Conversion of 2,5-dimethylfuran to 2-hydroxy-5-hydroperoxy-2,5-dimethyldihydrofuran,a true 1O2-derived reaction in aqueous 1O2 generating systems

It has been studied whether 2,5-dimethylfuran (DMF) is a specific ¹O₂ trapping agent in aqueous system. The exposure of DMF to aqueous ¹O₂ generating system (Rose Bengal photooxygenation system) gave 2-hydroxy-5-hydroperoxy-2,5-dimethyldihydrofuran (a hydrated form of endoperoxide, ¹O₂-derived reaction product) and cis-diacetylethylene (cis-DAE), while the bromine-catalyzed autoxidation of DMF afforded only trans-DAE. In Fenton system (·OH generating system) DMF was converted in the main to cis-DAE, but not to the hydrated form of endoperoxide. The exposure of DMF to acetaldehyde-xanthine oxidase system failed to detect the hydrated form of endoperoxide, but chiefly yielded a non-specific oxidation product, cis-DAE.     

Endogenous isoprene protects Phragmites australis leaves against singlet oxygen

The possible protective role of endogenous isoprene against oxidative stress caused by singlet oxygen (¹O₂) was studied in the isoprene‐emitting plant Phragmites australis. Leaves emitting isoprene and leaves in which isoprene synthesis was inhibited by fosmidomycin were exposed to increasing concentrations of ¹O₂ generated by Rose Bengal (RB) sensitizer at different light intensities. In isoprene‐emitting leaves, photosynthesis and H₂O₂ and malonyldialdehyde (MDA) contents were not affected by low to moderate ¹O₂ concentrations generated at light intensities of 800 and 1240 µmol m^?2 s^?1, but symptoms of damage and reactive oxygen accumulation started to be observed when high levels of ¹O₂ were generated by very high light intensity (1810 µmol m^?2 s^?1). A dramatic decrease in photosynthetic performance and an increase in H₂O₂ and MDA levels were measured in isoprene‐inhibited RB‐fed leaves, but photosynthesis was not significantly inhibited in leaves in which the isoprene leaf pool was reconstituted by fumigating exogenous isoprene. The inhibition of photosynthesis in isoprene‐inhibited leaves was linearly associated with the light intensity and with the consequently formed ¹O₂. Hence, physiological levels of endogenous isoprene may supply protection against ¹O₂. The protection mechanisms may involve a direct reaction of isoprene with ¹O₂. Moreover, as it is a small lipophilic molecule, it may assist hydrophobic interactions in membranes, resulting in their stabilization. The isoprene‐conjugated double bond structure may also quench ¹O₂ by facilitating energy transfer and heat dissipation. This action is typical of other isoprenoids, but we speculate that isoprene may provide a more dynamic protection mechanism as it is synthesized promptly when high light intensity produces ¹O₂.     

Lidocaine: a hydroxyl radical scavenger and singlet oxygen quencher

Lidocaine, a local anaesthetic, has been shown to reduce ventricular arrhythmias associated with myocardial infarction and ischemic myocardial injury and its protective effects has been attributed to its membrane stabilizing properties. Since oxygen radicals are known to be produced during ischemia induced tissue damage, we have investigated the possible antioxidant properties of lidocaine and found that lidocaine does not scavenge 0₂ ^–· radicals at 1 to 20 mM concentrations. However, lidocaine was found to be a potent scavenger of hydroxyl radicals and singlet oxygen. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic techniques. Lidocaine inhibited DMPO-OH adduct formation in a dose dependent manner. The amount of lidocaine needed to cause 50% inhibition of that rate was found to be approximately 80 M and at 300 M concentration it virtually eliminated the DMPO-OH adduct formation. The production of OH-dependent TBA reactive products of deoxyribose was also inhibited by lidocaine in a dose dependent manner. Lidocaine was also found to inhibit the ¹O₂-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation in a dose dependent manner. ¹O₂ was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers and was detected as TEMP-¹O₂ adduct by EPR spectroscopy. The amount of lidocaine required to cause 50% inhibition of TEMP-¹O₂ adduct formation was found to be 500 M. These results suggest that the protective effect of lidocaine on myocardial injury may, in part, be due to its reactive oxygen scavenging properties. These results may also explain the membrane stabilizing actions of lidocaine by scavenging OH · and ¹O₂ that are implicated in membrane lipid peroxidation.     

