Capacity of Tea Scavenging Singlet Oxygen Studied by Means of 1,3-Diphenylisobenzofuran as Fluorescence Probe

1 Introduction In recent years, the effects of reactive oxygen species(ROS) generated in the course of biological metabolism, such as superoxide(O_2~(-.)), hydrogen peroxide(H_2O_2), hydroxyl radical(HO~.) and singlet oxygen(~1O_2) on the human health have received more attention due to their vital roles in physiological functions. Normally, antioxidant molecules, superoxide dismutase and catalase in biological organism can scavenge excessive free radicals by a series of chemical reactions to keep the cells in a state of redox homeostasis[1].     

Quantification of singlet oxygen production in the reaction of superoxide with hydrogen peroxide using a selective chemiluminescent probe

A sensitive chemiluminescent probe that selectively reacts with singlet oxygen in the presence of superoxide and hydrogen peroxide has been used to quantify the production of singlet oxygen in the reaction of superoxide with hydrogen peroxide. The yield of singlet oxygen from this reaction was found to be low (0.2% relative to the initial superoxide concentration). No evidence for the formation of hydroxyl radical was observed in this reaction, ruling out the Haber-Weiss mechanism as a major singlet oxygen formation pathway. No singlet oxygen production was observed in the reaction of superoxide with 2-nitrobenzoic acid, which has a pKa similar to that of hydrogen peroxide, rendering the protonation of superoxide, followed by its disproportionation, an unlikely explanation for the formation of singlet oxygen in this system. The low yields of singlet oxygen and hydroxyl radical suggest that their formation in this reaction should be relatively unimportant in biological systems.     

Water Activation for Boosting Electrochemiluminescence

In conventional luminol electrochemiluminescence (ECL) systems, hydrogen peroxide and dissolved oxygen are employed as typical co-reactants to produce reactive oxygen species (ROS) for efficient ECL emission. However, the self-decomposition of hydrogen peroxide and limited solubility of oxygen in water inevitably restrict the detection accuracy and luminous efficiency of luminol ECL system. Inspired by ROS-mediated ECL mechanism, for the first time, we used cobalt-iron layered double hydroxide as co-reaction accelerator to efficiently activate water to generate ROS for enhancing luminol emission. Experimental investigations verify the formation of hydroxyl and superoxide radicals in the process of electrochemical water oxidation, which subsequently react with luminol anion radicals to trigger strong ECL signals. Finally, the detection of alkaline phosphatase has been successfully achieved with impressive sensitivity and reproducibility for practical sample analysis.     

Design of an Oxygen-Tolerant Photo-RAFT System for Protein-Polymer Conjugation Achieving High Bioactivity

Protein-polymer conjugates have significant potential in pharmaceutical and biomedical applications. To enable their widespread use, robust conjugation techniques are crucial. This study introduces a photo-initiated reversible addition-fragmentation chain-transfer (Photo-RAFT) polymerization system that exhibits excellent oxygen tolerance. This system allows for the synthesis of protein-polymer conjugates with high bioactivity under mild and aerobic conditions. Three photocatalytic systems utilizing Eosin Y (EY) as the photocatalyst with two different cocatalysts (ascorbic acid and triethanolamine) were investigated, each generating distinct reactive oxygen species (ROS) such as singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals. The impact of these ROS on three model proteins (lysozyme, albumin, and myoglobin) was evaluated, demonstrating varying bioactivities based on the ROS produced. The EY/TEOA system was identified as the optimal photo-RAFT initiating system, enabling the preparation of protein-polymer conjugates under aerobic conditions while maintaining high protein enzymatic activity. To showcase the potential of this approach, lysozyme-poly(dimethylaminoethyl acrylate) conjugates were successfully prepared and exhibited enhanced antimicrobial property against Gram-positive and Gram-negative bacteria.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS—IV. THE PHOTOLYSIS OF BENOXAPROFEN, AN ANTI-INFLAMMATORY DRUG WITH PHOTOTOXIC PROPERTIES

