Chemiluminescence Emission during Reactions between Superoxide and Selected Aliphatic and Aromatic Halocarbons in Aprotic Media

The reactions between superoxide free radical anion (.O⁻₂) with the halocarbons CCl₄, CHCl₃, BrCH₂CH₂Br(EDB), decachloro-biphenyl (DCBP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dimethyl sulphoxide (DMSO) results in the emission of chemiluminescence (CL). The chemiluminescence reactions are characterized as having biphasic second order kinetics, CL wavelengths between 350 nm and 650 nm, and exhibiting perturbation by chemicals reactive with singlet oxygen. These data suggest that singlet oxygen species are the excited state responsible for the light emissions. Polarographic studies confirm .O⁻₂ consumption and halide release in the reactions, while gas liquid chromatography and NBT reduction demonstrate the decomposition of the halocarbons into products. A chemiluminescent reaction mechanism is proposed involving reductive dehalogenation of the halocarbons and the generation of singlet oxygen. The significance of singlet oxygen generation is discussed with respect to a general mechanism for explaining the rapid initiation of lipid peroxidative membrane damage in halocarbon toxigenicity in animal and plant tissues.     

New Singlet Oxygen Source and Trapping Reagent for Peroxide Intermediates

Application of newly-developed water-soluble singlet oxygen sources in the oxidation of biologically important compounds and the electron-transfer process involving singlet oxygen has been reviewed. Particularly, oxidation products of tryptophan by chemically generated singlet oxygen were compared to those obtained in dye-sensitizd photooxygenation. The usefulness of trimethylsilyl cyanide as a trapping reagent for dipolar peroxide intermediates has been demonstrated in the photooxygenation of N-methylindoles, 2-(methoxymethylene)adamantane and adamantylideneadamantane in aprotic solvents. Based on these trapping reactions mechanism of singlet oxygen reaction of electron-rich enol ethers and enamines is discussed in light of theoretical calculation.     

New Singlet Oxygen Source and Trapping Reagent for Peroxide Intermediates

Application of newly-developed water-soluble singlet oxygen sources in the oxidation of biologically important compounds and the electron-transfer process involving singlet oxygen has been reviewed. Particularly, oxidation products of tryptophan by chemically generated singlet oxygen were compared to those obtained in dye-sensitizd photooxygenation. The usefulness of trimethylsilyl cyanide as a trapping reagent for dipolar peroxide intermediates has been demonstrated in the photooxygenation of N-methylindoles, 2-(methoxymethylene)adamantane and adamantylideneadamantane in aprotic solvents. Based on these trapping reactions mechanism of singlet oxygen reaction of electron-rich enol ethers and enamines is discussed in light of theoretical calculation.     

Irradiation of titanium dioxide generates both singlet oxygen and superoxide anion.

Although photoexcited TiO2 has been known to initiate various chemical reactions, such as the generation of reactive oxygen species, precise mechanism and chemical nature of the generated species remain to be elucidated. The present work demonstrates the generation of singlet oxygen by irradiated TiO2 in ethanol as measured by ESR spectroscopy using 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TMP) as a 1O2-sensitive trapping agent. Under identical conditions, the superoxide ion was also detected by spin trapping agent 5,5-dimethyl-pyrroline-N-oxide (DMPO). Kinetic analysis in the presence of both 4-oxo-TMP and DMPO revealed that singlet oxygen is produced directly at the irradiated TiO2 surface but not by a successive reaction involving superoxide anion. The basis for this view is the fact that DMPO added in the mixture increased the signals responsible for 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), a reaction product of 4-oxo-TMP and 1O2. The detailed mechanism for the generation of 1O2 and superoxide ion by irradiated TiO2 and reactions between these species and DMPO are discussed.     

Carbon nanodots with a controlled N structure by a solvothermal method for generation of reactive oxygen species under visible light

Carbon nanodots can function as photosensitizers that have the ability to generate reactive oxygen species such as singlet oxygen, hydroxy (OH) radicals, and superoxide ions. However, most of these can only be generated upon ultraviolet light excitation. Additionally, the mechanism of reactive oxygen species generation by carbon nanodots remains unclear. The development of carbon nanodots that can photosensitize under visible light irradiation is desirable for applications such as photodynamic therapy and pollutant decomposition under visible light. Here, we report novel carbon nanodot-based photosensitizers that generate reactive oxygen species under visible light; they were synthesized using a solvothermal method with two solvents (formamide and water) and amidol as the carbon source. Carbon nanodots from the solvothermal synthesis in formamide showed blue fluorescence, while those obtained in water showed green fluorescence. The photo-excited blue-fluorescent carbon nanodots produced OH radicals, superoxide ions, and singlet oxygen, and therefore could function as both type I and type II photosensitizers. In addition, photo-excited green-fluorescent carbon nanodots generated only singlet oxygen, therefore functioning as type II photosensitizers. It is proposed that the two photosensitizers have different origins of reactive oxygen species generation: the enrichment of graphitic N for blue-fluorescent carbon nanodots and molecular fluorophores for green-fluorescent carbon nanodots.     

