RESUMO
Although sphingomyelin (SM) is the most abundant phospholipid in the plasma, next to phosphatidylcholine (PC), its physiological function in plasma is unclear. Here we employed plasma from various genetic models of mice which naturally differ in their plasma SM/PC ratios, to study the role of SM as a modulator of LCAT, the enzyme responsible for HDL maturation and the synthesis of cholesteryl esters (CE) in normal plasma. Serine palmitoyltransferase deficient mice, and SM synthase deficient mice, both of which have below normal SM/PC ratios, showed significantly elevated LCAT activities when assayed with the endogenous substrates. On the other hand, LDL receptor knockout mice, and apo E knockout mice, both of which have high SM/PC ratios, had markedly reduced (-80%) LCAT activities. The LCAT levels in plasma, as assayed with an exogenous substrate, were similar in all groups, except for a 45% decrease in apo E knockout mice. Plasma samples with high SM/PC ratios had lower percentage of 20:4, 22:5, and 22:6 CE all of which are formed by LCAT, and a higher percentage of the atherogenic 18:1 CE which is mainly derived from the action of liver ACAT, showing that in vivo, the contribution of LCAT to plasma CE is reduced while that of liver ACAT is increased. These results show that SM is a physiological modulator of LCAT activity as well as plasma CE composition, and this may contribute to the previously reported pro-atherogenic effect of high plasma SM levels.
Assuntos
Ésteres do Colesterol/metabolismo , Colesterol/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Esfingomielinas/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Colesterol/sangue , Ésteres do Colesterol/sangue , Esterificação , Lipídeos/sangue , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , Fosfatidilcolinas/sangue , Fosfatidilcolinas/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Receptores de LDL/metabolismo , Serina C-Palmitoiltransferase/deficiência , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingomielinas/sangue , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.
Assuntos
Citocromos c/metabolismo , Mutação , Organofosfatos/química , Tirosina/metabolismo , Animais , Apoptose , Cardiolipinas/metabolismo , Caspases/metabolismo , Bovinos , Respiração Celular , Citocromos c/química , Citocromos c/genética , Camundongos , Fosforilação , Ligação Proteica , RatosRESUMO
Glioblastoma multiforme is the most frequent and aggressive primary brain tumor. A strong rationale to identify innovative approaches to treat these tumors is required since treatment failures result in local recurrences and median survivals range from 9 to 12 months. Glioma cells are reported to have less mitochondrial content compared to adjacent normal brain cells. Based on this difference, we suggest a new strategy, utilizing protection of normal brain cells by mitochondria-targeted electron scavengers and antioxidants-nitroxides-thus allowing for the escalation of the radiation doses. In this paper, we report that a conjugate of nitroxide with a hydrophobic cation, triphenyl-phosphonium (TPEY-Tempo), significantly protected brain endothelial cells from γ-irradiation-induced apoptosis while radiosensitizing brain tumor cells. Thus, TPEY-Tempo may be a promising adjunct in the treatment of glioblastoma with the potential to not only prolong survival but also to maintain quality of life and reduce treatment toxicity.
Assuntos
Apoptose/efeitos dos fármacos , Encéfalo/citologia , Mitocôndrias/efeitos da radiação , Fármacos Neuroprotetores/farmacologia , Óxidos de Nitrogênio/química , Compostos Organosselênicos/farmacologia , Apoptose/efeitos da radiação , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/ultraestrutura , Caspase 3/metabolismo , Linhagem Celular Transformada , Óxidos N-Cíclicos/metabolismo , Citocromos c/metabolismo , Relação Dose-Resposta à Radiação , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/efeitos da radiação , Células Endoteliais/ultraestrutura , Raios gama/efeitos adversos , Glioma/patologia , Glioma/ultraestrutura , Humanos , Mitocôndrias/metabolismo , Compostos Orgânicos/metabolismoRESUMO
Cytochrome c (cyt c), a mitochondrial intermembrane electron shuttle between complexes III and IV, can, upon binding with an anionic phospholipid, cardiolipin (CL), act as a peroxidase that catalyzes cardiolipin oxidation. H(2)O(2) was considered as a source of oxidative equivalents for this reaction, which is essential for programmed cell death. Here we report that peroxidase cyt c/CL complexes can utilize free fatty acid hydroperoxides (FFA-OOH) at exceptionally high rates that are approximately 3 orders of magnitude higher than for H(2)O(2). Similarly, peroxidase activity of murine liver mitochondria was high with FFA-OOH. Using EPR spin trapping and LC-MS techniques, we have demonstrated that cyt c/CL complexes split FFA-OOH predominantly via a heterolytic mechanism, yielding hydroxy-fatty acids, whereas H(2)O(2) (and tert-butyl hydroperoxide, t-BuOOH) undergo homolytic cleavage. Computer simulations have revealed that Arg(38) and His(33) are important for the heterolytic mechanism at potential FFA-OOH binding sites of cyt c (but not for H(2)O(2) or t-BuOOH). Regulation of FFA-OOH metabolism may be an important function of cyt c that is associated with elimination of toxic FFA-OOH and synthesis of physiologically active hydroxy-fatty acids in mitochondria.