Abstract— Benoxaprofen [2-(4-chlorophenyl)-α-methyl-5-benzoxazole acetic acid] is an anti-inflammatory drug that causes acute phototoxicity in many patients. Photolysis studies in organic solvents (ethanol, benzene, dimethylsulfoxide) showed that benoxaprofen underwent both Type I and Type II reactions. Irradiation of an anerobic solution of benoxaprofen in ethanol resulted in hydrogen abstraction from the solvent to yield hydroxyethyl and ethoxyl radicals. In the presence of oxygen, superoxide, singlet oxygen and hydroxyethyl radicals were detected. Photolysis of benoxaprofen in air-saturated benzene or dimethylsulfoxide gave superoxide. However, under anerobic conditions the drug yielded a carbon-centered radical in benzene that could not be identified. These findings suggest that both oxygen-dependent and oxygen-independent processes may be important in the phototoxic reactions of benoxaprofen.     

检测活性氧物种的氧杂蒽类光学探针的研究进展

活性氧物种在维持生物体的生理功能方面发挥着重要的作用.高于正常水平的活性氧物种会损伤蛋白质、DNA等生物分子,进而导致疾病.因此,活性氧物种的高选择性、高灵敏度检测研究对疾病的预防、诊断和治疗均具有重要意义.荧光探针因具有分析灵敏度高、样品时空分辨能力强等特点,已在该方面获得了广泛的应用.其中,具有发射波长长,光稳定性好,荧光量子产率高等优点的氧杂蒽类荧光探针已成为检测活性氧物种的研究热点.本论文主要总结了近五年来应用于活性氧物种检测的氧杂蒽类荧光探针的研究进展与成像分析,归纳了不同活性氧物种的识别单元,并展望了此类探针的发展趋势与应用前景.     

A Role for Hydrogen Peroxide in the Pro-apoptotic Effects of Photodynamic Therapy

Although the first reactive oxygen species (ROS) formed during irradiation of photosensitized cells is almost invariably singlet molecular oxygen (¹O₂), other ROS have been implicated in the phototoxic effects of photodynamic therapy (PDT). Among these are superoxide anion radical (^•O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (^•OH). In this study, we investigated the role of H₂O₂ in the pro-apoptotic response to PDT in murine leukemia P388 cells. A primary route for detoxification of cellular H₂O₂ involves the peroxisomal enzyme catalase. Inhibition of catalase activity by 3-amino-1,2,4-triazole led to an increased apoptotic response. PDT-induced apoptosis was impaired by addition of an exogenous recombinant catalase analog (CAT- skl) that was specifically designed to enter cells and more efficiently localize in peroxisomes. A similar effect was observed upon addition of 2,2'-bipyridine, a reagent that can chelate Fe⁺², a co-factor in the Fenton reaction that results in the conversion of H₂O₂ to ^•OH. These results provide evidence that formation of H₂O₂ during irradiation of photosensitized cells contributes to PDT efficacy.     

Hybrid TiO2–Ruthenium Nano‐photosensitizer Synergistically Produces Reactive Oxygen Species in both Hypoxic and Normoxic Conditions

Photodynamic therapy (PDT) is widely used to treat diverse diseases, but its dependence on oxygen to produce cytotoxic reactive oxygen species (ROS) diminishes the therapeutic effect in a hypoxic environment, such as solid tumors. Herein, we developed a ROS‐producing hybrid nanoparticle‐based photosensitizer capable of maintaining high levels of ROS under both normoxic and hypoxic conditions. Conjugation of a ruthenium complex (N3) to a TiO₂ nanoparticle afforded TiO₂‐N3. Upon exposure of TiO₂‐N3 to light, the N3 injected electrons into TiO₂ to produce three‐ and four‐fold more hydroxyl radicals and hydrogen peroxide, respectively, than TiO₂ at 160 mmHg. TiO₂‐N3 maintained three‐fold higher hydroxyl radicals than TiO₂ under hypoxic conditions via N3‐facilitated electron–hole reduction of adsorbed water molecules. The incorporation of N3 transformed TiO₂ from a dual type I and II PDT agent to a predominantly type I photosensitizer, irrespective of the oxygen content.     

