A mechanistic study of the photooxidation of A2E, a component of human retinal lipofuscin.

A major constituent of human retinal lipofuscin is A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-hexatrienyl]-pyridinium). Light transmitted by the lens is absorbed by A2E and the processes initiated by this absorption has been implicated in several maculopothies. The purpose of this study was to evaluate the dominant photochemical mechanisms involved in these reactions, whether through free radical or singlet oxygen intermediacy. The photodestruction of A2E occurs faster in water vs. chloroform and hydrogenated vs. perdeuterated methanol. Both results suggest a free radical mechanism. Product distributions indicate sequential oxygen addition rather than the addition of two oxygen atoms which would be expected if singlet oxygen was an intermediate. Finally, EPR trapping studies lead to the detection of superoxide as the primary intermediate in the photochemical reactions. It is concluded that if singlet oxygen is involved in these photochemical processes it is of minor importance.

Interaction of singlet molecular oxygen with melatonin and related indoles.

Singlet molecular oxygen (1O2) is one of the major agents responsible for (photo)oxidative damage in biological systems including human skin and eyes. It has been reported that the neural hormone melatonin (MLT) can abrogate 1O2-mediated cytotoxicity through its purported high antioxidant activity. We studied the interaction of MLT with 1O2 in deuterium oxide (D2O), acetonitrile and methanol by measuring the phosphorescence lifetime of 1O2 in the presence of MLT and related indoles for comparison. Rose bengal (RB) was used as the main 1O2 photosensitizer. The rate constant (kq) for the total (physical and chemical) quenching of 1O2 by MLT was determined to be 4.0 x 10(7) M(-1) s(-1) in D2O (pD 7), 6.0 x 10(7) M(-1) s(-1) in acetonitrile, and 6.1 x 10(7) M(-1) s(-1) in methanol-d1. The related indoles, tryptophan, 5-hydroxyindole, 5-methoxytryptamine, 5-hydroxytryptamine (5-OH-T, serotonin), 6-hydroxymelatonin (6-OH-MLT) and 6-chloromelatonin quenched 1O2 phosphorescence with similar kq values. We also compared the photosensitized photobleaching rate of MLT with that of other indoles, which revealed that MLT is the most sensitive to 1O2 bleaching. Hydroxylation of the indole moiety in 5-OH-T and 6-OH-MLT makes them more sensitive to photodegradation. In the absence of exogenous photosensitizers MLT itself can generate 1O2 with low quantum yield (0.1 in CH3CN) upon UV excitation. Thus, the processes we investigated may occur in the skin and eyes during physiological circadian rhythm (photo)signaling involving MLT and other indoles. Our results indicate that all the indoles studied, including MLT, are quite efficient yet very similar 1O2 quenchers. This directly shows that the exceptional antioxidant ability proposed for MLT is unsubstantiated when merely chemical mechanism(s) are considered in vivo, and it must predominantly involve humoral regulation that mobilizes other antioxidant defenses in living organisms.

Peroxidase- and nitrite-dependent metabolism of the anthracycline anticancer agents daunorubicin and doxorubicin.

