PHOTODYNAMIC LIPID PEROXIDATION BY THE PHOTOSENSITIZING NONSTEROIDAL ANTIINFLAMMATORY DRUGS SUPROFEN AND TIAPROFENIC ACID

Abstract The photochemistry of the photosensitizing nonsteroidal antiinflammatory drugs tiaprofenic acid and suprofen involves the intermediacy of short-lived species ( i.e. radicals). The data obtained in the present work strongly suggest that such intermediates may be responsible for the phototoxicity of 2-arylpropionic acids by inducing photodynamic lipid peroxidation at drug concentrations likely to be reached in the skin. This has been investigated using linoleic acid as a model lipid and determining the amount of hydroperoxides by measuring the spectrophotometric absorption at 233 nm, associated with the formation of dienic hydroperoxides. The major photoproducts of tiaprofenic acid and suprofen are derivatives bearing an ethyl side chain. Photoproducts of this type, due to the lack of polar moieties, are highly lipophilic and likely to accumulate in the lipid bilayer of cell membranes. Taking into account their ability to induce photodynamic lipid peroxidation and their marked photostability, it is conceivable that such photoproducts can participate in many catalytic cycles, playing a significant role in the mechanism of photosensitizatinn by tiprofenic acid and suprofen.     

Increased efficacy of in vitro Photofrin photosensitization of human oral squamous cell carcinoma by iron and ascorbate

Photofrin^®, a photosensitizer used in the photodynamic therapy of cancer, selectively localizes in cellular membranes. Upon exposure to visible light, Photofrin^® produces singlet oxygen (¹O₂), which reacts with membrane polyunsaturated fatty acids forming lipid hydroperoxides. Transition metals, such as Fe²⁺, catalyze the production of cytotoxic free radicals from lipid hydroperoxides. Ascorbate reduces ferric to ferrous iron, further augmenting lipid peroxidation. Therefore, to increase the efficacy of Photofrin^® photosensitization, we added 20 μM ferrous sulfate and 100 μM ascorbic acid, in an aqueous layer over SCC-25 oral squamous cell carcinoma cells during in vitro illumination. In electron paramagnetic resonance spin trapping experiments, using POBN (-(4-pyridyl-1-oxide)-N-tert-butylnitrone), we observed that the presence of this pro-oxidant combination greatly increases the production of membrane-derived lipid free radicals. The effect was time dependent but only partially concentration dependent. Trypan blue dye exclusion demonstrated that this increase in lipid radical formation correlated with cytotoxicity. These observations support the hypothesis that Photofrin^® photosensitization leads to lipid hydroperoxide formation, which increases the cell's susceptibility to iron-induced Fenton chemistry. The resulting free radical-mediated lipid peroxidation results in cell death. From these data we hypothesize that the efficacy of photodynamic therapy of superficial cancer might be increased by the topical application of the pro-oxidant combination of iron and ascorbate. Furthermore, their use will probably allow lower doses of Photofrin^® without compromising antitumor effect.     

TIN ETIOPURPURIN DICHLORIDE-SENSITIZED LIPID PHOTOOXIDATION OF ERYTHROCYTE MEMBRANES

Tin(IV) etiopurpurin dichloride (SnET2 x 2Cl) is a photosensitizer which has been shown to be an effective photodynamic agent for the treatment of transplantable animal tumors in vivo. The purpose of this study was to understand the effect of SnET2 x 2Cl on membrane lipid peroxidation. When erythrocyte membranes were exposed to visible light in the presence of SnET2 x 2Cl, lipid peroxidation was observed. An accumulation of lipid hydroperoxides and an increase in lipid fluorescence were also observed. Thin layer chromatography of lipid extracts from photooxidized membrane revealed photoperoxide products derived from phospholipid. Investigations into the mechanism(s) of lipid peroxidation by SnET2 x 2Cl and light-sensitized membranes were also performed. Results indicate that singlet oxygen (1O2) plays a major role in lipid peroxidation.     

Photosensitized oxidation of cholesterol in biological systems: reaction pathways, cytotoxic effects and defense mechanisms.