SINGLET OXYGEN INVOLVEMENT IN THE INACTIVATION OF CULTURED HUMAN FIBROBLASTS BY UVA (334 nm, 365 nm) AND NEAR-VISIBLE (405 nm) RADIATIONS

The UVA (320-380 nm) radiation inactivation of mammalian cells is dependent upon the presence of oxygen. In order to examine the intermediates involved, we have irradiated cells in the presence of chemical probes which are able to modify the activity of various oxygen species. We have also examined the possibility that UVA inactivates cultured human fibroblasts via generation of intracellular hydrogen peroxide. An iron scavenger (desferrioxamine) and a hydroxyl radical scavenger (dimethylsulfoxide) protect the cells against hydrogen peroxide. Diethyldithiocarbamate (a superoxide dismutase inhibitor) and aminotriazole (a catalase inhibitor) sensitize the cells to this oxidizing agent. These data support previous reports that hydrogen peroxide inactivates as a result of the iron-catalyzed generation of hydroxyl radical. None of these agents significantly alter the fluence-dependent inactivation of cell populations by radiation at 365 nm. In contrast, the cells are sensitized to radiation at 334, 365 and 405 nm in the presence of deuterium (an enhancer of singlet oxygen lifetime) and are protected against radiation at 365 nm by sodium azide (a quencher of singlet oxygen). These results are consistent with the conclusion that the generation of singlet oxygen, but not hydrogen peroxide or hydroxyl radical, plays an important role in the inactivation of cultured human cells by UVA and near-visible radiations.     

Quantitation of the Reactive Oxygen Species Generated by the UVA Irradiation of Ascorbic Acid-Glycated Lens Proteins

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein-bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol . 62, 454–463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/ cm² of absorbed UVA light (λ > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 μ M as determined by the superoxide dismutase (SOD)-depen-dent increase in hydrogen peroxide formation and of 52 μ M by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 μM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system.
Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N- dimethyl-4-nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H₂O₂ were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen.     

Role of molecular oxygen and its active forms in generation of electrochemiluminescence

A short review is dedicated to the role of molecular oxygen and its active forms in generation of electrochemiluminescence (ECL). It is shown at the example of such widely used luminogens as luminol, lucigenin, acridine esters, acridane, and indoles that the role of O₂ and its active forms, such as hydrogen peroxide, superoxide and hydroperoxide radicals in generation of ECL by organic compounds is largely reduced to oxidation of the initial luminogen or intermediates of its partial electrochemical preoxidation/prereduction. Such intermediates are most often particles in the doublet state (radical-cation, radical-anion, or free radicals) that form unstable emitter precursors of peroxide (dioxetanone) type under interaction with O₂, hydrogen peroxide, or other active oxygen forms. It is also shown that the product of secondary transformations of active oxygen forms is singlet oxygen that may also be a radiation source as a result of cooperative transitions.     

Towards tuning PDT relevant photosensitizer properties: comparative study for the free and Zn2+ coordinated meso-tetrakis[2,6-difluoro-5-(N-methylsulfamylo)phenyl]porphyrin

The spectroscopic, photochemical, and biological studies of 5,10,15,20-tetrakis[2,6-difluoro-5(N-methylsulfamylo)phenyl]porphyrinate Zn(II) (ZnF₂PMet) were carried out including absorption and fluorescence spectra, fluorescence quantum yields, triplet absorption spectra, triplet lifetimes, singlet oxygen quantum yield, and reactive oxygen species (ROS) detection under biological conditions and compared with its free-base analog (F₂PMet). Zinc coordination into the porphyrin ring results in decrease of hydrophobicity and in higher cellular uptake. F₂PMet localized specifically in endoplasmic reticulum and mitochondria while the ZnF₂PMet is more diffused all over the cell, bonded to membrane proteins, as assessed by fluorescence microscopy. Zn-porphyrin exhibits greater singlet oxygen quantum yield than its free-base analog. Studies with fluorescent probes confirm that the ZnF₂PMet produces mostly singlet oxygen, whereas F₂PMet generates more hydroxyl radicals as the ROS. F₂PMet is a more effective photosensitizer in vitro than its zinc complex, thus, the final photodynamic effect depends more on the nature of ROS than on the higher cellular uptake.     