The light-dependent cytotoxicity of benzo[a]pyrene: effect on human erythrocytes, Escherichia coli cells, and Haemophilus influenzae transforming DNA

In vitro, the photodynamic compound benzo[a]pyrene (BAP) generates singlet oxygen efficiently when irradiated in organic solvents. It also photogenerates superoxide anion radical in water and can act as a photoreducing agent in the absence of oxygen. In vivo, the hemolysis of human erythrocytes, the inactivation of Escherichia coli cells representing a series of strains differing in excision repair and catalase proficiency, and the inactivation of Haemophilus influenzae transforming DNA activity were used to characterize the phototoxicity of BAP in the presence of near-UV light (290-400 nm). The results are consistent with BAP behaving as a photosensitizer that generates both superoxide and singlet oxygen, and that damages chiefly membranes. DNA does not seem to be a major target in the phototoxic reactions investigated.     

Singlet Oxygen Generated in PS￠ò Particles Promotes the Superoxide Generation

The relationship between superoxide and singlet oxygen in PSⅡ particle of spinach under strong illumination was studied with spin-trap ESR technique. The generation of superoxide was increased under D2 environment which prolonged the half life of singlet oxygen. The generation of superoxide was decreased when histidine existed as a scavenger of singlet oxygen. It is highly possible that the superoxide generated in PSⅡ particle originates from singlet oxygen.     

Near infrared emission of singlet oxygen generated in the dark

Singlet oxygen generation is reported from (1) enzymatic reaction and (2) electron transfer reactions of the superoxide anion measured directly with an ultrasensitive near-IR emission spectrophotometer by monitoring the O₂(¹Δ_g) → O₂ (³Σ_g^?) transition at 1268 nm. Near-IR emission spectra from the myeloperoxidase and lactoperoxidase enzymatic systems show only emission of singlet oxygen at 1268nm. The lipoxygenase/Na–linoleate enzymatic reaction exhibits two emissions, 1268 nm and 1288 nm. The latter emission is identified as originating from a peroxy radical. Spectral and kinetic data giving evidence of singlet oxygen generation is obtained from the reaction of potassium superoxide solubilized by 18-crown-6-ether in acetonitrile with a series of organometallic coordination compounds.     

Effect of normobaric oxygen treatment on oxidative stress and enzyme activities in guinea pig heart

Normobaric oxygen (NBO) therapy is commonly applied for the treatment of various diseases, including myocardial infarctions, but its effectiveness is controversial. Potential adverse effects of hyperoxia are related to excessive formation of free radicals. In the present study we examined the effect of 60-h NBO treatment on lipid peroxidation (LPO), activity of manganese superoxide dismutase (Mn-SOD) and mitochondrial enzymes of energy metabolism in guinea pig heart. NBO treatment resulted in significant accumulation of thiobarbituric acid reactive substances and loss of Mn-SOD activity despite slight elevation of Mn-SOD protein content. Activity of electron transport chain complex III decreased significantly, while activity of complex IV was slightly elevated and citrate synthase was unchanged. LPO, inhibition of Mn-SOD and complex III activities were more pronounced when inhaled oxygen was partially enriched with superoxide radical. In contrast, when O(2) was enriched with oxygen cation (O(2)●+), LPO and loss of Mn-SOD activity were prevented. Complex III activity in the O(2)●+-treated group remained depressed but activities of complex IV and citrate synthase were elevated. These data suggest that NBO treatment is associated with myocardial oxidative damage and attenuation of antioxidant defense, but these adverse effects can be partially attenuated by inhalation of O(2) enriched with oxygen cation.     

