Singlet oxygen is essential for neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases.

Direct assessments of the antioxidant effects of the novel collagen peptide on reactive oxygen species using electron spin resonance spectroscopy.

In the present study, we evaluated the antioxidant effects of a pepsin-treated novel collagen peptide (P-NCP) on reactive oxygen species (ROS) such as hydroxyl radical (HO(•)), superoxide anion radical (O(2)(•-)), and singlet oxygen ((1)O(2)), and the effects on cell viability after ultraviolet ray (UV) irradiation of human fibroblasts. We confirmed, using electron spin resonance, that P-NCP directly inhibited HO(•) and (1)O(2). Furthermore, addition of P-NCP to fibroblasts inhibited cell death induced by UVA (400-315 nm) irradiation in a dose-dependent manner. In addition, the antioxidant effect on (1)O(2) was observed in the peptide fractions rich in Gly, Pro, Hyp, Glu, Ala, and Arg. We found that Gly, Hyp, Glu, and Ala directly scavenged (1)O(2). These results indicated that a peptide sequence including Gly, Hyp, Glu, and Ala could play a key role in the antioxidant effects of P-NCP on (1)O(2). It was suggested that P-NCP can inhibit photo-aging related to ROS owing to its antioxidant effects.

A preliminary assessment of singlet oxygen scavenging, cytotoxic and genotoxic properties of Geranium macrorrhizum extracts.

Strong radical-scavenging activity of Geranium macrorrhizum extracts isolated by using various solvent systems has been reported previously. This study aimed at expanding the knowledge on the bioactivities of antioxidatively active G. macrorrhizum butanol fraction, which was isolated from ethanolic extract (EB), and water fraction, which was isolated from water extract (WW) by measuring their singlet oxygen scavenging properties, as well as preliminary assessment of cytotoxicity and genotoxicity toward mammalian cells. The cytotoxicity (necrosis induction) of the extracts in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by antioxidants and stimulated by the prooxidant BCNU (N,N'-bis(2-chloroethyl)-N-nitrosourea). This indicates that the cytotoxicity of G. macrorrhizum extracts is at least partly attributed to their prooxidant action, presumably due to the formation of quinoidal products of their (auto)oxidation. The latter was evidenced by the nature of the peroxidase-catalyzed oxidation products, which supported DT-diaphorase-catalyzed oxidation of NADPH and participated in conjugation reactions with reduced glutathione. The genotoxic properties were studied using chromosome aberration (CA) and sister chromatid exchange (SCE) tests in human lymphocytes in vitro and Drosophila melanogaster somatic mutation and recombination test (SMART) in vivo. In the CA test, only the highest doses of both fractions significantly increased chromosome aberration frequency. In the SCE test, both fractions induced SCEs in a clear dose-dependent manner. G. macrorrhizum extracts were not genotoxic in the SMART test in vivo. Our data indicate that in spite of the possible beneficial (antioxidant) effects of Geranium extracts, the possibilities of their use as ingredients of functional foods and/or food supplements should be further examined due to their cyto- and genotoxic effects resulting mainly from the action of quercetin-derived components abundant in the extracts.

Edaravone directly reacts with singlet oxygen and protects cells from attack.

AIMS: Protective effects of edaravone, an approved medicine for acute brain infarction in Japan, on cell death induced by singlet oxygen (1O2) were examined. MAIN METHOD: The 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 FINDINGS: When rose Bengal (RB) in D2O was irradiated by a 514nm 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 525nm 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 100-400microM 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. SIGNIFICANCE: Since 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.

Effects of riboflavin photosensitization on daidzein and its photosensitized derivatives.