活性氧测定的基本原理与方法

综述了过氧化氢 (H2 O2 )、一重态氧 (1 O2 )、超氧阴离子自由基 (·O-2 )以及羟自由基 (·OH)等活性氧的测定方法。侧重介绍活性氧的化学反应法、捕捉法和直接测定法的基本原理和最新的进展情况 ,并比较了这几种方法的各自特点     

Recent advances in fluorescent probes for the detection of reactive oxygen species

Reactive oxygen species (ROS) have captured the interest of many researchers in the chemical, biological, and medical fields since they are thought to be associated with various pathological conditions. Fluorescent probes for the detection of ROS are promising tools with which to enhance our understanding of the physiological roles of ROS, because they provide spatial and temporal information about target biomolecules in in vivo cellular systems. ROS probes, designed to detect specific ROS with a high selectivity, would be desirable, since it is now becoming clear that each ROS has its own unique physiological activity. However, dihydro-compounds such as 2′,7′-dichlorodihydrofluorescein (DCFH), which have traditionally been used for detecting ROS, tend to react with a wide variety of ROS and are not completely photostable. Some attractive fluorescent probes that exhibit a high degree of selectivity toward specific ROS have recently been reported, and these selective probes are expected to have great potential for elucidating unknown physiological mechanisms associated with their target ROS. This review focuses on the design, detection mechanism, and performance of fluorescent probes for the detection of singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), hydroxyl radicals (^.OH), or superoxide anion (O₂ ^−.), a field in which remarkable progress has been achieved in the last few years.     

Oxidative modifications of the Photosystem II D1 protein by reactive oxygen species: from isolated protein to cyanobacterial cells

Action of reactive oxygen species (ROS) on the isolated D1 protein, a key component of Photosystem II (PSII) complex, was studied and compared with the effect of high irradiance on this protein in mildly solubilized photosynthetic membranes and cells of the cyanobacterium Synechocystis. Whereas singlet oxygen caused mainly protein modification reflected by shift of its electrophoretic mobility, action of hydrogen peroxide and superoxide resulted in generation of specific fragments. Hydroxyl radicals as the most ROS induced fast disappearance of the protein. The results substantiate the ability of ROS to cause direct scission of the D1 peptide bonds. Similar D1 modification, fragmentation and additionally cross-linking with other PSII subunits were observed during illumination or hydrogen peroxide treatment of mildly solubilized thylakoids. Peroxide-induced fragmentation did not occur in thylakoids of the strain lacking a ligand to the nonheme iron, confirming the role of this prosthetic group in the D1-specific cleavage. The D1 modification, fragmentation and cross-linking were suppressed by ROS scavengers, supporting the direct role of ROS in these phenomena. Identical symptoms of the ROS-induced D1 damage were detected in illuminated cells of Synechocystis mutants with a higher probability of ROS formation, documenting the relevance of the in vitro results for the situation in vivo.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS–XV. ANTHRALIN and ITS OXIDATION PRODUCT 1,8-DIHYDROXYANTHRAQUINONE

The photochemistry (Type I and II) of anthralin and its photo-oxidation product 1,8-dihydroxyanthraquinone (1,8-DHAQ) has been studied in ethanol, acetonitrile and dimethylsulfoxide using spin-trapping and direct detection of singlet oxygen (1O2) luminescence techniques. In ethanol, where it exists in its neutral form (AN), anthralin does not undergo either Type I or II reactions upon UV-irradiation. In contrast, irradiation of anthralin in acetonitrile, a solvent in which anthralin is partially converted to its corresponding mono-anion (AN-), generates both superoxide and singlet oxygen. Irradiation of anthralin in dimethylsulfoxide, where the AN- form is present in substantial quantity, generates superoxide and solvent derived radicals but no detectable singlet oxygen. UV-irradiation of 1,8-DHAQ in ethanol and acetonitrile produces both superoxide and singlet oxygen in significant yields. In dimethylsulfoxide, on the other hand, only superoxide and solvent derived radicals are observed. The 1O2 quantum yield for AN- and 1,8-DHAQ in acetonitrile were determined to be 0.14 and 0.88 relative to rose bengal in the same solvent. These findings suggest that the AN photosensitization occurs via Type I and II pathways, is solvent dependent and involves AN- as well as its oxidation product 1,8-DHAQ, which is a more potent generator of both singlet oxygen and superoxide.     