Cholesterol resembles other unsaturated lipids in being susceptible to peroxidative degradation when exposed to a sensitizing agent, exciting light of suitable wavelength and molecular oxygen. Selected hydroperoxides of cholesterol can be used as relatively convenient and reliable indicators of primary photochemical mechanisms, allowing a distinction to be made between free radical-mediated and singlet oxygen-mediated reactions. When generated in cell membranes, hydroperoxides of cholesterol and other lipids can have deleterious effects on membrane structure and function. Such damage may be exacerbated if these photoproducts undergo one-electron reduction to oxyl radicals which in turn initiate chain peroxidation reactions. Cells can resist these effects by using a membrane-based glutathione peroxidase to catalyze the two-electron reduction and detoxification of lipid hydroperoxides. Recent advances in our understanding of cholesterol photo-oxidation from the standpoints of (a) mechanistic information, (b) cytotoxicity and (c) cytoprotection are discussed in this article.     

Swallow-tailed perylene derivative: a new tool for fluorescent imaging of lipid hydroperoxides

A swallow-tailed perylene derivative including a triphenylphosphine moiety was synthesized and applied to the detection and the live-cell imaging of lipid hydroperoxides. The novel probe, named Spy-LHP, reacted rapidly and quantitatively with lipid hydroperoxides to form the corresponding oxide, Spy-LHPOx, which emits extremely strong fluorescence (Phi approximately 1) in the visible range (lambda(em) = 535 nm, 574 nm). Spy-LHP was highly selective for lipid hydroperoxides, and the addition of other reactive oxygen species (ROS) including hydrogen peroxides, hydroxyl radical, superoxide anion, nitric oxide, peroxynitrite, and alkylperoxyl radical, caused no significant increase in the fluorescence intensity. The probe exhibited good localization to cellular membranes and was successfully applied to the confocal laser scanning microscopy (CLSM) imaging of lipid hydroperoxides in live J774A.1 cells, in which lipid peroxidation was proceeded by the stimulation of 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH). These findings establish Spy-LHP as a promising new tool for investigating the physiology of lipid hydroperoxides.     

PROTECTIVE ACTION OF CAROTENOID PIGMENTS AGAINST PHOTODYNAMIC DAMAGE TO LIPOSOMES*

Abstract— When liposomes (a model membrane system) are subjected to a dye-sensitized photo-oxidation, lysis, as measured by glucose leakage or a change in light scattering, results. Before lysis occurs, the membrane lipids undergo peroxidative damage, as determined by the appearance of malondialdehyde. Carotenoids incorporated into the liposomal membrane protect against both lipid peroxidation and liposomal lysis. Other ¹O₂ quenchers and free radical absorbers also protect liposomes from photodynamic damage.     

IRON-SULFUR CENTERS AS ENDOGENOUS BLUE LIGHT SENSITIZERS IN CELLS: A STUDY WITH AN ARTIFICIAL NON-HEME IRON PROTEIN

The possible involvement of Fe-S clusters in photodynamic reactions as endogenous sensitizing chromophores in cells has been investigated, by using an artificial non-heme iron protein (ANHIP) derived from bovine serum albumin and ferredoxins isolated from spinach and a red marine algae. Ferredoxins and ANHIP, when exposed to visible light, generate singlet oxygen, as measured by the imidazole plus RNO method. Irradiation with intense blue light of the ANHIP-entrapped liposomes caused severe membrane-damage such as liposomal lysis and lipid peroxidation. In the presence of ANHIP, isocitrate dehydrogenase and fructose-1,6-diphosphatase were photoinactivated by blue light. However, all of these photosensitized reactions were significantly suppressed by a singlet oxygen (1O2) quencher, azide, but enhanced by a medium containing deuterium oxide. Further, the Fe-S proteins with the prosthetic groups destroyed did not initiate the blue light-induced reactions. In addition, the action spectrum for 1O2 generation from ANHIP was very similar to the visible absorption spectrum of Fe-S centers. The results obtained in this investigation appear consistent with the suggestion that Fe-S centers are involved in photosensitization in cells via a singlet oxygen mechanism.     