ESR Studies of a Series of Phthalocyanines. Mechanism of Phototoxicity. Comparative Quantitation of O2-using ESR Spin-Trapping and Cytochrome c Reduction Techniques

ESR experiments with 2,2,6,6-tetramethyl-4-piperi-done (4-oxo-TEMP) and the spin-trap 5,5-dimethyl pyrroline-N-oxide (DMPO) have been performed on a series of new phthalocyanines: the bis(tri-n-hexyl-siloxy) silicon phthalocyanine ([(nhex)₃SiO]₂SiPc), the hexadecachloro zinc phthalocyanine (ZnPcCl₁₆), the hexadecachloro aluminum phthalocyanine (AlPcCl₁₆), the hexadecachloro aluminum phthalocyanine sulfate (HSO₄A1PcCl₁₆), whose photocytotoxicity has been studied against various leukemic and melanotic cell lines. Type I and Type II pathways occur simultaneously in DMF although the Type II seems to be prevalent. These results are not changed when the bis(tri-n-hexylsiloxy) silicon phthalocyanine is entrapped into liposomes. By contrast, the Type I process is favored in membrane models for all the perchlori-nated phthalocyanines. This modified behavior may be accounted on a possible stacking of phthalocyanines in membranes and a preventing effect of axial ligands against aggregation in the case of the bis(tri-n-hexyl-siloxy) silicon phthalocyanine. The photodynamic action of zinc perchlorinated phthalocyanine is not dependent on singlet oxygen, phototoxicity of this molecule being essentially mediated by oxygen free radicals. Quantitation of the superoxide radical was accomplished, with good agreement, by two techniques: the cytochrome c reduction and the ESR quantitation based on the double integration of the first derivative of the ESR signal. The disproportionation of the superoxide radical or degradation of the spin-trap seem to be avoided in aprotic solvents such as DMF.     

Comparison of the photochemical and enzymic generation of excited states of oxygen on the induction of benzo(alpha)pyrene mono-oxygenase in liver cell cultures.

The photochemical generation of excited states of oxygen such as the superoxide ion(O-2) and singlet oxygen (1o2) by the mild illumination of culture medium containing riboflavin induces benzo(alpha)pyrene mono-oxygenase in 3 different cell lines derived from rat liver. Similar rates of O-2 generation can be produced by the action of xanthine oxidase on xanthine yet this system does not induce the mono-oxygenase. This result confirms that the mono-oxygenase induction is not mediated by O-2 is not mediated by O-2 and that 1O2 is the most likely candidate for stimulating the mono-oxygenase activity.     

Singlet oxygen production by human eosinophils

Human eosinophils, stimulated with phorbol myristate acetate, were found to produce 1268 nm chemiluminescence characteristic of singlet oxygen. Singlet oxygen generation required the presence of bromide ion. A bromide ion concentration of 100 microM, comparable to the total bromine content of whole blood, was sufficient for the eosinophils to generate measurable amounts of singlet oxygen. For the conditions used (10(7) cells/ml and 10 micrograms/ml phorbol myristate acetate), the duration of the singlet oxygen generation was brief, about 5 min, and the total yield of singlet oxygen was modest, 1.0 +/- 0.1 microM. The cells remained viable after the singlet oxygen production ceased. This is the first demonstration of singlet oxygen production from living cells. The singlet oxygen generated by eosinophils likely results from a peroxidase-catalyzed mechanism, since a purified eosinophil peroxidase-hydrogen peroxide-bromide system was also shown to produce singlet oxygen. The unique properties of eosinophil peroxidase are illustrated by the fact that at p2H 7.0 and with 100 microM bromide, eosinophil peroxidase generated 20 +/- 2% of the theoretical yield of singlet oxygen, whereas under identical conditions, myeloperoxidase and lactoperoxidase produced only 1.0 +/- 0.1% and -0.1 +/- 0.1%, respectively.     

Bromine derivatives of amino acids as intermediates in the peroxidase-catalyzed formation of singlet oxygen

Recently, J. R. Kanofsky et al. (1988, J. Biol. Chem. 263, 9692-9696) reported that human eosinophils generated modest amounts of singlet oxygen. In the mechanism proposed, hypobromous acid (made from the peroxidase-catalyzed oxidation of bromide ion) reacted with hydrogen peroxide to form singlet oxygen. In contrast, human neutrophils, which generate both hypochlorous acid and hydrogen peroxide, do not make singlet oxygen. The failure of human neutrophils to generate singlet oxygen is due in part to the trapping of hypochlorous acid by endogenous amines. In this paper, I show that amino acids are much more effective traps for hypochlorous acid than for hypobromous acid. Glycine totally inhibits singlet oxygen generation from a model enzyme system composed of chloroperoxidase, hydrogen peroxide, and chloride ion, but causes only a 35% reduction in singlet oxygen generation from an analogous enzyme system containing bromide ion instead of chloride ion. The products of the reaction of hypobromous and glycine (presumably an equilibrium mixture of N-bromoglycine, N,N-dibromoglycine, and hypobromous acid) retain the ability to react with hydrogen peroxide to form singlet oxygen. In contrast, the products of the reaction of hypochlorous acid and glycine do not react with hydrogen peroxide to produce singlet oxygen. Similar results were obtained for L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, and L-tyrosine. Thus, bromine derivatives of amino acids may act as intermediates in the peroxidase-catalyzed generation of singlet oxygen.     