Assuntos
Antioxidantes/metabolismo , Cardiolipinas/metabolismo , Citocromos c/metabolismo , Ácidos Graxos/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias Hepáticas/enzimologia , Animais , Armoracia/enzimologia , Modelos Moleculares , Murinae , Oxirredução , Ligação ProteicaRESUMO
In mitochondrial apoptosis, the formation of cytochrome c-cardiolipin complex ([CL-cyt c]) with peroxidase properties is an early event in the cascade of reactions that leads to cell death. Herein, we report the synthesis of a new prodrug, (2-hydroxyamino-vinyl)-triphenyl-phosphonium (HVTP), which compartmentalizes exclusively into mitochondria, undergoes a [CL-cyt c]-catalyzed bioactivation to nitric oxide (NO), inhibits peroxidase activity, and protects cells from apoptosis.
Assuntos
Apoptose , Citocromos c/metabolismo , Hidroxilaminas/metabolismo , Mitocôndrias/enzimologia , Doadores de Óxido Nítrico/metabolismo , Óxido Nítrico/metabolismo , Compostos Organofosforados/metabolismo , Peroxidases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/metabolismo , Catálise/efeitos dos fármacos , Compartimento Celular , Citoproteção/efeitos dos fármacos , Dactinomicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Hidroxilaminas/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Oxirredução/efeitos dos fármacos , Peroxidases/antagonistas & inibidores , Fosfatidilserinas/metabolismoRESUMO
Cardiolipin (CL), a unique mitochondrial phospholipid synthesized by CL synthase (CLS), plays important, yet not fully understood, roles in mitochondria-dependent apoptosis. We manipulated CL levels in HeLa cells by knocking down CLS using RNA interference and selected a clone of CL-deficient cells with approximately 45% of its normal content. ESI-MS analysis showed that the CL molecular species were the same in CL-deficient and CL-sufficient cells. CL deficiency did not change mitochondrial functions (membrane potential, reactive oxygen species generation, cellular ATP levels) but conferred resistance to apoptosis induced by actinomycin D (ActD), rotenone, or gamma-irradiation. During ActD-induced apoptosis, decreased CL peroxidation along with suppressed cytochrome (cyt) c release was observed in CL-deficient cells, whereas Bax translocation to mitochondria remained similar to that in CL-sufficient HeLa cells. The amounts of loosely bound cyt c (releasable under high ionic strength conditions) were the same in CL-deficient and CL-sufficient cells. Given that CL peroxidation during apoptosis is catalyzed by CL/cyt c complexes and CL oxidation products are essential for cyt c release from mitochondria, our results suggest that CL deficiency prevents adequate assembly of productive CL/cyt c complexes and CL peroxidation, resulting in increased resistance to apoptosis.
Assuntos
Apoptose/fisiologia , Cardiolipinas/fisiologia , Citocromos c/metabolismo , Peroxidação de Lipídeos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , Imunofluorescência , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , RNA Interferente Pequeno/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Frações SubcelularesRESUMO
Bax/Bak activation and cardiolipin peroxidation are essential for cytochrome c release during apoptosis, yet, the link between them remains elusive. We report that sequence of events after exposure of mouse embryonic fibroblast (MEF) cells to actinomycin D followed the order: Bax translocation-->superoxide production-->cardiolipin peroxidation. Genetic ablation of Bax/Bak inhibited actinomycin D induced superoxide production and cardiolipin peroxidation. Rotenone caused robust superoxide generation but did not trigger cardiolipin peroxidation in Bax/Bak double knockout MEF cells. This suggests that, in addition to participating in ROS generation, Bax/Bak play another specific role in cardiolipin oxidation. In isolated mitochondria, recombinant Bax enhanced succinate induced cardiolipin oxidation and cytochrome c release. Mitochondrial peroxidase activity, likely involved in cardiolipin peroxidation, was enhanced upon incubation with recombinant Bax. Thus, cardiolipin peroxidation may be causatively and time-dependently related to Bax/Bak effects on ROS generation and peroxidase activation of cytochrome c.