EPR spin-trapping evidence for the direct,one-electron reduction of tert-butylhydroperoxide to the tert-butoxyl radical by copper(II): paradigm for a previously overlooked reaction in the initiation of lipid peroxidation

Lipid peroxidation is often initiated using Cu(II) ions. It is widely assumed that Cu(II) oxidizes preformed lipid hydroperoxides to peroxyl radicals, which propagate oxidation of the parent fatty acid via hydrogen atom abstraction. However, the oxidation of alkyl hydroperoxides by Cu(II) is thermodynamically unfavorable. An alternative means by which Cu(II) ions could initiate lipid peroxidation is by their one-electron reduction of lipid hydroperoxides to alkoxyl radicals, which would be accompanied by the generation of Cu(III). We have investigated by EPR spectroscopy, in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide, the reactions of various Cu(II) chelates with tert-butylhydroperoxide. Spectra contained signals from the tert-butoxyl, methyl, and methoxyl radical adducts. In many previous studies, the signal from the methoxyl adduct has been assigned incorrectly to the tert-butylperoxyl adduct, which is now known to be unstable, releasing the tert-butoxyl radical upon decomposition. This either is trapped by 5,5-dimethyl-1-pyrroline N-oxide or undergoes beta-scission to the methyl radical, which either is trapped or reacts with molecular oxygen to give, ultimately, the methoxyl radical adduct. By using metal chelates that are known to be specific in either their oxidation or reduction of tert-butylhydroperoxide (the Cu(II) complex of bathocuproine disulfonic acid and the Fe(II) complex of diethylenetriaminepentaacetic acid, respectively) for comparison, we have been able to deduce, from the relative concentrations of the three radical adducts, that the Cu(II) complexes tested each reduce tert-butylhydroperoxide directly to the tert-butoxyl radical. These findings suggest that a previously overlooked reaction, namely the direct reduction of preformed lipid hydroperoxides to alkoxyl radicals by Cu(II), may be responsible for the initiation of lipid peroxidation by Cu(II) ions.     

Linoleic acid peroxidation vs. isomerization: a biomimetic model of free radical reactivity in the presence of thiols

Biomimetic models of free radical-induced transformation of polyunsaturated fatty acids, such as micelles and liposomes, have been used for the study of lipid peroxidation and lipid isomerization. Free radical reactivity of thiol compounds is the common link between the two processes, since lipid peroxidation is inhibited by thiols, due to their H-donation ability, whereas lipid isomerization is catalysed by S-centered radicals. In this paper the two processes are compared for the first time, in solution and under biomimetic conditions, demonstrating that hydroperoxides and trans lipids are formed to comparable extents as a result of oxidative free radical conditions. The biomimetic model of micelles of linoleic acid, prepared by addition of a non-ionic surfactant (TWEEN(?)-20) and 2-mercaptoethanol as the amphiphilic thiol, was irradiated by ionizing radiation up to 400 Gy under various conditions. In air-equilibrated solutions, the cis-trans isomerization process was observed with a catalytic cycle of 370 together with a substantial amount of hydroperoxides (LOOH). The effect of micelle size was also studied in order to envisage the effect of the supramolecular organization on the outcome of the two processes, and in particular, for the positional preference of the double bond isomerization.     

Antioxidant and immunity activities of Fufang Kushen Injection Liquid

We investigated the effects of Fufang Kushen Injection Liquid (FFKSIL) on gastric immunity and oxidant-antioxidant status during N-methyl-N′-nitro-N-nitroso-guanidine (MNNG)-induced gastric carcinogenesis. The extent of lipid peroxidation and the levels of reduced glutathione (GSH) and activities of the GSH-dependent enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were used to monitor the peroxidative balance. Enhanced lipid peroxidation in the gastric cancer animals was accompanied by significant decreases in the activities of GSH, GPx, GST and GR. Administration of FFKSIL significantly enhanced serum IgA, IgG, IgM, IL-2, IL-4 and IL-10 levels, decreased serum IL-6 and TNF-α levels, lowered the levels of lipid peroxides and enhanced GSH levels and activities of GSH-dependent enzymes. Our results suggest that FFKSIL blocks experimental gastric carcinogenesis by protecting against carcinogen-induced oxidative damage and improving immunity activity.     