Oxidation of the anticancer anthracyclines doxorubicin (DXR) and daunorubicin (DNR) by lactoperoxidase(LPO)/H(2)O(2) and horseradish peroxidase(HRP)/H(2)O(2) systems in the presence and absence of nitrite (NO(2)(-)) has been investigated using spectrophotometric and EPR techniques. We report that LPO/H(2)O(2)/NO(2)(-) causes rapid and irreversible loss of anthracyclines' absorption bands, suggesting oxidative degradation of their chromophores. Both the initial rate and the extent of oxidation are dependent on both NO(2)(-) concentration and pH. The initial rate decreases when the pH is changed from 7 to 5, and the reaction virtually stops at pH 5. Oxidation of a model hydroquinone compound, 2,5-di-tert-butylhydroquinone, by LPO/H(2)O(2) is also dependent on NO(2)(-); however, in contrast to DNR and DXR, this oxidation is most efficient at pH 5, indicating that LPO/H(2)O(2)/NO(2)(-) is capable of efficiently oxidizing simple hydroquinones even in the neutral form. Oxidation of anthracyclines by HRP/H(2)O(2)/NO(2)(-) is substantially less efficient relative to that by LPO/H(2)O(2)/NO(2)(-) at either pH 5 or pH 7, most likely due to the lower rate of NO(2)(-) metabolism by HRP/H(2)O(2). EPR measurements show that interaction of anthracyclines and 2,5-di-tert-butylhydroquinone with LPO/H(2)O(2)/NO(2)(-) generates the corresponding semiquinone radicals presumably via one-electron oxidation of their hydroquinone moieties. The possible role of the (*)NO(2) radical, a putative LPO metabolite of NO(2)(-), in oxidation of these compounds is discussed. Because in vivo the anthracyclines may co-localize with peroxidases, H(2)O(2), and NO(2)(-) in tissues, their oxidation via the proposed mechanism is likely. These observations reveal a novel, peroxidase- and nitrite-dependent mechanism for the oxidative transformation of the anticancer anthracyclines, which may be pertinent to their biological activities in vivo.

Biological effects of menadione photochemistry: effects of menadione on biological systems may not involve classical oxidant production.

Because cell-mediated reduction of menadione leads to the generation of reactive oxygen species (ROS), this quinone is widely used to investigate the effects of ROS on cellular functions. We report that A549 human lung epithelial cells exposed to menadione demonstrate a dose-dependent increase in both intracellular calcium ([Ca(2+)](i)) and ROS formation. The concentrations of menadione required to initiate these two events are markedly different, with ROS detection requiring higher levels of menadione. Modulators of antioxidant defences (e.g. buthionine sulphoximine, 3-amino-1,2,4-triazole) have little effect on the [Ca(2+)](i) response to menadione, suggesting that ROS formation does not account for menadione-dependent alterations in [Ca(2+)](i). Additional evidence suggests that menadione photochemistry may be responsible for the observed [Ca(2+)](i) effects. Specifically: (a) EPR studies with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) show that light exposure (maximum effect at 340 nm) stimulates menadione-dependent formation of the DMPO/(.)OH spin adduct that was not sensitive to antioxidant interventions; (b) DMPO inhibits menadione and light-dependent increases in [Ca(2+)](i); and (c) light (maximum effect at 340 nm) augments the deleterious effects of menadione on cell viability as determined by (51)Cr release. These photo effects do not appear to involve formation of singlet oxygen by menadione, but rather are the result of the oxidizing chemistry initiated by menadione in the triplet state. This work demonstrates that menadione species generated by photo-irradiation can exert biological effects on cellular functions and points to the potential importance of photochemistry in studies of menadione-mediated cell damage.

Investigation of the mechanism of action of microperoxidase-11, (MP11), a potential anti-cataract agent, with hydrogen peroxide and ascorbate.

The interaction of hydrogen peroxide, ascorbate and microperoxidase-11 (MP11), a ferriheme undecapeptide derived from cytochrome c, has been investigated using spectrophotometry, oxymetry, electron paramagnetic resonance (EPR), and mass spectroscopy techniques. It is shown that in 50 m M phosphate pH 7. 0-7.4 in the absence of other reactants H(2)O(2)induces a concentration-dependent decrease in absorption at the Soret band (399 nm) of the microperoxidase, with concomitant H(2)O(2)decomposition and oxygen evolution. The reaction causes irreversible heme degradation, concomitant with loss of enzymatic activity. Ascorbate effectively protects MP11 from degradation and inhibits oxygen evolution. At ascorbate concentrations greater than that of H(2)O(2), microperoxidase degradation is almost completely prevented. Mass spectrometry showed that H(2)O(2)oxidizes the microperoxidase to a monooxygenated product, which did not form if ascorbate was included in the reaction system. There appears to be a 1:1 relationship between H(2)O(2)degradation and ascorbate oxidation. EPR experiments revealed that an ascorbate radical was formed during the reaction. These reactions may be described by a scheme where a putative 'compound I' of the microperoxidase is reduced by ascorbate back to the original redox state (ferric) of the peroxidase in two one-electron steps, concomitantly with oxidation of the ascorbate to an ascorbate radical or in one two-electron transfer step forming dehydroascorbate. In the absence of ascorbate, the 'compound I' reacts further with the peroxide causing microperoxidase degradation and partial oxygen evolution. These observations are relevant to the interaction of ferrihemes with H(2)O(2)and ascorbic acid and may be pertinent for the potential application of MP11 as an anti-cataract agent.