Photosensitized compounds from daidzein were studied in a riboflavin model system under visible light irradiation by high-performance liquid chromatography (HPLC). As the period of light irradiation increased, concentration of daidzein decreased significantly (P < 0.05) and new peaks of daidzein derivatives were observed and changed during photosensitization. Three new peaks from photosensitized daidzein were tentatively identified as 7-, 3', 4'-trihydroxyisoflavone (or 3'-hydroxydaidzein) and 2 dimmers of daidzein by a combination of HPLC-mass spectrometry (MS) and retention times of standard compounds by HPLC. Addition of sodium azide and removal of headspace oxygen treatment affected the formation of newly formed peaks. The type I pathway of riboflavin photosensitization played more important roles than type II pathways on the formation of daidzein derivatives. Practical Application: Isoflavones are important phytochemicals found in soy foods. Generally, many foods containing soy ingredients are displayed under visible light irradiation. Also, riboflavin can be found in many foods containing vegetables. The results of this study can be used to understand the stability and changes of isoflavone aglycones in soy and soy-based foods under visible light irradiation.

Changes of headspace volatiles in milk with riboflavin photosensitization.

Effects of fluorescent light, riboflavin, ascorbic acid, sodium azide, and butylated hydroxyanisole (BHA) on the volatiles in milk at 4 degrees C were determined using a combination of headspace-solid phase microextraction (HS-SPME), gas chromatography (GC), and mass spectrometry (MS). Pentanal, hexanal, heptanal, and dimethyl disulfide were formed only in the milk stored under light and increased significantly as the duration of light exposure increased from 0 to 8 h and the concentration of added riboflavin increased from 5 to 50 ppm (P < 0.05). As fat content in milk increased, peak areas of pentanal, hexanal, and heptanal increased significantly (P < 0.05) while those of dimethyl disulfide did not change significantly (P > 0.05). Sodium azide prevented the formation of dimethyl disulfide in milk, implying that dimethyl disulfide can be formed through singlet oxygen oxidation (type II pathway). Addition of ascorbic acid and BHA reduced the formation of hexanal, heptanal, and dimethyl disulfide significantly (P < 0.05). Generation mechanisms of pentanal seem to be different from those of hexanal and heptanal in milk. Both singlet oxygen oxidation (type II pathway) and free radicals (type I pathway) play important roles in the formation of light-induced volatiles in milk.

Singlet oxygen scavenging activity of non-steroidal anti-inflammatory drugs.

It has long been known that singlet oxygen ((1)O2) is generated during inflammatory processes. Once formed in substantial amounts, (1)O2 may have an important role in mediating the destruction of infectious agents during host defense. On the other hand, (1)O2 is capable of damaging almost all biological molecules and is particularly genotoxic, which gives a special relevance to the scavenging of this ROS throughout anti-inflammatory treatments. Considering that the use of non-steroidal anti-inflammatory drugs (NSAIDs) constitutes a first approach in the treatment of persistent inflammatory processes (due to their ability to inhibit cyclooxygenase), a putative scavenging activity of NSAIDs for (1)O2 would also represent a significant component of their therapeutic effect. The aim of the present study was to evaluate the scavenging activity for (1)O2 by several chemical families of NSAIDs. The results suggested that the pyrazole derivatives (dipyrone and aminopyrine) are, by far, the most potent scavengers of (1)O2 (much more potent compared to the other tested NSAIDs), displaying IC(50)-values in the low micromolar range. There was a lack of activity for most of the arylpropionic acid derivatives tested, with only naproxen and indoprofen displaying residual activities, as for the oxazole derivative, oxaprozin. On the other hand, the pyrrole derivatives (tolmetin and ketorolac), the indolacetic acid derivatives (indomethacin, and etodolac), as well as sulindac and its metabolites (sulindac sulfide and sulindac sulfone) displayed scavenging activity in the high micromolar range. Thus, the scavenging effect observed for dipyrone and aminopyrine will almost certainly contribute to their healing effect in the treatment of prolonged or chronic inflammation, while that of the other studied NSAIDs may have a lower contribution, though these assumptions still require further in vivo validation.

Tocochromanols, plastoquinol, and other biological prenyllipids as singlet oxygen quenchers-determination of singlet oxygen quenching rate constants and oxidation products.