Assuntos
Apoptose , Cardiolipinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Citocromos c/metabolismo , Dactinomicina/farmacologia , Fibroblastos , Camundongos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Peroxidases/metabolismo , Transporte Proteico , Rotenona/farmacologia , Proteína X Associada a bcl-2/genéticaRESUMO
PURPOSE: To evaluate the in vitro radioprotective effect of the mitochondria-targeted hemigramicidin S-conjugated 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (hemi-GS-TEMPO) 5-125 in gamma-irradiated mouse embryonic cells and adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells BEAS-2B and explore the mechanisms involved in its radioprotective effect. METHODS AND MATERIALS: Cells were incubated with 5-125 before (10 minutes) or after (1 hour) gamma-irradiation. Superoxide generation was determined by using dihydroethidium assay, and lipid oxidation was quantitated by using a fluorescence high-performance liquid chromatography-based Amplex Red assay. Apoptosis was characterized by evaluating the accumulation of cytochrome c in the cytosol and externalization of phosphatidylserine on the cell surface. Cell survival was measured by means of a clonogenic assay. RESULTS: Treatment (before and after irradiation) of cells with 5-125 at low concentrations (5, 10, and 20 mum) effectively suppressed gamma-irradiation-induced superoxide generation, cardiolipin oxidation, and delayed irradiation-induced apoptosis, evaluated by using cytochrome c release and phosphatidylserine externalization. Importantly, treatment with 5-125 increased the clonogenic survival rate of gamma-irradiated cells. In addition, 5-125 enhanced and prolonged gamma-irradiation-induced G(2)/M phase arrest. CONCLUSIONS: Radioprotection/mitigation by hemi-GS-TEMPO likely is caused by its ability to act as an electron scavenger and prevent superoxide generation, attenuate cardiolipin oxidation in mitochondria, and hence prevent the release of proapoptotic factors from mitochondria. Other mechanisms, including cell-cycle arrest at the G(2)/M phase, may contribute to the protection.
Assuntos
Apoptose/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Raios gama , Mitocôndrias/metabolismo , Lesões por Radiação/prevenção & controle , Animais , Cardiolipinas/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Óxidos N-Cíclicos/metabolismo , Citocromos c/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/efeitos da radiação , Etídio/metabolismo , Fase G2/efeitos dos fármacos , Fase G2/fisiologia , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Polietilenoglicóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/farmacologia , Superóxidos/metabolismo , Fatores de TempoRESUMO
PURPOSE: To determine whether cytochrome c (cyt c) content and associated cardiolipin oxidation can be determinants of cell sensitivity to irradiation-induced apoptosis. METHODS AND MATERIALS: The small interfering RNA (siRNA) approach was used to engineer HeLa cells with lowered contents of cyt c (14%, HeLa 1.2 cells). Cells were treated by gamma-irradiation (in doses of 5-40 Gy). Lipid oxidation was characterized by electrospray ionization mass spectrometry analysis and fluorescence high-performance liquid chromatography-based Amplex Red assay. Release of a proapoptotic factor (cyt c, Smac/DIABLO) was detected by Western blotting. Apoptosis was revealed by caspase-3/7 activation and phosphatidylserine externalization. RESULTS: Irradiation caused selective accumulation of hydroperoxides in cardiolipin (CL) but not in other phospholipids. HeLa 1.2 cells responded by a lower irradiation-induced accumulation of CL oxidation products than parental HeLa cells. Proportionally decreased release of a proapoptotic factor, Smac/DIABLO, was detected in cyt c-deficient cells after irradiation. Caspase-3/7 activation and phosphatidylserine externalization were proportional to the cyt c content in cells. CONCLUSIONS: Cytochrome c is an important catalyst of CL peroxidation, critical to the execution of the apoptotic program. This new role of cyt c in irradiation-induced apoptosis is essential for the development of new radioprotectors and radiosensitizers.
Assuntos
Apoptose/fisiologia , Cardiolipinas/metabolismo , Citocromos c/metabolismo , Mitocôndrias/efeitos da radiação , Tolerância a Radiação/fisiologia , Proteínas Reguladoras de Apoptose , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Ativação Enzimática , Fluorescência , Raios gama , Células HeLa/metabolismo , Células HeLa/efeitos da radiação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peroxidação de Lipídeos/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredução , Fosfatidilserinas/metabolismo , RNA Interferente Pequeno/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration-dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at the sn-2 position, generating the corresponding PUFA-lyso PtdCho. This specificity for PUFA-PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species.