Photodynamic action of benzo[a]pyrene in K562 cells

Benzo[a]pyrene (BaP) is ubiquitously distributed in the environment, being considered the most phototoxic element among polycyclic aromatic hydrocarbon (PAHs). In presence of oxygen, PAHs can act as a photosensitizer by means of promoting photo-oxidation of biological molecules (photodynamic action, PDA). Thus, the present study analyzed the photodynamic action of BaP under UVA irradiation (BaP + UVA) and its oxidative effects on K562 cells. The evaluation of BaP kinetics showed that the highest intracellular concentration occurred after 18 h of incubation. Cell viability, reactive oxygen species (ROS) generation, lipid peroxidation, DNA damage (breaks and DNA-protein cross-link [DNAPC]) were assessed after exposure to BaP, UVA and BaP plus UVA irradiation (BaP + UVA). Cell viability was decreased just after exposure to BaP + UVA. Lipid peroxidation and DNA breaks increased after BaP + UVA exposure, while the DNAPC increased after BaP, UVA and BaP + UVA exposure, suggesting that BaP + UVA effects were a consequence of both type II (producing mainly singlet oxygen) and type I (producing others ROS) mechanisms of PDA.     

Photodynamic effects induced by meso-tetrakis[4-(carboxymethyleneoxy)phenyl] porphyrin on isolated Sarcoma 180 ascites mitochondria.

Using mitochondria isolated from Sarcoma 180 ascites tumour in Swiss mice as a model system, we have evaluated the ability of a novel porphyrin, meso-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin (H₂T4CPP), to induce damage on photosensitization. Oxidative damage to mitochondria, one of the primary and crucial targets of the photodynamic effect, is assessed by measuring products of lipid peroxidation such as thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH), besides the loss of activity of the mitochondrial marker enzyme succinate dehydrogenase (SDH). Analysis of product formation, the effect of deuteration and selective inhibition by scavengers of reactive oxygen species (ROS) show that the damage observed is due mainly to singlet oxygen (¹O₂) and to a minor extent to hydroxyl radicals (^•OH). The ¹O₂ generation and triplet lifetime of this porphyrin have also been estimated. Fluorescence spectroscopy, used to ascertain the binding of this porphyrin to the mitochondrial proteins, shows a rapid association within 0–2 h and a decline thereafter. Confocal microscopy reveals intracellular localisation of this porphyrin in cells in vitro. Our overall results suggest that the porphyrin H₂T4CPP, due to its ability to bind to mitochondrial protein components and to generate ROS upon photoexcitation, may have potential applications in photodynamic therapy.     

Cholesterol Peroxidation as a Special Type of Lipid Oxidation in Photodynamic Systems

Like other unsaturated lipids in cell membranes and lipoproteins, cholesterol (Ch) is susceptible to oxidative modification, including photodynamic oxidation. There is a sustained interest in the pathogenic properties of Ch oxides such as those generated by photooxidation. Singlet oxygen (¹O₂)‐mediated Ch photooxidation (Type II mechanism) gives rise to three hydroperoxide (ChOOH) isomers: 5α‐OOH, 6α‐OOH and 6β‐OOH, the 5α‐OOH yield far exceeding that of the others. 5α‐OOH detection is relatively straightforward and serves as a definitive indicator of ¹O₂ involvement in a reaction, photochemical or otherwise. Like all lipid hydroperoxides (LOOHs), ChOOHs can disrupt membrane or lipoprotein structure/function on their own, but subsequent light‐independent reactions may either intensify or attenuate such effects. Such reactions include (1) one‐electron reduction to redox‐active free radical intermediates, (2) two‐electron reduction to redox‐silent alcohols and (3) translocation to other lipid compartments, where (1) or (2) may take place. In addition to these effects, ChOOHs may act as signaling molecules in reactions that affect cell fates. Although processes a‐c have been well studied for ChOOHs, signaling activity is still poorly understood compared with that of hydrogen peroxide. This review focuses on these various aspects Ch photoperoxidation and its biological consequences.     