Chemiluminescence response of the human polymorphonuclear neutrophil to lipopolysaccharides

Polymorphonuclear neutrophils (PMN) respond to a variety of stimuli with a sequence of reactions that lead to the production of "active oxygen" species, including H2O2, free radicals, such as superoxide (O2-.) and hydroxyl (HO.), and singlet molecular oxygen (1O2). Some of these can oxidize (5-amino-2,3-dihydrophthalazine 1,4-dione) (luminol) to the ground state aminophthalate ion; this reaction sequence is accompanied by the generation of a photon and forms the basis for the chemiluminescence (CL) response. In this work we used a dedicated photon counting instrument to record CL from PMN incubated with bacterial lipopolysaccharide (LPS). We have studied the CL response to the LPS from Escherichia coli strains 026:B6 and 055:B5, as well as Salmonella minnesota RE 595 and have determined that CL requires heat-labile serum factors, these most likely being intact components of the complement system.     

Scope and limitations of the TEMPO/EPR method for singlet oxygen detection: the misleading role of electron transfer

For many biological and biomedical studies, it is essential to detect the production of ¹O₂ and quantify its production yield. Among the available methods, detection of the characteristic 1270-nm phosphorescence of singlet oxygen by time-resolved near-infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with a singlet oxygen probe. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6-tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been widely employed, it can produce misleading data. This is demonstrated by the present study, in which the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method are compared for a set of well-known photosensitizers. The results reveal that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizer is efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP⁺ radical cation, followed by deprotonation and reaction with molecular oxygen, gives rise to an EPR-detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological, and medical studies.     

Water induced dismutation of superoxide anion generates singlet molecular oxygen

Direct spectroscopic measurement of 1268 nm singlet oxygen emission from KO2 suspensions at room temperature in three non-protonic solvents--CCl4, Cl2FCCClF2, and C6F14 by the action of water is reported. The results clearly show that the singlet oxygen generation is due to a water induced reaction, and suggest that one role of the enzyme superoxide dismutase may be the protection of biological structures, for example, lipid membranes, from degradation by singlet oxygen.     

A possible mechanism of the generation of singlet molecular oxygen in nadph-dependent microsomal lipid peroxidation.

A simplified system, consisting of NADPH, Fe3+-ADP, EDTA, liposomes, NADPH-cytochrome c reductase and Tris - HCl buffer (pH 6.8), has been employed in studies of the generation of singlet oxygen in NADPH-dependent microsomal lipid peroxidation. The light emitted by the system involves 1deltag type molecular oxygen identifiable by its characteristic emission spectrum and its behavior with beta-carotene. The generation of another excited species (a compound in the triplet state) could be demonstrated in this system by changes of light intensity and emission spectra which arise from photosensitizer (9,10-dibromoanthracene sulfonate, eosin, Rose-Bengal)-mediated energy transfers. Chemiluminescence in the visible region was markedly quenched by various radical trappers and by an inhibitor of NADPH-cytochrome c reductase, but not by superoxide dismutase. During the early stage of lipid peroxidation, the intensity of chemiluminescence was proportional to the square of the concentration of lipid peroxide. These characteristics suggest that singlet oxygen and a compound in the triplet state (probably a carbonyl compound) are generated by a self-reaction of lipid peroxy radicals.     

Light-dependent spin trapping of hydroxyl radical from human erythrocytes.

The generation of reactive oxygen species from human erythrocytes has previously been demonstrated. Furthermore, erythrocytic protoporphyrin IX has been shown to generate superoxide and singlet oxygen when exposed to light. These findings suggest that a component of erythrocytic reactive oxygen species production may be light-dependent. By inhibiting erythrocyte superoxide dismutase, catalase, and glutathione peroxidase with N,N-diethyldithiocarbamate or sodium cyanide, we demonstrate the light-dependent generation of hydroxyl radical in human erythrocytes using spin trapping/Electron Spin Resonance spectroscopy. This finding may be significant in tissues where blood is exposed to light, such as in the eye.