Assuntos
Lipase/antagonistas & inibidores , Lipoproteínas/metabolismo , Esfingomielinas/farmacologia , Humanos , Hidrólise , Técnicas In Vitro , Lipoproteínas/química , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Lipossomos , Fosfatidilcolinas/metabolismo , Proteínas Recombinantes/metabolismo , Esfingomielinas/metabolismo , Especificidade por SubstratoRESUMO
Although marine oysters contain abundant amounts of ether-linked aminophospholipids, the structural identification of the various molecular species has not been reported. We developed a normal-phase silica liquid chromatography/negative-ion electrospray ionization/quadrupole multiple-stage linear ion-trap mass spectrometric (NPLC-NI-ESI/Q-TRAP-MS(3)) method for the structural elucidation of ether molecular species of serine and ethanolamine phospholipids from marine oysters. The major advantages of the approach are (i) to avoid incorrect selection of isobaric precursor ions derived from different phospholipid classes in a lipid mixture, and to generate informative and clear MS(n) product ion mass spectra of the species for the identification of the sn-1 plasmanyl or plasmenyl linkages, and (ii) to increase precursor ion intensities by "concentrating" lipid molecules of each phospholipid class for further structural determination of minor molecular species. Employing a combination of NPLC-NI-ESI/MS(3) and NPLC-NI-ESI/MS(2), we elucidated, for the first time, the chemical structures of docosahexaenoyl and eicosapentaenoyl plasmenyl phosphatidylserine (PS) species and differentiated up to six isobaric species of diacyl/alkylacyl/alkenylacyl phosphatidylethanolamine (PE) in the US pacific oysters. The presence of a high content of both omega-3 plasmenyl PS/plasmenyl PE species and multiple isobaric molecular species isomers is the noteworthy characteristic of the marine oyster. The simple and robust NPLC-NI-ESI/MS(n)-based methodology should be particularly valuable in the detailed characterization of marine lipid dietary supplements with respect to omega-3 aminophospholipids.
Assuntos
Ostreidae/química , Éteres Fosfolipídicos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Suplementos Nutricionais/análise , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/isolamento & purificação , Fosfatidilserinas/química , Fosfatidilserinas/isolamento & purificação , Éteres Fosfolipídicos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/economiaRESUMO
Probucol inhibits the proliferation of vascular smooth muscle cells in vitro and in vivo, and the drug reduces intimal hyperplasia and atherosclerosis in animals via induction of heme oxygenase-1 (HO-1). Because the succinyl ester of probucol, succinobucol, recently failed as an antiatherogenic drug in humans, we investigated its effects on smooth muscle cell proliferation. Succinobucol and probucol induced HO-1 and decreased cell proliferation in rat aortic smooth muscle cells. However, whereas inhibition of HO-1 reversed the antiproliferative effects of probucol, this was not observed with succinobucol. Instead, succinobucol but not probucol induced caspase activity and apoptosis, and it increased mitochondrial oxidation of hydroethidine to ethidium, suggestive of the participation of H(2)O(2) and cytochrome c. Also, succinobucol but not probucol converted cytochrome c into a peroxidase in the presence of H(2)O(2), and succinobucol-induced apoptosis was decreased in cells that lacked cytochrome c or a functional mitochondrial complex II. In addition, succinobucol increased apoptosis of vascular smooth muscle cells in vivo after balloon angioplasty-mediated vascular injury. Our results suggest that succinobucol induces apoptosis via a pathway involving mitochondrial complex II, H(2)O(2), and cytochrome c. These unexpected results are discussed in light of the failure of succinobucol as an antiatherogenic drug in humans.