Oxidation of biological electron donors and antioxidants by a reactive lactoperoxidase metabolite from nitrite (NO2-): an EPR and spin trapping study.

We report that a lactoperoxidase (LPO) metabolite derived from nitrite (NO2-) catalyses one-electron oxidation of biological electron donors and antioxidants such as NADH, NADPH, cysteine, glutathione, ascorbate, and Trolox C. The radical products of the reaction have been detected and identified using either direct EPR or EPR combined with spin trapping. While LPO/H2O2 alone generated only minute amounts of radicals from these compounds, the yield of radicals increased sharply when nitrite was also present. In aerated buffer (pH 7) the nitrite-dependent oxidation of NAD(P)H by LPO/H2O2 produced superoxide radical, O2*-, which was detected as a DMPO/*O2H adduct. We propose that in the LPO/H2O2/NO2-/biological electron donor systems the nitrite functions as a catalyst because of its preferential oxidation by LPO to a strongly oxidizing metabolite, most likely a nitrogen dioxide radical *NO2, which then reacts with the biological substrates more efficiently than does LPO/H2O2 alone. Because both nitrite and peroxidase enzymes are ubiquitous our observations point at a possible mechanism through which nitrite might exert its biological and cytotoxic action in vivo, and identify some of the physiological targets which might be affected by the peroxidase/H2O2/nitrite systems.

Electron paramagnetic resonance and spin trapping investigations of the photoreactivity of human lens proteins.

Oxidation of cysteine, glutathione and ascorbate by photoexcited proteins from normal and cataractous lenses was investigated using electron paramagnetic resonance in combination with spin trapping. We report that illumination of these proteins in pH 7 buffer with light > 300 nm in the presence of thiols (RSH) and a spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), afforded DMPO/S-cysteine and DMPO/SG adducts, suggesting the formation of the corresponding thiyl radicals. In a nonbuffered aqueous solution, illumination of the proteins and glutathione also produced superoxide detected as a DMPO/O2H adduct. Irradiation of these proteins in the presence of ascorbate generated ascorbate radical. We conclude that chromophores present in the natural normal and cataractous lenses are capable of initiating photooxidative processes involving endogenous thiols and ascorbic acid. This observation may be pertinent to UV-induced development of cataract.

Lactoperoxidase-catalyzed oxidation of melanin by reactive nitrogen species derived from nitrite (NO2-): an EPR study.

The reaction of synthetic DOPA melanin (DM) with lactoperoxidase (LPO), hydrogen peroxide, and nitrite (NO2-) has been investigated using EPR. We observed that in the presence of nitrite LPO/H2O2 generated large amount of melanin radicals, as evidenced by a strong, up to 11-fold, increase in the intensity of the melanin EPR signal. In contrast, when nitrite was omitted the increase was much less, ca. 30%, which, nevertheless, indicates that DM can be metabolized directly by LPO/H2O2. When the nitrite was present, the concentration of melanin radicals was linearly dependent on [NO2-] (for [NO2-] <5 mM), and increased when [LPO] and [H2O2] increased (at constant [NO2-]). We propose that the mechanism for the generation of melanin radicals by the LPO/H2O2/nitrite system involves oxidation of NO2- by LPO/H2O2 to a reactive metabolite, most likely the nitrogen dioxide radical (.NO2), which subsequently reacts with melanin 5,6-dihydroxyindole subunits producing the respective semiquinone radicals. Because melanin and .NO2 generating systems (nitrite, peroxidase enzymes, hydrogen peroxide) may coexist in cells in vivo, our results suggest that melanin could function as a natural scavenger of this highly reactive nitrogen species. This property may be relevant to the physiological functions of the melanin pigments in vivo.