Ultraviolet irradiation of titanium dioxide in aqueous dispersion generates singlet oxygen

Abstract

We previously reported that irradiation of titanium dioxide (TiO₂) in ethanol generates both singlet oxygen (¹O₂) and superoxide anion (O₂^·-) as measured by EPR spectroscopy. The present study describes the production of reactive oxygen species upon irradiation of TiO₂ in aqueous suspension as determined by EPR spectroscopy using 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TMP) and 5,5- dimethyl-pyrroline-N-oxide (DMPO). Photoproduction of ¹O₂ by suspended TiO₂, detected as 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (4-oxo-TEMPO), was measured in water and deuterium oxide (D₂O) in the presence or absence of sodium azide (NaN₃) and under air or argon atmospheres. Production of a DMPO-OH adduct was examined in 4-oxo-TMP containing medium in the presence or absence of dimethyl sulfoxide (DMSO). The signal for the DMPO spin adduct of superoxide anion was not observed in aqueous conditions. Kinetic analysis revealed that ¹O₂ was produced at the surface of irradiated TiO₂ in aqueous suspension as was observed in ethanol. Kinetic analysis revealed that the formation of DMPO-OH adduct reflects oxidation of DMPO by ¹O₂ rather than the trapping of the hydroxyl radical produced by the reaction of photo-exited TiO₂ and water. The production of large amounts of ¹O₂ by TiO₂ in aqueous suspension as compared to those in ethanol and possible formation of hydroxyl radical in aqueous suspension but not in alcohol, suggest that irradiation of TiO₂ in aqueous environments is biologically more important than that in non-aqueous media.     

Role of reactive oxygen species in cardiac preconditioning: Study with photoactivated rose bengal in isolated rat hearts

Oxygen radical scavengers have been shown to prevent the development of ischemic preconditioning, suggesting that reactive oxygen species (ROS) might be involved in this phenomenon. In the present study, we have investigated whether direct exposure to ROS produced by photoactivated Rose Bengal (RB) could mimic the protective effects of ischemic preconditioning.

Methods In vitro generation of ROS from photoactivated RB in a physiological buffer was first characterised by ESR spectroscopy in the presence of 2,2,6,6-tetramethyl-1-piperidone (oxoTEMP) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In a second part of the study, isolated rat hearts were exposed for 2.5 min to photoactivated RB. After 5 min washout, hearts underwent 30 min no-flow normothermic ischemia followed by 30 min of reperfusion.

Results and Conclusions The production of singlet oxygen (¹O₂) by photoactivated RB in the perfusion medium was evidenced by the ESR detection of the nitroxyl radical oxoTEMPO. Histidine completely inhibited oxoTEMPO formation. In addition, the use of DMPO has indicated that (i) superoxide anions (O^·-₂) are produced directly and (ii) hydroxyl radicals (HO^·) are formed indirectly from the successive O^·-₂ dismutation and the Fenton reaction. In the perfusion experiments, myocardial post-ischemic recovery was dramatically impaired in hearts previously exposed to the ROS produced by RB photoactivation (¹O₂, O^·-₂, H₂O₂ and HO^·) as well as when ¹O₂ was removed by histidine (50 mM) addition. However, functional recovery was significantly improved when hearts were exposed to photoactivated RB in presence of superoxide dismutase (10⁵ IU/L) and catalase (10⁶ IU/L).