Assuntos
Apoptose/efeitos dos fármacos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Probucol/análogos & derivados , Animais , Aorta/citologia , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocromos c/metabolismo , Fragmentação do DNA , Complexo II de Transporte de Elétrons/metabolismo , Indução Enzimática/efeitos dos fármacos , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Peróxido de Hidrogênio/metabolismo , Masculino , Metaloporfirinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Probucol/farmacologia , Protoporfirinas/farmacologia , Coelhos , RatosRESUMO
Ascorbate is a vital reductant/free radical scavenger in the CNS, whose content defines - to a large extent - the redox status and the antioxidant reserves. Quick, reliable and specific methods for its measurement in brain samples are highly desirable. We have developed a new high-throughput screening assay for measurements of ascorbate using a fluorescence plate-reader. This assay is based on a direct reaction of ascorbate with a nitroxide radical conjugated with a fluorogenic acridine moiety, 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl radical (AC-TEMPO), yielding fluorescent hydroxylamine product (AC-TEMPO-H). The reaction was monitored over time using fluorescence and electron spin resonance techniques. The appearance of fluorescent AC-TEMPO-H was linear within the range of 3.75-75µM AscH(-) in the sample (0.5-10µM AscH(-) in the well). Assay was validated with high performance liquid chromatography method. The concentration of ascorbate in murine tissue samples, including brain samples after traumatic brain injury and hemorrhagic shock, was measured.
Assuntos
Ácido Ascórbico/química , Química Encefálica , Ensaios de Triagem em Larga Escala/métodos , Animais , Ácido Ascórbico/fisiologia , Química Encefálica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Ionizing radiation triggers mitochondrial overproduction of H(2)O(2) with concomitant induction of intrinsic apoptosis, whereby clearance of H(2)O(2) upon overexpression of mitochondrial catalase increases radioresistance in vitro and in vivo. As an alternative to gene therapy, we tested the potential of Mn((III))-porphyrin complexes to clear mitochondrial H(2)O(2). We report that triphenyl-[(2E)-2-[4-[(1Z,4Z,9Z,15Z)-10,15,20-tris(4-aminophenyl)-21,23-dihydroporphyrin-5-yl]phenyl]iminoethyl]phosphonium-Mn((III)) compartmentalizes preferentially into mitochondria of mouse embryonic cells, reacts with H(2)O(2), impedes γ-ray-induced mitochondrial apoptosis, and increases the survival of mice exposed to whole body irradiation with γ-rays.
RESUMO
The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of haemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids that blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its haem-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 h before through 24 h after the irradiation.
Assuntos
Morte Celular/efeitos dos fármacos , Citocromo-c Peroxidase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Mitocôndrias/efeitos dos fármacos , Protetores contra Radiação/farmacologia , Animais , Morte Celular/efeitos da radiação , Espectroscopia de Ressonância de Spin Eletrônica , Inibidores Enzimáticos/química , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/enzimologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Protetores contra Radiação/químicaRESUMO
We have shown previously that single-walled carbon nanotubes can be catalytically biodegraded over several weeks by the plant-derived enzyme, horseradish peroxidase. However, whether peroxidase intermediates generated inside human cells or biofluids are involved in the biodegradation of carbon nanotubes has not been explored. Here, we show that hypochlorite and reactive radical intermediates of the human neutrophil enzyme myeloperoxidase catalyse the biodegradation of single-walled carbon nanotubes in vitro, in neutrophils and to a lesser degree in macrophages. Molecular modelling suggests that interactions of basic amino acids of the enzyme with the carboxyls on the carbon nanotubes position the nanotubes near the catalytic site. Importantly, the biodegraded nanotubes do not generate an inflammatory response when aspirated into the lungs of mice. Our findings suggest that the extent to which carbon nanotubes are biodegraded may be a major determinant of the scale and severity of the associated inflammatory responses in exposed individuals.
Assuntos
Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidade , Neutrófilos/enzimologia , Peroxidase/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/patologia , Animais , Humanos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Nanotubos de Carbono/ultraestrutura , Neutrófilos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Espectrofotometria Infravermelho , Análise Espectral RamanRESUMO
Manganese superoxide dismutase (MnSOD) is vital to the protection of mitochondria and cells against oxidative stress. Earlier, we demonstrated that catalytically active homo-tetramer of MnSOD can be stabilized by oxidative cross-linking. Here we report that this effect may be translated into increased radioresistance of mouse embryonic cells (MECs) by pre-exposure to oxidative stress. Pre-treatment of MECs with antimycin A, rotenone or H(2)O(2) increased their survival after irradiation. Using MnSOD siRNA, we show that MECs with decreased MnSOD levels displayed a lowered ability to preconditioning. Thus oxidative preconditioning may be used for targeted regulation of MnSOD.
