RESUMO
Selected singlet oxygen photosensitizers have been examined from the perspective of obtaining a molecule that is sufficiently stable under conditions currently employed to study singlet oxygen behavior in single mammalian cells. Reasonable predictions about intracellular sensitizer stability can be made based on solution phase experiments that approximate the intracellular environment (e.g., solutions containing proteins). Nevertheless, attempts to construct a stable sensitizer based solely on the expected reactivity of a given functional group with singlet oxygen are generally not sufficient for experiments in cells; it is difficult to construct a suitable chromophore that is impervious to all of the secondary and/or competing degradative processes that are present in the intracellular environment. On the other hand, prospects are reasonably positive when one considers the use of a sensitizer encapsulated in a specific protein; the local environment of the chromophore is controlled, degradation as a consequence of bimolecular reactions can be mitigated, and genetic engineering can be used to localize the encapsulated sensitizer in a given cellular domain. Also, the option of directly exciting oxygen in sensitizer-free experiments provides a useful complementary tool. These latter systems bode well with respect to obtaining more accurate control of the "dose" of singlet oxygen used to perturb a cell; a parameter that currently limits mechanistic studies of singlet-oxygen-mediated cell signaling.
Assuntos
Oxigênio/química , Fármacos Fotossensibilizantes/química , Oxigênio Singlete/química , Animais , Bovinos , Corantes Fluorescentes/química , Fulerenos/química , Engenharia Genética , Células HeLa , Humanos , Fotodegradação , Albumina Sérica/química , Transdução de SinaisRESUMO
A tetrafluoro-substituted fluorescein derivative covalently linked to a 9,10-diphenyl anthracene moiety has been synthesized, and its photophysical properties have been characterized. This compound, denoted Aarhus Sensor Green (ASG), has distinct advantages for use as a fluorescent probe for singlet molecular oxygen, O2(a(1)Δg). In the least, ASG overcomes several limitations inherent to the use of the related commercially available product called Singlet Oxygen Sensor Green (SOSG). The functional behavior of both ASG and SOSG derives from the fact that these weakly fluorescent compounds rapidly react with singlet oxygen via a π2 + π4 cycloaddition to irreversibly yield a highly fluorescent endoperoxide. The principal advantage of ASG over SOSG is that, at physiological pH values, both ASG and the ASG endoperoxide (ASG-EP) do not themselves photosensitize the production of singlet oxygen. As such, ASG better fits the requirement of being a benign probe. Although ASG readily enters a mammalian cell (i.e., HeLa) and responds to the presence of intracellular singlet oxygen, its behavior in this arguably complicated environment requires further investigation.
Assuntos
Antracenos/química , Antracenos/síntese química , Corantes Fluorescentes/química , Oxigênio Singlete/química , Animais , Reação de Cicloadição , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Luz , Fármacos Fotossensibilizantes/químicaRESUMO
Carotenoids, and ß-carotene in particular, are important natural antioxidants. Singlet oxygen, the lowest excited state of molecular oxygen, is an intermediate often involved in natural oxidation reactions. The fact that ß-carotene efficiently quenches singlet oxygen in solution-phase systems is invariably invoked when explaining the biological antioxidative properties of ß-carotene. We recently developed unique microscope-based time-resolved spectroscopic methods that allow us to directly examine singlet oxygen in mammalian cells. We now demonstrate that intracellular singlet oxygen, produced in a photosensitized process, is in fact not efficiently deactivated by ß-carotene. This observation requires a re-evaluation of ß-carotene's role as an antioxidant in mammalian systems and now underscores the importance of mechanisms by which ß-carotene inhibits radical reactions.
Assuntos
Antioxidantes/química , Oxigênio Singlete/química , beta Caroteno/química , Células HeLa , HumanosRESUMO
The development of efficient and selective luminescent probes for reactive oxygen species, particularly for singlet molecular oxygen, is currently of great importance. In this study, the photochemical behavior of Singlet Oxygen Sensor Green(®) (SOSG), a commercially available fluorescent probe for singlet oxygen, was examined. Despite published claims to the contrary, the data presented herein indicate that SOSG can, in fact, be incorporated into a living mammalian cell. However, for a number of reasons, caution must be exercised when using SOSG. First, it is shown that the immediate product of the reaction between SOSG and singlet oxygen is, itself, an efficient singlet oxygen photosensitizer. Second, SOSG appears to efficiently bind to proteins which, in turn, can influence uptake by a cell as well as behavior in the cell. As such, incorrect use of SOSG can yield misleading data on yields of photosensitized singlet oxygen production, and can also lead to photooxygenation-dependent adverse effects in the system being investigated.