Further studies are now required to determine whether the cardioprotective effects of Rose Bengal in presence of O^·-₂ and H₂O₂ scavengers are due to singlet oxygen or to other species produced by Rose Bengal degradation.     

No or little production of singlet molecular oxygen in HOCl or HOClH2O2 a model system for myeloperoxidase/H2O2/Cl−

The HOCl in chlorine-water oxidizes DPF to cis-DBE in parallel to the HOCl concentration. The addition of H₂O₂ produces singlet molecular oxygen, and a bimol collision above pH 6.0, but not in the pH region 3.0 to 4.0. The DPF conversion to cis-DBE is initiated by a 1,2-position attack of OH^? and Cl⁺, followed by the HCl elimination. The oxidation potency of HOCl is much greater than the singlet molecular oxygen generated in chlorine-water/H₂O₂ solution, on the pH range 6.0 to 8.0 where both HOCl and OCl^? are present.     

Chemically shifted singlet oxygen spectrum

The estimated light emission spectrum was determined for a singlet oxygen (¹O₂)-producing system, NaOCl + H₂O₂, alone and in the presence of tryptophan and bovine serum albumin. Tryptophan and bovine serum albumin caused a decrease in the red emission of ¹O₂ and an increase in the amount of shorter wavelength light. This effect was due to chemiluminescence rather than fluorescence. Arachidonic acid caused a similar spectral shift, while guanosine demonstrated a late chemiluminescent reaction of predominantly short wavelength light in the presence of ¹O₂.     

Edaravone directly reacts with singlet oxygen and protects cells from attack

AimsProtective effects of edaravone, an approved medicine for acute brain infarction in Japan, on cell death induced by singlet oxygen (¹O₂) were examined.Main methodThe ¹O₂ scavenging activity was examined by direct analysis of near-infrared luminescence in a cell-free system and by fluorospectrometry in the presence of cells. The protective effects of edaravone on ¹O₂-induced cell death were examined, using rat neuronal B50 cells. Cell death was evaluated by mitochondrial respiration (MTT assay), confocal microscopy and time-lapse imaging. The chemical reaction of edaravone with ¹O₂ was examined by production analysis using high performance liquid chromatography (HPLC).Key findingsWhen rose Bengal (RB) in D₂O was irradiated by a 514 nm laser beam, the signal of ¹O₂ was observed. Edaravone suppressed the ¹O₂ signal more potently than azide, a ¹O₂ scavenger. When B50 cells were irradiated by 525 nm green light in the RB solution, production of ¹O₂ and induction of cell death were observed. The fluorospectrometric study and the MTT assay revealed that 100–400 µM edaravone suppressed the ¹O₂ production and attenuated cell death in a concentration-dependent manner. Confocal microscopy and the time-lapse imaging revealed that edaravone prevented the impairment of membrane integrity and the progression of cell death induced by ¹O₂. The HPLC study revealed that edaravone chemically reacted with ¹O₂ and changed another compound.SignificanceSince ¹O₂ is possibly involved in post-ischemic neuronal damage, the clinically approved curative effects of edaravone on acute brain infarction might be attributed to its potent ¹O₂ scavenging activity.     

Singlet oxygen inhibits ATPase and proton translocation activity of the thylakoid ATP synthase CF1CFo

Singlet oxygen (¹O₂) produced in plants during photosynthesis has a strong damaging effect not only on both photosystems but also on the whole photosynthetic machinery. This is also applicable for the adenosine triphosphate (ATP) synthase. Here we describe the impact of ¹O₂ generated by the photosensitizer Rose Bengal on the ATP hydrolysis and ATP-driven proton translocation activity of CF1CFo. Both activities were reduced dramatically within 1 min of exposure. Interestingly, it is shown that oxidized thylakoid ATP synthase is more susceptible to ¹O₂ than CF1CFo in its reduced state, a new insight on the mechanism of ¹O₂ interaction with the γ subunit.     