Singlet oxygen quenching rate constants for tocopherol and tocotrienol homologues have been determined in organic solvents of different polarities, as well as for other biological prenyllipids such as plastoquinol, ubiquinol, and alpha-tocopherolquinol. The obtained results showed that the quenching activity of tocochromanols was mainly due to the chromanol ring of the molecule and the activity increased with the number of the methyl groups in the ring and solvent polarity. Among prenylquinols, alpha-tocopherolquinol was the most active scavenger of singlet oxygen followed by ubiquinol and plastoquinol. The oxidation products of tocopherols were identified as 8a-hydroperoxy-tocopherones which are converted to the corresponding tocopherolquinones under acidic conditions. The primary oxidation products of prenylquinols, containing unsaturated side chains, were the corresponding prenylquinones that were further oxidized to hydroxyl side-chain derivatives. In the case of plastochromanol, the gamma-tocotrienol homologue found in some seed oils, mainly the hydroxyl derivatives were formed, although 8a-hydroperoxy-gamma-tocopherones were also formed to a minor extent, both from plastochromanol and from its hydroxyl, side-chain derivatives. The obtained results were discussed in terms of the activity of different prenyllipids as singlet oxygen scavengers in vivo.

Photoprotection by porcine eumelanin against singlet oxygen production.

Melanin, a major pigment found in retinal pigment epithelium (RPE) cells, is considered to function in dual roles, one protective and one destructive. By quenching free radical species and reactive oxygen species (ROS) melanin counteracts harmful redox stress. However, melanin is also thought to be capable of creating ROS. In this destructive role, melanin increases redox strain in the cell. This study uses readily available eumelanin extracted from porcine RPE cells as a more authentic model than synthetic melanin to determine specific mechanisms of melanin activity with regard to singlet oxygen in the presence and absence of rose bengal, a singlet-oxygen photosensitizer. Optical detection of singlet-oxygen was determined by monitoring the bleaching of p-nitrosodimethylaniline in the presence of histidine. Production of singlet oxygen in aqueous oxygen-saturated solutions of rose bengal without eumelanin was readily accomplished. In contrast, detection of singlet oxygen in oxygen-saturated solutions of eumelanin without rose bengal failed, consistent with results of others. However, a significant decrease in singlet oxygen production by rose bengal was observed in the presence of eumelanin. After correction for light absorption and chemical bleaching of eumelanin, the results show that eumelanin also provides a photoprotective mode arising from chemistry, that is, not just the physical process of light absorption followed by energy dissipation as heat.

Reactivity towards singlet oxygen of propofol inside liposomes and neuronal cells.

Singlet oxygen (1O2), a reactive oxygen species, has been found to be implicated in many cellular events and pathological disorders. Herein, we investigated the reactivity of 1O2 towards the anaesthetic agent propofol (PPF) encapsulated within DMPC liposomes. By time resolved luminescence, the rate constant of 1O2 quenching by PPF was evaluated, depending on the location of the sensitizer, with following values: 1.35+/-0.05x10(7) M(-1) s(-1) for deuteroporphyrin (as embedded source) and 0.8+/-0.04x10(7) M(-1) s(-1) for uroporphyrin (as external source), respectively. The nature of the oxidation product, resulting from the reaction of 1O2 with PPF, was determined using absorption and HPLC techniques. Finally, the in vitro protective effect of PPF towards the 1O2-induced neuronal cell toxicity was evaluated in terms of cell viability.

Induction of phr gene expression in E. coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation.

We have observed that He-Ne laser irradiation of E. coli strain KY706/pPL-1 leads to induction of photolyase gene, phr. The magnitude of induction was found to depend on the He-Ne laser fluence, fluence rate and post-irradiation incubation period in the nutrient medium. The optimum values for fluence and fluence rate were 7x10(3) J/m(2) and 100 W/m(2), respectively, and the induction of phr gene was observed to saturate beyond an incubation period of approximately 2 h. Experiments carried out with singlet oxygen quenchers and with D(2)O suggest that the effect is mediated via singlet oxygen. Photoreactivation studies carried out after UVC exposure of both the He-Ne laser-exposed as well as unexposed cells showed a larger surviving fraction in the He-Ne laser pre-irradiated cells. This can be attributed to He-Ne laser irradiation-induced induction of phr expression. However, since even without photoreactivating light He-Ne laser pre-irradiated cells show higher survival against UVC radiation it appears that He-Ne laser irradiation induces both light-dependent as well as dark DNA repair processes.