Assuntos
Corantes Fluorescentes/análise , Fármacos Fotossensibilizantes/análise , Oxigênio Singlete , Artefatos , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Luz , Microscopia de Fluorescência , Processos Fotoquímicos/efeitos da radiação , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Análise de Célula Única/métodos , Oxigênio Singlete/análise , Oxigênio Singlete/metabolismo , Soluções/química , Soluções/metabolismo , Análise EspectralRESUMO
Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti-inflammatory activity. However, knowledge of their activity at a molecular level is limited. The objective of this study was to identify biological targets of phenethyl isothiocyanate (PEITC) using an affinity purification approach. An analogue of PEITC was synthesized to enable conjugation to a solid-phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was the major protein captured from cell lysates. Site-directed mutagenesis and mass spectrometry showed that PEITC covalently modified the N-terminal proline residue of MIF. This resulted in complete loss of catalytic tautomerase activity and disruption of protein conformation, as determined by impaired recognition by a monoclonal antibody directed to the region that receptors and interacting proteins bind to MIF. The conformational change was supported by in silico modeling. Monoclonal antibody binding to plasma MIF was disrupted in humans consuming watercress, a major dietary source of PEITC. The isothiocyanates have significant potential for development as MIF inhibitors, and this activity may contribute to the biological properties of these phytochemicals.
Assuntos
Citocinas/metabolismo , Isotiocianatos/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Humanos , Inflamação , Células Jurkat , Modelos Biológicos , Modelos Químicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação ProteicaRESUMO
Nitric oxide (NO(*)) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO(*) and S-nitrosated thiol proteins. MitoSNO1-induced NO(*) production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO(*) generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO(*) donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.
Assuntos
Mitocôndrias/metabolismo , Traumatismo por Reperfusão/prevenção & controle , S-Nitrosotióis/farmacologia , Compostos de Sulfidrila/metabolismo , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Linhagem Celular , Complexo I de Transporte de Elétrons/metabolismo , Células HeLa , Coração/efeitos dos fármacos , Coração/fisiopatologia , Humanos , Técnicas In Vitro , Masculino , Espectrometria de Massas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/fisiologia , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Óxido Nítrico/metabolismo , Nitrosação/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologia , S-Nitrosotióis/síntese química , S-Nitrosotióis/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
Piperidine nitroxides such as TEMPOL have been widely used as antioxidants in vitro and in vivo. MitoTEMPOL is a mitochondria-targeted derivative of TEMPOL designed to protect mitochondria from the oxidative damage that they accumulate, but once there is rapidly reduced to its hydroxylamine, MitoTEMPOL-H. As little is known about the antioxidant efficacy of hydroxylamines, this study has assessed the antioxidant activity of both MitoTEMPOL and MitoTEMPOL-H. The hydroxylamine was more effective at preventing lipid-peroxidation than MitoTEMPOL and decreased oxidative damage to mitochondrial DNA caused by menadione. In contrast to MitoTEMPOL, MitoTEMPOL-H has no superoxide dismutase activity and its antioxidant actions are likely to be mediated by hydrogen atom donation. Therefore, even though MitoTEMPOL is rapidly reduced to MitoTEMPOL-H in cells, it remains an effective antioxidant. Furthermore, as TEMPOL is also reduced to a hydroxylamine in vivo, many of its antioxidant effects may also be mediated by its hydroxylamine.
Assuntos
Antioxidantes/farmacologia , Óxidos N-Cíclicos/farmacologia , Hidroxilamina/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Animais , DNA Mitocondrial/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Oxirredução/efeitos dos fármacos , Reação em Cadeia da Polimerase , Ratos , Marcadores de Spin , Superóxido Dismutase/metabolismoRESUMO
Mitochondrial oxidative damage is thought to contribute to a wide range of human diseases; therefore, the development of approaches to decrease this damage may have therapeutic potential. Mitochondria-targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death have been developed. These compounds contain antioxidant moieties, such as ubiquinone, tocopherol, or nitroxide, that are targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation. Because of the large mitochondrial membrane potential, the cations are accumulated within the mitochondria inside cells. There, the conjugated antioxidant moiety protects mitochondria from oxidative damage. Here, we outline some of the work done to date on these compounds and how they may be developed as therapies.
