Antioxidant nanoreactor based on superoxide dismutase encapsulated in superoxide-permeable vesicles

We designed and tested an antioxidant nanoreactor based on encapsulation of Cu,Zn superoxide dismutase in amphiphilic copolymer nanovesicles, the membranes of which are oxygen permeable. The nanovesicles, made of poly(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline), successfully encapsulated the protein during their self-assembling process, as proved by confocal laser-scanning microscopy and fluorescence-correlation spectroscopy. Electron paramagnetic resonance spectroscopy and circular dichroism analyses showed that no structural changes appeared in the protein molecules once inside the inner space of the nanovesicles. The function of this antioxidant nanoreactor was tested by pulse radiolysis, which demonstrated that superoxide dismutase remains active inside the nanovesicles and detoxifies the superoxide radical in situ. The membrane of our triblock copolymer nanovesicles plays a double role, both to shield the sensitive protein and to selectively let superoxide and dioxygen penetrate to its inner space. This simple and robust hybrid system provides a selective shielding of sensitive enzymes from proteolytic attack and therefore a new direction for developing drug delivery applications.     

POTENTIATION OF OXIDATIVE DAMAGE TO PROTEINS BY ULTRAVIOLET-A AND PROTECTION BY ANTIOXIDANTS

We have studied the damage of alcohol dehydrogenase (ADH) and glyceraldehyde 3-phosphate dehydrogenase (GAPD) induced by Fe++/EDTA + H2O2 in combination with UV-A (main output at 365 nm). Enzyme inactivation, formation of hydroxyl radicals (measured in the absence of enzymes), increase in protein carbonyls, oxidation of sulfhydryl (SH) groups, loss of native protein fluorescence, and enhanced protease degradation were used to determine protein damage. Hydroxyl radical production was greatly enhanced by the combination of UV-A with Fe++/EDTA + H2O2. The combined treatment increased protein carbonyls but decreased native protein fluorescence and SH groups. The combined treatment caused turbidity in GAPD but not in ADH, whereas trypsin susceptibility was increased more in ADH than in GAPD. These measurements of protein oxidation correlated well with enzyme activities. Glyceraldehyde 3-phosphate dehydrogenase and dithiothreitol were most protective against such damage, while hydroxyl radical and singlet oxygen scavengers were partially effective. Superoxide dismutase had no effect. Thus, UV-A potentiation of protein damage induced by FE++/EDTA + H2O2 appeared to involve hydroxyl radicals and perhaps singlet oxygen but not superoxide radicals. The damage to proteins induced by combination of UV-A with physiological oxidants, iron ions and H2O2 may be relevant to UV-A-induced skin and tissue damage.     

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.     

Theory of stabilizing thermally stable polymers

It has been discovered that chemical compounds generated in some polymeric samples or articles and possessing high activity in the reactions with oxygen and free radicals as well as yielding inert products in the above reactions may stabilize the polymeric materials by inhibiting oxidative (and even thermal) degradation. The phenomenon described has both theoretical and experimental evidence.     

The microenvironment effect on the generation of reactive oxygen species by Pd-bacteriopheophorbide

Generation of reactive oxygen species (ROS) is the hallmark of important biological processes and photodynamic therapy (PDT), where ROS production results from in situ illumination of certain dyes. Here we test the hypothesis that the yield, fate, and efficacy of the species evolved highly depend on the dye's environment. We show that Pd-bacteriopheophorbide (Pd-Bpheid), a useful reagent for vascular targeted PDT (VTP) of solid tumors, which has recently entered into phase II clinical trials under the code name WST09 (trade name TOOKAD), forms appreciable amounts of hydroxyl radicals, superoxide radicals, and probably hydrogen peroxide in aqueous medium but not in organic solvents where singlet oxygen almost exclusively forms. Evidence is provided by pico- and nanosecond time-resolved spectroscopies, ESR spectroscopy with spin-traps, time-resolved singlet oxygen phosphorescence, and chemical product analysis. The quantum yield for singlet oxygen formation falls from approximately 1 in organic solvents to approximately 0.5 in membrane-like systems (micelles or liposomes), where superoxide and hydroxyl radicals form at a minimal quantum yield of 0.1%. Analysis of photochemical products suggests that the formation of oxygen radicals involves both electron and proton transfer from (3)Pd-Bpheid at the membrane/water interface to a colliding oxygen molecule, consequently forming superoxide, then hydrogen peroxide, and finally hydroxyl radicals, with no need for metal catalysis. The ability of bacteriochlorophyll (Bchl) derivatives to form such radicals upon excitation at the near infrared (NIR) domain opens new avenues in PDT and research of redox regulation in animals and plants.     