HEMOLYSIS OF HUMAN ERYTHROCYTES BY ACTIVATED OXYGEN SPECIES

Abstract— The hemolysis of human erythrocytes by irradiation at 254 nm has been studied. Neither superoxide radicals nor singlet oxygen play a significant rôle and it is likely that the major species involved are hydroxyl radicals and, indirectly, carbonate anion or formate radicals. Similarly, when erythrocytes are treated with a system commonly used as source of superoxide radicals (photoreduction of riboflavin) it has been demonstrated that O^-₂ does not participate in lysis, but that singlet oxygen (possibly with hydroxyl radicals) is a major oxygen species involved in destruction of the cell membrane.     

Spectroscopic properties and photodynamic effects of new lipophilic porphyrin derivatives: efficacy, localisation and cell death pathways

Photodynamic therapy (PDT) and photodynamic diagnostics (PDD) of cancer are based on the use of non-toxic dyes (photosensitisers) in combination with harmless visible light. This paper reports physicochemical properties, cell uptake, localisation as well as photodynamic efficiency of two novel lipophilic porphyrin derivatives, suitable for use as PDT sensitisers. Both compounds are characterised by high quantum yield of singlet oxygen generation which was measured by time-resolved phosphorescence. Photodynamic in vitro studies were conducted on three cancer cell lines. Results of cell survival tests showed negligible dark cytotoxicity but high phototoxicity. The results also indicate that cell death is dependent on energy dose and time following light exposure. Using confocal laser scanning microscopy both compounds were found to localise in the cytoplasm around the nucleus of the tumour cells. The mode of cell death was evaluated based on the morphological changes after differential staining. In summary, good photostability, high quantum yield of singlet oxygen and biological effectiveness indicate that the examined lipophilic porphyrin derivatives offer quite interesting prospects of photodynamic therapy application.     

The Photochemistry of the Retinoids as Studied by Steady-State and Pulsed Methods

The retina and retinal pigment epithelium contain a number of retinoids in a metabolic pathway that eventually forms the visual pigments. This study investigates the photochemistry of those retinoids that may contribute to light-induced damage to the retina. These include retinal (RAL), retinol (ROL), retinylpalmitate (ROLpal) and the protonated Schiff-base of retinal (RAL.,). Their photochemistry was followed by both EPR spin-trapping techniques and the direct detection of singlet oxygen via its luminescence at 1270 nm. Irradiation (>300 nm) of RAL, ROL in methanol (MeOH) or RALpal in dimeth-ylformamide, produces free radicals from both solvents. Illumination of RAL., in MeOH containing NADH with light above 400 nm (and even above 455 nm) generates the superoxide radical. We also determined that the quantum yields for singlet oxygen sensitization by RAL, ROL or RALpal in MeOH are 0.05, 0.03 and 4.01, respectively. These values are at least 75% less than those previously found using chemical methods. These observations indicate that a major photochemical process for these retinoids may be an electron (or hydrogen) process that will lead to radical products, and that the singlet oxygen mechanism is of relatively minor importance in protic solvents. These results may explain the action spectra obtained from light-induced damage to the retina.     

DOUBLY ALLYLIC HYDROPEROXIDE FORMED IN THE REACTION BETWEEN STEROL 5,7-DIENES AND SINGLET OXYGEN

Abstract Ergosterol and 7-dehydrocholesterol, common 5,7-conjugated diene sterols, react with photochemically produced singlet oxygen very efficiently to yield, in parallel pathways, the corresponding 5,8-endoperoxides and the 7β-hydroperoxy-5,8(9),22-trienol or -5, 8(9)-dienol, respectively. The hydroperoxides decompose in an acid-catalyzed reaction to generate hydrogen peroxide and the 5, 7, 9(1 1), 22-tetraenol or 5, 7, 9(11) trienol, respectively, with 1:l stochiometry. The molar ratio of endoperoxide to hydroperoxide was constant (16:5) with two different reaction solvents, two different photosensitizers, and at all time points between 5 min and 3 h from the start of irradiation. Ergosterol did not react with either hydrogen peroxide or superoxide ion under our reaction conditions. Inhibition studies with nitrogen, 2,5-dimethylfuran, βcarotene, and tert -butanol confirmed the involvement of singlet oxygen in these reactions. The unstable hydroperoxide would be expected to have undesirable biological consequences if formed in vivo .