Lactoperoxidase-catalyzed oxidation of the anticancer agent mitoxantrone by nitrogen dioxide (NO2.) radicals.

Mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl] amino]-9,10-anthracenedione, MXH2] is a novel anticancer agent frequently employed in the chemotherapy of leukemia and breast cancer. Earlier studies have shown that metabolic oxidation to reactive 1,4-quinone or/and 5,8-diiminequinone intermediates may be an important mechanism of activation of this agent, pertinent to its cytotoxic action in vivo. Here we report that in the presence of nitrite ions (NO2-), MXH2 undergoes oxidation by the mammalian enzyme lactoperoxidase (LPO) and hydrogen peroxide and that the process proceeds at a rate that is proportional to NO2- concentration. In contrast, when MXH2 was exposed to LPO/H2O2 in the absence of nitrite, oxidation of the drug was either completely absent or markedly inhibited. These experiments were carried out using concentrated solutions of MXH2 (approximately 100 microM) at near neutral pH where dimers of the drug predominate. We propose that oxidation of MXH2 is mediated by an LPO/ H2O2 metabolite of NO2-, most likely the .NO2 radical. Because in mitoxantrone therapy the drug is administered intravenously, it is directly exposed to nitrogen oxides and other free radicals produced by blood components. It is therefore possible that the ability of mitoxantrone to react with the nitrogen dioxide radical may be relevant to the biological action of the drug in vivo.

Acid-catalyzed oxidation of the anticancer agent mitoxantrone by nitrite ions.

Mitoxantrone (1,4-dihydroxy-5,8-bis[2-[(2-hydroxyethyl)amino]ethyl]amino-9,10-anth rac enedione; MXH2) is a novel anticancer agent that is useful in the treatment of leukemia and breast cancer. In contrast to other anthracenedione-based agents, this drug causes fewer side effects, mainly because it is resistant to metabolic reduction. We investigated the interaction between MXH2 and inorganic nitrite (NO2-) in aqueous solutions and found that this drug undergoes acid-catalyzed oxidation by nitrite. The rate of this reaction measured versus [NaNO2] at constant pH or versus pH at constant [NaNO2] was found to be directly proportional to the actual HNO2 concentration, indicating HNO2 to be the major oxidizing species. Involvement of .NO and/or NO2. radicals as minor oxidants is suggested based on the dependence of the rate of oxidation on the presence of air. Spectrophotometric and electron paramagnetic resonance analyses indicate that early products of the reaction are identical to those generated by oxidation of MXH2 by a horseradish peroxidase/hydrogen peroxide system. The major product is hexahydronaphtho[2,3-f]quinoxaline-7,12-dione, which is formed by intramolecular cyclization of one alkylamino side chain in the oxidized, diiminoquinone MX(N) form of the drug. This study shows that MXH2 effectively scavenges HNO2 and possibly other nitrogen oxides. Because these reactive forms of nitrogen may be present in vivo, this property of the drug may be relevant to its biological or perhaps anticancer activities.

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 (RALsb). 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 dimethylformamide, produces free radicals from both solvents. Illumination of RALsb 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 < 0.01, 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.

Free radical reactions photosensitized by the human lens component, kynurenine: an EPR and spin trapping investigation.