A novel functional imidazole fluorescent ionic liquid: simple and efficient fluorescent probes for superoxide anion radicals

Novel imidazole fluorescent ionic liquids with anthracene groups (ImS-FILA) were synthesized for the first time to act as fluorescent probes. They were developed for the determination of superoxide anion radicals (O₂ ^?-) in an aqueous system. O₂ ^?- was produced by pyrogallol autoxidation. The fluorescence of ImS-FILA was quenched by superoxide anion radicals. The π-bond structure of the fluorescent molecules was oxidized and damaged. This method is very simple and sensitive. The linear range of sensitivity was 1–70 μM ImS-FILA, and the detection limit for reactive oxygen species was 0.1 μM. This method was used to detect superoxide radicals in papaya and garlic, with satisfactory results. Further work is needed to demonstrate the utility of this method in detecting reactive oxygen species in a biological aqueous system.

Instantaneous carbon-carbon bond formation using a microchannel reactor with a catalytic membrane

Instantaneous catalytic carbon-carbon bond forming reactions were achieved in a microchannel reactor having a polymeric palladium complex membrane. The catalytic membrane was constructed inside the microchannel via self-assembling complexation at the interface between the organic and aqueous phases flowing laminarly, where non-cross-linked polymer-bound phosphine and ammonium tetrachloropalladate dissolved, respectively. A palladium-catalyzed coupling reaction of aryl halides and arylboronic acids was performed using the microchannel reactor to give quantitative yields of biaryls within 4 s of retention time in the defined channel region.     

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.     

内毒素性休克时血清的特异荧光研究

本文以大肠杆菌内毒素攻击介导的感染性多系统脏器功能衰竭家兔为模型，探讨了血清的特异荧光。应用发光理论讨论了模型血清的分子光谱，以及感染性多脏衰竭进程与特异荧光的关系。同时，还制定了血液、心、肝、肺、肾各组织器官中脂过氧化物代谢产物丙二醛和超氧化物歧化酶变化情况，结果具有明显的相关性和一致性。荧光法可作为感染性多脏器衰竭的早期诊断预示的一种方法。     

芯片毛细管电泳评估脂质体介导超氧化物歧化酶减低细胞内氧化应激

超氧化物歧化酶(SOD)可用作抗氧化的药物。它能催化并清除细胞内的活性氧组分(ROS),保护细胞免受自由基的氧化破坏。但是由于SOD分子量较大,难以透过细胞膜进入细胞内,显著降低了SOD的药效。本研究用激光共聚焦荧光显微镜拍摄的荧光图像说明,纳米脂质体可介导SOD进入细胞。用芯片毛细管电泳激光诱导荧光分析法(MCE-LIF)测定单细胞中ROS和谷胱甘肽(GSH)的荧光信号强度,评估了用脂质体包裹的SOD与细胞作用的抗氧化效果。用脂质体包裹的SOD与肝癌细胞共培养2h,与直接用SOD作用于肝癌细胞相比较,细胞内ROS明显降低,GSH明显提高。实验结果说明,用脂质体包裹SOD是一种减低细胞内氧化应激的有效给药途径。     

Production of O Radicals from Cavitation Bubbles under Ultrasound

In the present review, the production of O radicals (oxygen atoms) in acoustic cavitation is focused. According to numerical simulations of chemical reactions inside a bubble using an ODE model which has been validated through studies of single-bubble sonochemistry, not only OH radicals but also appreciable amounts of O radicals are generated inside a heated bubble at the violent collapse by thermal dissociation of water vapor and oxygen molecules. The main oxidant created inside an air bubble is O radicals when the bubble temperature is above about 6500 K for a gaseous bubble. However, the concentration and lifetime of O radicals in the liquid water around the cavitation bubbles are unknown at present. Whether O radicals play some role in sonochemical reactions in the liquid phase, which are usually thought to be dominated by OH radicals and H2O2, should be studied in the future.     