Assuntos
Antioxidantes/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Antioxidantes/farmacologia , Humanos , Mitocôndrias/metabolismo , Estresse OxidativoRESUMO
Piperidine nitroxides such as TEMPOL act as antioxidants in vivo due to their interconversion among nitroxide, hydroxylamine, and oxoammonium derivatives, but the mechanistic details of these reactions are unclear. As mitochondria are a significant site of piperidine nitroxide metabolism and action, we synthesized a mitochondria-targeted nitroxide, MitoTEMPOL, by conjugating TEMPOL to the lipophilic triphenylphosphonium cation. MitoTEMPOL was accumulated several hundred-fold into energized mitochondria where it was reduced to the hydroxylamine by direct reaction with ubiquinol. This reaction occurred by transfer of H() from ubiquinol to the nitroxide, with the ubisemiquinone radical product predominantly dismutating to ubiquinone and ubiquinol, together with a small amount reacting with oxygen to form superoxide. The piperidine nitroxides TEMPOL, TEMPO, and butylTEMPOL reacted similarly with ubiquinol in organic solvents but in mitochondrial membranes the rates varied in the order: MitoTEMPOL > butylTEMPOL > TEMPO > TEMPOL, which correlated with the extent of access of the nitroxide moiety to ubiquinol within the membrane. These findings suggest ways of using mitochondria-targeted compounds to modulate the coenzyme Q pool within mitochondria in vivo, and indicate that the antioxidant effects of mitochondria-targeted piperidine nitroxides can be ascribed to their corresponding hydroxylamines.
Assuntos
Hidroxilamina/química , Mitocôndrias/metabolismo , Óxido Nítrico/química , Ubiquinona/análogos & derivados , Antioxidantes/química , Bioquímica/métodos , Cátions , Cromatografia Líquida de Alta Pressão , Óxidos N-Cíclicos/química , Espectroscopia de Ressonância de Spin Eletrônica , Espectrometria de Massas/métodos , Modelos Químicos , Estresse Oxidativo , Marcadores de Spin , Superóxidos/metabolismo , Fatores de Tempo , Ubiquinona/químicaRESUMO
Mitochondrial oxidative damage contributes to a range of degenerative diseases. Ubiquinones have been shown to protect mitochondria from oxidative damage, but only a small proportion of externally administered ubiquinone is taken up by mitochondria. Conjugation of the lipophilic triphenylphosphonium cation to a ubiquinone moiety has produced a compound, MitoQ, which accumulates selectively into mitochondria. MitoQ passes easily through all biological membranes and, because of its positive charge, is accumulated several hundred-fold within mitochondria driven by the mitochondrial membrane potential. MitoQ protects mitochondria against oxidative damage in vitro and following oral delivery, and may therefore form the basis for mitochondria-protective therapies.
Assuntos
Mitocôndrias/metabolismo , Compostos Organofosforados/metabolismo , Quinonas/química , Ubiquinona/análogos & derivados , Administração Oral , Animais , Cátions , Membrana Celular/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Potenciais da Membrana , Doenças Mitocondriais/terapia , Modelos Biológicos , Modelos Químicos , Oxigênio/metabolismo , Ubiquinona/metabolismoRESUMO
Lipophilic monocations can pass through phospholipid bilayers and accumulate in negatively-charged compartments such as the mitochondrial matrix, driven by the membrane potential. This property is used to visualize mitochondria, to deliver therapeutic molecules to mitochondria and to measure the membrane potential. In theory, lipophilic dications have a number of advantages over monocations for these tasks, as the double charge should lead to a far greater and more selective uptake by mitochondria, increasing their therapeutic potential. However, the double charge might also limit the movement of lipophilic dications through phospholipid bilayers and little is known about their interaction with mitochondria. To see whether lipophilic dications could be taken up by mitochondria and cells, we made a series of bistriphenylphosphonium cations comprising two triphenylphosphonium moieties linked by a 2-, 4-, 5-, 6- or 10-carbon methylene bridge. The 5-, 6- and 10-carbon dications were taken up by energized mitochondria, whereas the 2- and 4-carbon dications were not. The accumulation of the dication was greater than that of the monocation methyltriphenylphosphonium. However, the uptake of dications was only described by the Nernst equation at low levels of accumulation, and beyond a threshold membrane potential of 90-100 mV there was negligible increase in dication uptake. Interestingly, the 5- and 6-carbon dications were not accumulated by cells, due to lack of permeation through the plasma membrane. These findings indicate that conjugating compounds to dications offers only a minor increase over monocations in delivery to mitochondria. Instead, this suggests that it may be possible to form dications within mitochondria that then remain within the cell.