We have undertaken electron paramagnetic resonance and spin trapping investigations of the photochemistry of kynurenine (KN), a natural component of the human eye and close analog of the principal chromophore in the young human lens 3-OH-kynurenine O-glucoside (3HKG). 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was employed as a spin trap. We found that upon UV irradiation (> 300 nm) KN photoreduces oxygen to superoxide radical (in DMSO) and nitromethane (CH3NO2) to a nitromethane radical anion (CH3NO2.-) (in air-free buffers, pH 7 and 9.5). KN also sensitized photooxidation of cysteine, NADH, EDTA, azide, and ascorbate; oxygen greatly accelerated this process. Oxidation of cysteine, NADH, and EDTA was accompanied by superoxide radical formation. Cysteinyl and azidyl radicals were detected as DMPO adducts. We also observed that KN undergoes photodegradation to a product(s) whose photosensitizing capacity is greater than that of KN itself. We postulate that: (i) 3HKG may be able to photoinitiate free radical reactions in vivo, and (ii) oxygen is an important factor determining the yields of free radical processes initiated by lenticular chromophores.

One-electron reduction of arenediazonium compounds by physiological electron donors generates aryl radicals. An EPR and spin trapping investigation.

Arenediazonium compounds (ArN2+) are strong oxidizing agents, which upon one-electron reduction decompose, releasing aryl radicals (Ar.). The present studies were undertaken to determine whether reductive fragmentation of ArN2+ can be induced by biologically relevant electron donors. We found that 4-X-Ph-N2+ (where X: -NO2, -Br, -Cl, -OMe and -N(Et)2) decomposes to the respective aryl radicals when reduced by ascorbate, NADH, potassium ferrocyanide, catechol or p-hydroquinone in aqueous solutions. Radical identification was based on analysis of the EPR spectra of spin adducts formed by reaction of these radicals with spin traps 2-methyl-2-nitrosopropane (MNP), 3,5-dibromo-4-nitrosobenzene sulphonate (DBNBS) or 5,5-dimethyl-1-pyrroline N-oxide (DMPO). This study shows that reduction of arenediazonium ions can be a convenient method for generating aryl radicals in aqueous solutions. In addition, this investigation confirms that biological reducing agents are capable of inducing fragmentation of ArN2+ into aryl radicals. This reaction may be pertinent to some biological actions of arenediazonium compounds.

Photochemical sensitization by azathioprine and its metabolites. Part 3. A direct EPR and spin-trapping study of light-induced free radicals from 6-mercaptopurine and its oxidation products.

Sunlight has been implicated in the high incidence of skin cancer found in patients receiving 6-mercaptopurine (PSH) in the form of its pro-drug azathioprine. In this study we have used EPR spectroscopy in conjunction with the spin-trapping technique to determine whether PSH and its metabolic or photochemical oxidation products generate highly reactive free radicals upon UV irradiation. When an aqueous anaerobic solution (pH 5 or 9) of PSH (pKa = 7.7) and either 2-methyl-2-nitrosopropane (MNP) or nitromethane (NM) were irradiated (lambda > 300 nm) with a Xe arc lamp, the corresponding purine-6-thiyl (PS.) radical adduct and the reduced form of the spin trap (MNP/H. or CH3NO2.-) were observed. However, no radical adducts were detected when PSH and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were irradiated (lambda = 320 nm) in oxygen-free buffer. These findings suggest that PSH does not photoionize but that instead MNP and NM are reduced by direct electron transfer from excited state PSH, 1.3(PSH)*. In aerobic solution, oxygen can act as an electron acceptor and the O2.- and PS. radicals are formed and trapped by DMPO. 6-Mercaptopurine did photoionize when irradiated with a Nd:YAG laser at 355 nm as evidenced by the appearance of the DMPO/H.(eq- + H+) adduct, which decreased in intensity in the presence of N2O. 1.3(6-Mercaptopurine)* oxidized ascorbate, formate and reduced glutathione to the corresponding ascorbyl, CO2.- or glutathiyl radicals. The photochemical behavior of 6-thioxanthine and 6-thiouric acid was similar to PSH. However, the excited states of these metabolic oxidation products exhibited stronger reducing properties than 1.3(PSH)*.(ABSTRACT TRUNCATED AT 250 WORDS)

Photochemistry of 2-mercaptopyridines. Part 2. An EPR and spin-trapping investigation using 2-methyl-2-nitrosopropane and aci-nitromethane as spin traps in aqueous solutions.