Evidence of singlet oxygen participation in the chlorophyll-sensitized photooxidation of indoleacetic Acid

Chlorophyll-sensitized photooxidation of indoleacetic acid (IAA)—with chlorophyll extracted from Pisum sativum L. cv. Alaska W.R.—was determined in the presence of deuterium oxide and known quenchers of singlet oxygen (¹O₂) to explore the involvement of ¹O₂ in the reaction. O₂ uptake was measured in light in a buffered aqueous micellar system containing Triton X-100, KCl, chlorophyll, and IAA. The rate of O₂ uptake was zero in darkness. The reaction was stimulated by deuterium oxide and inhibited by sodium azide indicating that ¹O₂ participated in IAA photooxidation. Both mannitol and superoxide dismutase failed to inhibit O₂ uptake suggesting that neither the hydroxyl radical nor the superoxide anion played a significant role in the reaction.     

Measurement of rate constants for quenching singlet oxygen with a Cypridina luciferin analog (2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one) and sodium azide

The rate constants for [¹O₂] [MCLA] and [¹O₂][NaN₃] were measured by quenching the near-infrared emission (¹Δ_g→³∑_g) in steady state with MCLA and NaN₃, respectively. ¹O₂ was constantly generated by energy transfer to O₂ from Ar laser-excited Rose Bengal. The Stern—Volmer plots yielded the second-order rate constants of 2.94 × 10⁹ M^?1 S^?1 and 3.83 × 10⁸ M^?1 S^?1 for quenching ¹O₂ with MCLA and NaN₃ in water at pH 5.4, respectively. The ¹O₂ + MCLA reaction emitted light with maximum at 465 nm at pD 4.5 identical to the O₂^? + MCLA reaction.     

Quenching of singlet oxygen by D-α-tocopherol in human granulocytes

The ability of D-α-tocopherol to act as a quencher of ¹O₂ (singlet oxygen) was tested with a biological source of ¹O₂, namely the phagocytosis activated myeloperoxidase contained in the homogenate of human circulating polymorphonuclear leukocytes.With this system, the ¹O₂ quenching efficiency of exogenously added D-α-tocopherol was estimated from its inhibitory effect on the luminol amplified chemiluminescence. This inhibitory effect was dose dependent. D-α-tocopherol was also efficient in quenching the chemiluminescence generated through the H₂O₂-horseradish system. In both systems the quenching effect may be almost entirely “physical”, since very little tocopherol was destroyed when compared to the relatively large amount of H₂O₂ consumed.     

Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions

Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (λ>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (¹O₂) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of ¹O₂. In addition, the generation of ¹O₂ was verified further in the presence of D₂O and specific singlet oxygen quencher — 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN₃). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of ¹O₂ by PBPs are as follows: APC > C-PC > R-PE.     

Determination of H2O2-dependent generation of singlet oxygen from human saliva with a novel chemiluminescence probe

Singlet oxygen (¹O₂) has been shown to play an important role in salivary defense system, but its generation process and level from human saliva remain uncertain due to the lack of a reliable detection method. We have previously reported 4,4′(5′)-bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene (BAET) as a novel chemiluminescence probe for ¹O₂. In this work, the probe is successfully used to characterize H₂O₂-dependent generation of ¹O₂ from saliva in real time. However, the yield of ¹O₂ is found to be very low, for example, being about 0.13 nmol from 200 μL saliva in the presence of 1 mM of hydrogen peroxide over a 5-s reaction period. The result is also compared with that obtained with another ¹O₂ probe 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), demonstrating that, besides ¹O₂, the other reactive oxygen species such as hydroxyl radical may also be involved in the reaction of saliva with H₂O₂. Furthermore, the present study shows that the selectivity of BAET for ¹O₂ is much higher than that of CLA and thus BAET is highly suited for the detection of ¹O₂ in the presence of other reactive oxygen species in biological systems.     