Assuntos
Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Superóxido Dismutase/metabolismo , Animais , Antimicina A/farmacologia , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Embrião de Mamíferos/citologia , Camundongos , Espécies Reativas de Oxigênio/farmacologia , Rotenona/farmacologia , Fatores de TempoRESUMO
Generation of reactive oxygen species by damaged respiratory chain followed by the formation of cytochrome c (cyt c)-cardiolipin (CL) complex with peroxidase activity are early events in apoptosis. By quenching the peroxidase activity of cyt c-CL complexes in mitochondria, nitric oxide can exert anti-apoptotic effects. Therefore, mitochondria-targeted pro-drugs capable of gradual nitric oxide radical (NO*) release are promising radioprotectants. Here we demonstrate that (2-hydroxyamino-vinyl)-triphenyl-phosphonium effectively accumulates in mitochondria, releases NO* upon mitochondrial peroxidase reaction, protects mouse embryonic cells from irradiation-induced apoptosis and increases their clonogenic survival after irradiation. We conclude that mitochondria-targeted peroxidase-activatable NO-donors represent a new interesting class of radioprotectors.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Hidroxilaminas/farmacologia , Óxido Nítrico/metabolismo , Compostos Organofosforados/farmacologia , Animais , Apoptose/efeitos da radiação , Biotransformação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Raios gama , Hidroxilaminas/farmacocinética , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doadores de Óxido Nítrico/farmacocinética , Doadores de Óxido Nítrico/farmacologia , Compostos Organofosforados/farmacocinética , Protetores contra Radiação/farmacocinética , Protetores contra Radiação/farmacologiaRESUMO
The critical role of mitochondria in programmed cell death leads to the design of mitochondriotropic agents as a strategy in regulating apoptosis. For anticancer therapy, stimulation of proapoptotic mitochondrial events in tumor cells and their suppression in surrounding normal cells represents a promising paradigm for new therapies. Different approaches targeting regulation of components of mitochondrial antioxidant system such as Mn-SOD demonstrated significant antitumor efficiency, particularly in combination therapy. This review is focused on a newly discovered early stage of mitochondria-dependent apoptosis - oxidative lipid signaling involving a mitochondria-specific phospholipid cardiolipin (CL). Cytochrome c (cyt c) acts as a CL-specific peroxidase very early in apoptosis. At this stage, the hostile events are still secluded within the mitochondria and do not reach the cytosolic targets. CL oxidation process is required for the release of pro-apoptotic factors into the cytosol. Manipulation of cyt c interactions with CL, inhibition of peroxidase activity, and prevention of CL peroxidation are prime targets for the discovery of anti-apoptotic drugs acting before the "point-of-no-return" in the fulfillment of the cell death program. Therefore, mitochondria-targeted disruptors and inhibitors of cyt c/CL peroxidase complexes and suppression of CL peroxidation represent new strategies in anti-apoptotic drug discovery.
Assuntos
Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Citocromos c/antagonistas & inibidores , Mitocôndrias/fisiologia , Neoplasias/tratamento farmacológico , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Cardiolipinas/farmacologia , Cardiolipinas/fisiologia , Cardiolipinas/uso terapêutico , Sequência Conservada , Citocromos c/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Mitocôndrias/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de SequênciaRESUMO
Removal of excessive mitochondrial reactive oxygen species by electron scavengers and antioxidants is a promising therapeutic strategy to reduce the detrimental effects of radiation exposure. Here we exploited triphenylphosphonium (TPP) cation as a means to target nitroxide radicals to mitochondria. We synthesized a library of TPP-conjugated nitroxides and tested their radioprotective effects in gamma-irradiated mouse embryo cells and human epithelial BEAS-2B cells. Cells were incubated with conjugates either before or after irradiation. We found that [2-(1-oxyl-2,2,6,6-tetramethyl-piperidin-4-ylimino)-ethyl]-triphenyl-phosphonium (TPEY-Tempo) significantly blocked radiation-induced apoptosis as revealed by externalization of phosphatidylserine on the cell surface and inhibition of cytochrome c release from mitochondria. Using electron paramagnetic resonance, we showed that TPEY-Tempo was integrated into cells and mitochondria, where it underwent one-electron reduction to hydroxylamine. TPEY-Tempo acted as an electron scavenger that prevented superoxide generation and cardiolipin oxidation in mitochondria. Finally, TPEY-Tempo increased the clonogenic survival rate of irradiated cells. The cellular integration efficiencies of nonradioprotective TPP conjugates, including Mito-Tempo (Alexis, San Diego, CA), were markedly lower, although these homologues were integrated into isolated succinate-energized mitochondria to a similar extent as TPEY-Tempo. We conclude that mitochondrial targeting of TPP-conjugated nitroxides represents a promising approach for the development of novel radioprotectors.