins leading to TNF-α elevation in the periphery and, subsequently, in brain following cancer chemotherapy. We hypothesize that such processes play a central role in CICI. The current study tested the notion that O2(-)⢠is involved and likely responsible for Dox-induced plasma protein oxidation and TNF-α release. Addition of O2(-)⢠as the potassium salt (KO2) to plasma resulted in significantly increased oxidative damage to proteins, indexed by protein carbonyl (PC) and protein-bound HNE levels. We then adapted this protocol for use in cell culture. Incubation of J774A.1 macrophage culture using this KO2-18crown6 protocol with 1 and 10 µM KO2 resulted in dramatically increased levels of TNF-α produced. These findings, together with our prior results, provide strong evidence that O2(-)⢠and its resulting reactive species are critically involved in Dox-induced plasma protein oxidation and TNF-α release.