High optical-throughput spectroscopic singlet oxygen and photosensitizer luminescence dosimeter for monitoring of photodynamic therapy

We report a high light-throughput spectroscopic dosimeter system that is able to noninvasively measure luminescence signals of singlet oxygen (¹O₂) produced during photodynamic therapy (PDT) using a CW (continuous wave) light source. The system is based on a compact, fiber-coupled, high collection efficiency spectrometer (>50% transmittance) designed to maximize optical throughput but with sufficient spectral resolution (~7 nm). This is adequate to detect ¹O₂ phosphorescence in the presence of strong luminescence background in vivo. This system provides simultaneous acquisition of multiple spectral data points, allowing for more accurate determination of luminescence baseline via spectral fitting and thus the extraction of ¹O₂ phosphorescence signal based solely on spectroscopic decomposition, without the need for time-gating. Simultaneous collection of photons at different wavelengths improves the quantum efficiency of the system when compared to sequential spectral measurements such as filter-wheel or tunable-filter based systems. A prototype system was tested during in vivo PDT tumor regression experiments using benzoporphyrin derivative (BPD) photosensitizer. It was found that the treatment efficacy (tumor growth inhibition rate) correlated more strongly with ¹O₂ phosphorescence than with PS fluorescence. These results indicate that this high photon-collection efficiency spectrometer instrument may offer a viable option for real-time ¹O₂ dosimetry during PDT treatment using CW light.     

Involvement of singlet oxygen in the fungal degradation of lignin

The possible involvement of singlet oxygen (¹O₂) in the degradation of lignin by $\text{Phanerochaete}\text{chrysosporium}$ was examined. Ligninolytic cultures and photochemically generated ¹O₂ gave the same oxidation products from the lignin substructure model compound 1,2-bis(3-methoxy-4-alkoxyphenyl)propan-1,3-diol. Fluorescence and near UV absorbance of the specific ¹O₂ trapping agent anthracene-9,10-bisethanesulfonic acid (AES) disappeared in ligninolytic cultures, indicating that ¹O₂ was produced. AES strongly inhibited oxidation of ¹⁴C-lignin, but not ¹⁴C-glucose, to ¹⁴CO₂ in cultures, and also strongly suppressed oxidation of the model compound. These results indicate the ¹O₂ plays an integral role in lignin biodegradation.     

Glow discharge in singlet oxygen

Currently, there is no experimental data on the plasma balance in gas mixtures with a high content of singlet delta oxygen O₂(¹Δ_g). These data can be obtained by studying the parameters of an electric discharge in singlet oxygen produced by a chemical generator. The O₂(¹Δ_g) molecules significantly change the kinetics of electrons and negative ions in plasma. Hence, the discharge conditions at low and high. O₂(¹Δ_g) concentrations are very different. Here, the parameters of the positive column of a glow discharge in a gas flow from a chemical singlet-oxygen generator are studied. It is experimentally shown that, at an O₂(¹Δ_g) concentration of 50% and at pressures of 1.5 and 2 torr, the electric field required to sustain the discharge is considerably lower than in the case when all of the oxygen molecules are in the ground state. A theoretical model of the glow discharge is proposed whose predictions are in good agreement with the experimental data.     

Membrane transport of singlet oxygen monitored by dipole potential measurements

The efficiency of photodynamic reactions depends on 1), the penetration depth of the photosensitizer into the membrane and 2), the sidedness of the target. Molecules which are susceptible to singlet oxygen (¹O₂) experience less damage when separated from the photosensitizer by the membrane. Since ¹O₂ lifetime in the membrane environment is orders of magnitude longer than the time required for nonexcited oxygen (O₂) to cross the membrane, this observation suggests that differences between the permeabilities or membrane partition of ¹O₂ and O₂ exist. We investigated this hypothesis by releasing ¹O₂ at one side of a planar membrane while monitoring the kinetics of target damage at the opposite side of the same membrane. Damage to the target, represented by dipole-modifying molecules (phloretin or phlorizin), was indicated by changes in the interleaflet dipole potential difference Δφ_b. A simple analytical model allowed estimation of the ¹O₂ interleaflet concentration difference from the rate at which Δφ_b changed. It confirmed that the lower limit of ¹O₂ permeability is ∼2 cm/s; i.e., it roughly matches O₂ permeability as predicted by Overton's rule. Consequently, the membrane cannot act as a barrier to ¹O₂ diffusion. Differences in the reaction rates at the cytoplasmic and extracellular membrane leaflets may be attributed only to ¹O₂ quenchers inside the membrane.     