Applications on the monitoring of oxidative modification of LDL by capillary electrophoresis: a comparison with spectrophotometer assay

The aim of this work is the application of Stocks and Miller capillary electrophoresis (CE) method in order to evaluate the human LDL susceptibility to Cu²⁺-induced oxidation. Lipid peroxidation determines a change in the relative electrophoretic mobility (REM) of lipoprotein that can be monitored by capillary electrophoresis using uncoated fused silica capillaries and tricine-methylglucamine as electrophoretic buffer.We have evaluated the differences in the susceptibility to oxidation of LDL subjected to different preparations (dialysis or gel filtration, after ultracentrifugation, to remove EDTA), and different storage (4 °C for 1 week or lyophilization) by measuring REM and lipid hydroperoxides (ROOH) with a spectrophotometer assay. Our results indicate that gel filtration is a more convenient procedure than dialysis for the isolation of LDL and that lyophilised samples are less prone to oxidation than those stored at 4 °C. Moreover, REM appears to be a more sensitive and easier method than spectrophotometer assay both to monitor the oxidative modification of LDL and to evaluate oxidative state of native LDL.     

SOME RELATIONSHIPS BETWEEN ULTRAVIOLET LIGHT AND HEME-PROTEIN-INDUCED PEROXIDATIVE LIPID BREAKDOWN IN LIPOSOMES, AS REFLECTED BY FLUORESCENCE CHANGES: THE EFFECT OF NEGATIVE SURFACE CHARGE

Abstract— The water soluble, photolabile nitrene precursor,azidonaphthalene–2,7-disulfonic acid (ANDS) was encapsulated in small unilamellar, isoelectric (egg PC) or negatively charged (egg PC + dihexadecylphosphate) liposomes. The individual and combined effects of heme-proteins and UV irradiation on the fluorescence of these vesicles under aerobic conditions were studied. Consistent with the catalytic action of heme-proteins on lipid peroxidation and peroxide decomposition, addition of cytochrome c (positively charged) or catalase (negatively charged) to the vesicles elicited immediate formation of a fluorescence band at 470 nm, characteristic of Schiff bases that form from aldehyde byproducts of decomposing hydroperoxides. Ultraviolet irradiation of liposomes for 5 min caused no significant changes in the fluorescence spectrum, in spite of the radiolysis of ANDS inside the vesicles with consequent formation of nitrene radicals. When isoelectric vesicles were irradiated with UV light in the presence of cytochrome c or catalase, Schiff base formation was further increased by2–3 fold, which effect was not observed in the absence of internal ANDS, or in the presence of negative surface charge on the vesicles. These findings suggest that (a) UV irradiation, by itself, cannot trigger lipid decomposition even when it is assisted by photoproduced nitrene radicals, (b) there is a ternary synergism between UV light, heme-proteins and nitrene radicals in promoting peroxidative lipid breakdown, and (c) negative surface charge inhibits the above synergism, which effect is unlikely to be due to electrostatic interaction between the vesicles and the protein or the ANDS.     

A highly water-soluble C60-NVP copolymer: a potential material for photodynamic therapy

Water-soluble C60 polymers, which have the highest solubility of fullerene reported to date (7.8 mM of C60 in water), were prepared via copolymerization, and showed significant visible light-induced O2*- generation and DNA cleavage, indicating the potential of these water-soluble fullerene derivatives as agents for photodynamic therapy.     

Photodynamic effects induced by meso-tetrakis[4-(carboxymethyleneoxy)phenyl] porphyrin on isolated Sarcoma 180 ascites mitochondria

Using mitochondria isolated from Sarcoma 180 ascites tumour in Swiss mice as a model system, we have evaluated the ability of a novel porphyrin, meso-tetrakis[4-(carboxymethyleneoxy)phenyl]porphyrin (H₂T4CPP), to induce damage on photosensitization. Oxidative damage to mitochondria, one of the primary and crucial targets of the photodynamic effect, is assessed by measuring products of lipid peroxidation such as thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH), besides the loss of activity of the mitochondrial marker enzyme succinate dehydrogenase (SDH). Analysis of product formation, the effect of deuteration and selective inhibition by scavengers of reactive oxygen species (ROS) show that the damage observed is due mainly to singlet oxygen (¹O₂) and to a minor extent to hydroxyl radicals (^•OH). The ¹O₂ generation and triplet lifetime of this porphyrin have also been estimated. Fluorescence spectroscopy, used to ascertain the binding of this porphyrin to the mitochondrial proteins, shows a rapid association within 0–2 h and a decline thereafter. Confocal microscopy reveals intracellular localisation of this porphyrin in cells in vitro. Our overall results suggest that the porphyrin H₂T4CPP, due to its ability to bind to mitochondrial protein components and to generate ROS upon photoexcitation, may have potential applications in photodynamic therapy.     