The Action of Peroxyl Radicals,Powerful Deleterious Reagents,Explains Why Neither Cholesterol Nor Saturated Fatty Acids Cause Atherogenesis and Age‐Related Diseases

Cells respond to alterations in their membrane structure by activating hydrolytic enzymes. Thus, polyunsaturated fatty acids (PUFAs) are liberated. Free PUFAs react with molecular oxygen to give lipid hydroperoxide molecules (LOOHs). In case of severe cell injury, this physiological reaction switches to the generation of lipid peroxide radicals (LOO^.). These radicals can attack nearly all biomolecules such as lipids, carbohydrates, proteins, nucleic acids and enzymes, impairing their biological functions. Identical cell responses are triggered by manipulation of food, for example, heating/grilling and particularly homogenization, representing cell injury. Cholesterol as well as diets rich in saturated fat have been postulated to accelerate the risk of atherosclerosis while food rich in unsaturated fatty acids has been claimed to lower this risk. However, the fact is that LOO^. radicals generated from PUFAs can oxidize cholesterol to toxic cholesterol oxides, simulating a reduction in cholesterol level. In this review it is shown how active LOO^. radicals interact with biomolecules at a speed transcending usual molecule–molecule reactions by several orders of magnitude. Here, it is explained how functional groups are fundamentally transformed by an attack of LOO^. with an obliteration of essential biomolecules leading to pathological conditions. A serious reconsideration of the health and diet guidelines is required.     

Spin trapping reactions with nitric oxides II. Acylalkyl nitroxides

Radical trapping by nitric oxide in the presence of oxygen has been studied by ESR spectroscopy. The oxygen was found to react much faster with alkyl radicals than nitric oxide, it suppresses formation of dialkyl nitroxides and initiates creation of acyl nitroxides through reactions between peroxy radicals and nitric oxide.     

BLUE LIGHT PERCEPTION BY ENDOGENOUS REDOX COMPONENTS OF THE PLANT PLASMA MEMBRANE

b-Type cytochromes of the higher plant plasma membrane may be reduced by irradiation with actinic blue light (light-induced absorbance change). Although this reaction has been reported to depend on the presence of an exogenous oxygen-scavenging system, significant cytochrome reduction was obtained in bean hook (Phaseolus vulgaris L. cv. “Limburgse Vroege”) plasma membranes without any addition. An endogenous oxygen-consuming reaction is apparently sufficient to achieve a proper redox balance. A blue light-mediated absorbance change with absorbance minima at 450 and 475 nm precedes cytochrome b reduction and indicates the presence of a flavoprotein in the plasma membrane fraction. Cytochrome b reduction by blue light in the absence of an oxygen scavenger is highly sensitive to flavin photosensitizers. Glucose oxidase, which has previously been used to lower the oxygen concentration in membrane samples, was demonstrated to have a photosensitizing effect. Inhibitors of flavin photochemical reactions (KI and phenylacetic acid) were highly effective in preventing cytochrome b reduction. These results indicate that the blue light-mediated reaction probably involves an endogenous plasma membrane flavoprotein as the photoreceptor. As plasma membrane NADH-dependent oxidoreductases potentially are flavoproteins these experiments raise the question whether a plasma membrane cytochrome b and a flavin-enzyme may cooperate in blue light reactions. Evidence is also discussed, suggesting the possible involvement of oxygen radicals in the blue light-induced cytochrome b reduction.     

ESR study on the antioxidant activity of TAK-218 in biological model membranes

TAK-218 has a 2,3-dihydrobenzofuran-5-amine (coumaran) structure which resembles alpha-tocopherol, and is a promising candidate as an agent for central nervous system (CNS) trauma and ischemia. The radical scavenging activity of TAK-218 was studied using electron spin resonance (ESR) spectroscopy. TAK-218 exhibited a more potent scavenging activity towards the hydroxyl radical than did the well-known hydroxyl radical scavengers, mannitol and dimethylsulfoxide. Towards the superoxide radical, TAK-218 showed equal potency to glutathione. TAK-218 reacted rapidly with stable radicals, such as galvinoxyl and 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH), and gave the quinone as a two-electron oxidized product in analogy with alpha-tocopherol. To exhibit an excellent antioxidative activity in living systems, the compounds should not only have the intrinsic radical scavenging activity but also good distribution in the biological lipid-bilayer membrane. To examine the antioxidant activity of TAK-218, the inhibition of lipid peroxidation by alpha-tocopherol and TAK-218 in liposomal membranes was studied using an ESR spin-label technique. Both alpha-tocopherol and TAK-218 completely inhibited lipid peroxidation by radicals generated in an aqueous layer using a water-soluble radical initiator, 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH). At a high incubation temperature (45 degrees C), alpha-tocopherol scavenged radicals more effectively than TAK-218 on the surface of the membrane, while TAK-218 scavenged radicals more effectively in the interior of the membrane. The difference between TAK-218 and alpha-tocopherol for radical scavenging in the membrane system derives from the different distribution pattern of these compounds. TAK-218 can penetrate the membrane freely and can scavenge the radical in the membrane interior. Furthermore, TAK-218 was shown to inhibit lipid peroxidation initiated by a lipid soluble radical initiator, 2,2'-azobis-(2,4-dimethylvaleronitrile) (AMVN), in a membrane more effectively than alpha-tocopherol.     