On the mechanism of photosensitized luminescence of singlet oxygen dimols in air-saturated pigment solutions

Luminescence of singlet oxygen dimols (¹O₂)₂ was studied in aerobic solutions of a nonfluorescent photosensitizer phenalenone in CCl₄ and C₆F₆ using a setup with a mechanical phosphoroscope and relatively low rates of photosensitizer excitation. The luminescence spectrum was found to resemble those reported in our previous papers dealing with dimol luminescence in solutions of porphyrins and other organic dyes. The main maximum was located at 703–706 nm, and two much weaker bands at 640 and 770–780 nm. These data suggest that dimol luminescence arises owing to interaction of two ¹O₂ molecules and one ground-state pigment molecule. Light is emitted by the dimol-pigment contact complexes, which are formed as a result of ¹O₂ collisions with metastable, probably triplet, intermediates appearing in ¹O₂ reaction with pigment molecules. It is proposed that this mechanism of dimol luminescence might be of general importance for photochemical, chemical, and biological systems where singlet oxygen is generated. However, the luminescence of this type dominates at relatively low rates of ¹O₂ generation. According to the literature data, at high ¹O₂ generation rates the prevalent type of dimol luminescence has the main maximum at 635–637 nm and is caused by direct collisions of two ¹O₂ molecules.     

Production of singlet oxygen in a non-self-sustained discharge

The production of O₂(a¹Δ_g) singlet oxygen in non-self-sustained discharges in pure oxygen and mixtures of oxygen with noble gases (Ar or He) was studied experimentally. It is shown that the energy efficiency of O₂(a¹Δ_g production can be optimized with respect to the reduced electric field E/N. It is shown that the optimal E/N values correspond to electron temperatures of 1.2–1.4 eV. At these E/N values, a decrease in the oxygen percentage in the mixture leads to an increase in the excitation rate of singlet oxygen because of the increase in the specific energy deposition per O₂ molecule. The onset of discharge instabilities not only greatly reduces the energy efficiency of singlet oxygen production but also makes it impossible to achieve high energy deposition in a non-self-sustained discharge. A model of a non-self-sustained discharge in pure oxygen is developed. It is shown that good agreement between the experimental and computed results for a discharge in oxygen over a wide range of reduced electric fields can be achieved only by taking into account the ion component of the discharge current. The cross section for the electron-impact excitation of O₂(a¹Δ_g and the kinetic scheme of the discharge processes with the participation of singlet oxygen are verified by comparing the experimental and computed data on the energy efficiency of the production of O₂(a¹Δ_g and the dynamics of its concentration. It is shown that, in the dynamics of O₂(a¹Δ_g molecules in the discharge afterglow, an important role is played by their deexcitation in a three-body reaction with the participation of O(³P) atoms. At high energy depositions in a non-self-sustained discharge, this reaction can reduce the maximal attainable concentration of singlet oxygen. The effect of a hydrogen additive to an Ar: O₂ mixture is analyzed based on the results obtained using the model developed. It is shown that, for actual electron beam current densities, a significant energy deposition in a non-self-sustained discharge in the mixtures under study can be achieved due to the high rate of electron detachment from negative ions. In this case, however, significant heating of the mixture can lead to a rapid quenching of O₂(a¹Δ_g molecules by atomic hydrogen.