Antioxidant properties of MitoTEMPOL and its hydroxylamine

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

Aarhus Sensor Green: A Fluorescent Probe for Singlet Oxygen

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(a¹Δ_g). 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

Antioxidant β-carotene does not quench singlet oxygen in mammalian cells

Carotenoids, and β-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 β-carotene efficiently quenches singlet oxygen in solution-phase systems is invariably invoked when explaining the biological antioxidative properties of β-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 β-carotene. This observation requires a re-evaluation of β-carotene's role as an antioxidant in mammalian systems and now underscores the importance of mechanisms by which β-carotene inhibits radical reactions.Fil: Bosio, Gabriela Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; Argentina. University Aarhus; DinamarcaFil: Breitenbach, Thomas. University Aarhus; DinamarcaFil: Parisi, Julieta Marcia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Reigosa, Miguel Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Blaikie, Frances H.. University Aarhus; DinamarcaFil: Pedersen, Brian W.. University Aarhus; DinamarcaFil: Silva, Elsa F. F.. University Aarhus; Dinamarca. Universidad de Coimbra; PortugalFil: Martire, Daniel Osvaldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Ogilby, Peter R.. University Aarhus; Dinamarc

Intracellular singlet oxygen photosensitizers: On the road to solving the problems of sensitizer degradation, bleaching and relocalization

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.Fil: Da Silva, Elsa F. F.. Universidad de Coimbra; Portugal. University Aarhus; DinamarcaFil: Pimenta, Frederico M.. University Aarhus; DinamarcaFil: Pedersen, Brian W.. University Aarhus; DinamarcaFil: Blaikie, Frances H.. University Aarhus; DinamarcaFil: Bosio, Gabriela Natalia. University Aarhus; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas; ArgentinaFil: Breitenbach, Thomas. University Aarhus; DinamarcaFil: Westberg, Michael. University Aarhus; DinamarcaFil: Bregnhøj, Mikkel. University Aarhus; DinamarcaFil: Etzerodt, Michael. University Aarhus; DinamarcaFil: Arnaut, Luis G.. Universidad de Coimbra; PortugalFil: Ogilby, Peter R.. University Aarhus; Dinamarc

Antioxidant β‑Carotene Does Not Quench Singlet Oxygen in Mammalian Cells

Carotenoids, and β-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 β-carotene efficiently quenches singlet oxygen in solution-phase systems is invariably invoked when explaining the biological antioxidative properties of β-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 β-carotene. This observation requires a re-evaluation of β-carotene’s role as an antioxidant in mammalian systems and now underscores the importance of mechanisms by which β-carotene inhibits radical reactions

Accumulation of lipophilic dications by mitochondria and cells

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

Direct Modification of the Proinflammatory Cytokine Macrophage Migration Inhibitory Factor by Dietary Isothiocyanates*

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