BENZOPHENONE-SENSITIZED LIPID PEROXIDATION IN LINOLEATE MICELLES

Abstract— Benzophenone-photosensitized peroxidation in linoleic acid micelles (Type I reaction) has been investigated and quantum yields of both sensitizer photolysis and lipid peroxidation have been obtained. Benzophenone as well as its derivative, benzophenone-4'-heptyl-4-pentanoic acid (BHPA) have been utilized as sensitizers in this study. A quantum yield of 1.2 for hydroperoxide formation, compared with a benzophenone disappearance quantum yield of 0.41 (in the same linoleate micellar solution) was obtained; yields of 1.3 and 0.6 were found for comparable systems in which BHPA was the sensitizer. Although such data are consistent with the peroxidation mechanism of chain reaction, it is necessary to know the yields of initiating species to arrive at chain reaction lengths in these systems. Such yields have been determined by flash photolysis. The dependencies of sensitizer photolysis and hydroperoxide yields on linoleate concentration have also been determined in order to elucidate the effects of micelle formation or molecular organization on the peroxidative process. Additionally, for comparison in a saturated surfactant (lipid) system hydrogen abstraction was investigated by photolysis techniques in sodium dodecyl sulfate alone using these two sensitizers.     

近红外发光的聚集诱导发光分子在抗菌光动力治疗中的应用

抗生素的误用和滥用,使越来越多的耐药细菌出现,对人类构成致命威胁。近年来,聚集诱导发光材料的发展和生物学科的交叉融合,为治疗细菌感染提供了许多创新思路。相对于紫外/可见光,近红外(NIR)光具有优异的组织深度渗透性和安全性等独特优势,有利于构建光动力抗菌平台进行深度治疗。随着对聚集诱导发光分子(AIEgens)设计及应用的不断探索,AIEgens在光动力抗菌治疗中表现出巨大的应用潜力。本文综述了NIR发光的AIEgens通过光动力疗法治疗细菌感染的研究进展,讨论了不同结构的聚集诱导发光材料存在的主要问题以及该领域当前的挑战和前景。     

Phthalocyanine-sensitized lipid peroxidation in cell membranes: use of cholesterol and azide as probes of primary photochemistry

Various phthalacyanine (Pc) derivatives of phototherapeutic interest have been shown to be efficient type II (singlet oxygen, 1O2) sensitizers in aqueous and non-aqueous solutions. However, primary Pc photochemistry in biological environments, e.g. cell membranes, has not been studied in a definitive manner. To address this question, we used endogenous cholesterol in the erythrocyte ghost as a mechanistic reporter lipid Membranes sensitized with chloroaluminum Pc tetrasulfonate (AlPcS) and exposed to white light at 10 degrees C underwent lipid peroxidation, as indicated by the accumulation of hydroperoxides and thiobarbituric acid reactivity. Specific analysis of cholesterol photo-products by thin layer chromatography and high performance liquid chromatography revealed the presence of 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), with much smaller amounts of 3 beta-hydroxycholest-5-ene-7 alpha-hydroperoxide (7 alpha-OOH) and 5 alpha-cholest-6-en-3 beta, 5-diol and cholest-5-en-3 beta, 7 alpha-diol (5 alpha-OH and 7 alpha-OH). Identification of 5 alpha-OOH as a major photoproduct provides unambiguous evidence for large scale 1O2 intermediacy. Azide inhibited lipid peroxidation in a dose-dependent fashion, providing additional support for a type II mechanism. However, the 1O2 quenching constant from Stern-Volmer analysis was approximately 50 times lower than that determined for a non-membrane probe, lactate dehydrogenase. The latter value agreed with literature values. A probable explanation is that membrane-bound dye generates most of the 1O2 involved in lipid peroxidation. Although azide can intercept any 1O2 escaping into (or formed in) the medium, it has limited access to 1O2 generated on the membrane and reacting (or being quenched) near its site of origin.