Assuntos
Membranas Intracelulares/metabolismo , Lipídeos/química , Mitocôndrias/metabolismo , Compostos Organofosforados/metabolismo , Compostos de Terfenil/metabolismo , Trifosfato de Adenosina/metabolismo , Algoritmos , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Cátions Bivalentes/química , Cátions Bivalentes/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Ionóforos/farmacologia , Células Jurkat , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/fisiologia , Nigericina/farmacologia , Oniocompostos/química , Oniocompostos/metabolismo , Compostos Organofosforados/química , Cloreto de Potássio/farmacologia , Ratos , Rotenona/farmacologia , Radioisótopos de Rubídio/metabolismo , Compostos de Terfenil/química , Trítio/metabolismo , Compostos de Tritil/química , Compostos de Tritil/metabolismo , Desacopladores/farmacologiaRESUMO
The protonmotive force (Deltap) across the mitochondrial inner membrane drives ATP synthesis. In addition, the energy stored in Deltap can be dissipated by proton leak through the inner membrane, contributing to basal metabolic rate and thermogenesis. Increasing mitochondrial proton leak pharmacologically should decrease the efficiency of oxidative phosphorylation and counteract obesity by enabling fatty acids to be oxidised with decreased ATP production. While protonophores such as 2,4-dinitrophenol (DNP) increase mitochondrial proton leak and have been used to treat obesity, a slight increase in DNP concentration above the therapeutically effective dose disrupts mitochondrial function and leads to toxicity. Therefore we set out to develop a less toxic protonophore that would increase proton leak significantly at high Deltap but not at low Deltap. Our design concept for a potential self-limiting protonophore was to couple the DNP moiety to the lipophilic triphenylphosphonium (TPP) cation and this was achieved by the preparation of 3-(3,5-dinitro-4-hydroxyphenyl)propyltriphenylphosphonium methanesulfonate (MitoDNP). TPP cations accumulate within mitochondria driven by the membrane potential (Deltapsi), the predominant component of Deltap. Our hypothesis was that MitoDNP would accumulate in mitochondria at high Deltapsi where it would act as a protonophore, but that at lower Deltapsi the accumulation and uncoupling would be far less. We found that MitoDNP was extensively taken into mitochondria driven by Deltapsi. However MitoDNP did not uncouple mitochondria as judged by its inability to either increase respiration rate or decrease Deltapsi. Therefore MitoDNP did not act as a protonophore, probably because the efflux of deprotonated MitoDNP was inhibited.
Assuntos
Dinitrofenóis/metabolismo , Ionóforos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Prótons , Desacopladores/metabolismo , Animais , Respiração Celular/fisiologia , Dinitrofenóis/química , Potenciais da Membrana/fisiologia , Estrutura Molecular , Ratos , Desacopladores/químicaRESUMO
Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.
Assuntos
Carbono/química , Proteínas de Transporte/metabolismo , Peroxidação de Lipídeos , Proteínas de Membrana/metabolismo , Mitocôndrias Hepáticas/metabolismo , Óxidos de Nitrogênio/química , Superóxidos/metabolismo , Animais , Óxidos N-Cíclicos , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Canais Iônicos , Proteínas Mitocondriais , Ratos , Espécies Reativas de Oxigênio , Marcadores de Spin , Proteína Desacopladora 1RESUMO
The selective manipulation of the expression and replication of mitochondrial DNA (mtDNA) within mammalian cells has proven difficult. In progressing towards this goal we synthesized a novel mitochondria-targeted DNA-alkylating reagent. The active alkylating moiety [(11aS)-8-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (DC-81)], irreversibly alkylates guanine bases in DNA (with a preference for AGA triplets), preventing its expression and replication. To target this compound to mitochondria it was covalently coupled to the lipophilic triphenylphosphonium (TPP) cation to form a derivative referred to as mitoDC-81. Incorporation of this lipophilic cation led to the rapid uptake of mitoDC-81 by mitochondria, driven by the large membrane potential across the inner membrane. This compound efficiently alkylated isolated supercoiled, relaxed-circular or linear plasmid DNA and isolated mtDNA. However mitoDC-81 did not alkylate mtDNA within isolated mitochondria or cells, even though it accessed the mitochondrial matrix at concentrations up to 100-fold higher than those required to alkylate isolated DNA. This surprising finding suggests that mtDNA within intact mitochondria may not be accessible to this class of alkylating reagent. This inability to alkylate mtDNA in situ has significant implications for the design of therapies for mtDNA diseases and for studies on the packaging, expression and turnover of mtDNA in general.
Assuntos
Alquilantes/metabolismo , Benzodiazepinas/síntese química , Benzodiazepinas/metabolismo , DNA Mitocondrial/metabolismo , Iodetos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Compostos Organofosforados/síntese química , Compostos Organofosforados/metabolismo , Alquilantes/síntese química , Alquilantes/química , Animais , Benzodiazepinas/química , Cátions/química , Cátions/metabolismo , Respiração Celular/fisiologia , Humanos , Iodetos/química , Estrutura Molecular , Compostos Organofosforados/química , Ratos , Células Tumorais CultivadasRESUMO
Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.