Chemical and photochemical electron transfer of new helianthrone derivatives: aspects of their photodynamic activity

Helianthrones 2-4 are a new class of synthetic photosensitizers, which have a molecular skeleton related to that of hypericin. We established that irradiation of heliantrones with visible light leads to the formation of semiquinone radicals and reactive oxygen species. The structures of the paramagnetic anion species produced by electron transfer were calculated on the density functional level and investigated by cyclovoltammetry, UV/vis, and EPR/ENDOR spectroscopy. As with hypericin, the pi system of the helianthrones was found to be considerably deviated from planarity, and, upon electron transfer, deprotonation in the bay region occurs. The structure of the semiquinone radicals was found to be identical in THF, DMF, and aqueous buffered solutions regardless of the means by which reduction was achieved. Semiquinone radicals can be formed via self-electron transfer between the excited state and the ground state or via electron transfer from an electron donor to the excited state of helianthrone. Therefore, the presence of an electron donor significantly enhanced the photogeneration of semiquinone and superoxide radical. The kinetic studies showed that no significant photochemical destruction of helianthrones occurred upon irradiation. Generation of superoxide and singlet oxygen upon irradiation of helianthrones was established by spin trapping techniques. This shows that both type I and type II mechanisms are of importance for the photodynamic action of these compounds.     

含硒杂环化合物的合成和对超氧阴离子作用的ESR研究

引言硒作为人体必需的微量元素,其主要生化作用与谷胱甘肽过氧化物酶密切相关.在体内,该酶可以催化许多过氧化物的分解,并且与谷胱甘肽一起组成一个强有力的细胞防御体系,发挥其抗氧化作用.近几年来,人们发现了一种小分子有机硒化物2-苯基-1,2-苯并异硒唑酮-3(简称Ebselen)具有类似谷胱甘肽过氧化物酶的活性.它能有效地抑制活性氧自由基的产生而引起的细胞损伤,具有良好的抗炎活性,且毒性极低(大鼠LD_(50)>6180mg/os).因而,普遍认为Ebselen及其类似物是一类具有广谱抗炎活性的潜在药物,在医药领域具有广阔的应用前景.     

Free Radicals as a Double-Edged Sword: The Cancer Preventive and Therapeutic Roles of Curcumin

Free radicals, generally composed of reactive oxygen species (ROS) and reactive nitrogen species (RNS), are generated in the body by various endogenous and exogenous systems. The overproduction of free radicals is known to cause several chronic diseases including cancer. However, increased production of free radicals by chemotherapeutic drugs is also associated with apoptosis in cancer cells, indicating the dual nature of free radicals. Among various natural compounds, curcumin manifests as an antioxidant in normal cells that helps in the prevention of carcinogenesis. It also acts as a prooxidant in cancer cells and is associated with inducing apoptosis. Curcumin quenches free radicals, induces antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), and upregulates antioxidative protein markers–Nrf2 and HO-1 that lead to the suppression of cellular oxidative stress. In cancer cells, curcumin aggressively increases ROS that results in DNA damage and subsequently cancer cell death. It also sensitizes drug-resistant cancer cells and increases the anticancer effects of chemotherapeutic drugs. Thus, curcumin shows beneficial effects in prevention, treatment and chemosensitization of cancer cells. In this review, we will discuss the dual role of free radicals as well as the chemopreventive and chemotherapeutic effects of curcumin and its analogues against cancer.     

Spectroscopic studies on the photostability and photoactivity of metallo-tetraphenylporphyrin in micelles

Inspired by natural photosynthesis, we employed polymeric micelles to enhance the water solubility and photostability of hydrophobic metallo-tetraphenylporphyrin (metallo-TPP) by complexation with poly(ethylene glycol)-block-poly(4-vinylpyridine) (PEG-b-P4VP) via axial coordination. The structure and photochemical properties of the complex micelles were characterized by UV-visible spectroscopy, fluorescence spectroscopy, and laser light scatting. The photostability and electron transfer ability of metalloporphyrins in the micelles were investigated under continuous irradiation. The results show that metallo-TPPs entrapped in the micellar cores possess higher photostability and better electron transfer ability. The hydrophobic metalloporphyrins remains active inside the micelles which is reminiscent of chlorophyll protein complex in photosynthesis. The use of micelle thus may provide a promising system on designing photocatalysts for targeting applications in solar energy conversion and photodynamic tumor therapy (PDT).