Compounds possessing a pyridine-2-thione moiety show antimicrobial, antifungal and anticancer activities. Some of them are also photochemically active and upon UV irradiation generate free radicals. In this work, employing EPR and the spin traps 2-methyl-2-nitrosopropane (MNP) and aci-nitromethane (NM), we investigated the photochemistry in aqueous solutions of N-hydroxypyridine-2-thione (used here as a sodium salt, 2-S-PyrNONa), and pyridine-2-thione (2-S-PryH), as well as photochemistry of the respective disulfides, 2,2'-dithiobis(pyridine N-oxide) [(2-S-PyrN-->O)2] and 2,2'-dithiodipyridine [(2-S-Pyr)2]. We found that UV irradiation of 2-S-PyrNONa and of 2-S-PyrH in the presence of MNP and NM generates EPR signals of reduced spin traps in addition to signals of MNP and NM adducts with aryl-thiyl radicals, 2-.S-PyrN-->O and 2-.S-Pyr. The identification of the aromatic thiyl radicals was based on comparison of EPR spectra of spin adducts generated by irradiation of 2-S-PyrNONa and 2-S-PyrH with those produced by UV photolysis of the respective disulfides (2-S-PyrN-->O)2 and (2-S-Pyr)2. It is concluded that pyridine-2-thione and N-hydroxypyridine-2-thione possess a photoreducing capacity and generate aromatic thiyl radicals upon UV activation. This property may be relevant to biological action of agents containing the pyridine-2-thione moiety.

Photocytotoxicity of curcumin.

Curcumin, bis(4-hydroxy-3-methoxyphenyl)-1,6-diene-3,5-dione, is a yellow-orange dye derived from the rhizome of the plant Curcuma longa. Curcumin has demonstrated phototoxicity to several species of bacteria under aerobic conditions (Dahl, T. A., et al., 1989, Arch. Microbiol. 151 183), denoting photodynamic inactivation. We have now found that curcumin is also phototoxic to mammalian cells, using a rat basophilic leukemia cell model, and that this phototoxicity again requires the presence of oxygen. The spectral and photochemical properties of curcumin vary with environment, resulting in the potential for multiple or alternate pathways for the exertion of photodynamic effects. For example, curcumin photogenerates singlet oxygen and reduced forms of molecular oxygen under several conditions relevant to cellular environments. In addition, we detected carbon-centered radicals, which may lead to oxidation products (see accompanying paper). Such products may be important reactants in curcumin's phototoxicity since singlet oxygen and reduced oxygen species alone could not explain the biological results, such as the relatively long lifetime (t1/2 = 27 s) of the toxicant responsible for decreased cell viability.

Spectral and photochemical properties of curcumin.

Curcumin, bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, is a natural yellow-orange dye derived from the rhizome of Curcuma longa, an East Indian plant. In order to understand the photobiology of curcumin better we have studied the spectral and photochemical properties of both curcumin and 4-(4-hydroxy-3-methoxy-phenyl)-3-buten-2-one (hC, half curcumin) in different solvents. In toluene, the absorption spectrum of curcumin contains some structure, which disappears in more polar solvents, e.g. ethanol, acetonitrile. Curcumin fluorescence is a broad band in acetonitrile (lambda max = 524 nm), ethanol (lambda max = 549 nm) or micellar solution (lambda max = 557 nm) but has some structure in toluene (lambda max = 460, 488 nm). The fluorescence quantum yield of curcumin is low in sodium dodecyl sulfate (SDS) solution (phi = 0.011) but higher in acetonitrile (phi = 0.104). Curcumin produced singlet oxygen upon irradiation (lambda > 400 nm) in toluene or acetonitrile (phi = 0.11 for 50 microM curcumin); in acetonitrile curcumin also quenched 1O2 (kq = 7 x 10(6) M-1 s-1). Singlet oxygen production was about 10 times lower in alcohols and was hardly detectable when curcumin was solubilized in a D2O micellar solution of Triton X-100. In SDS micelles containing curcumin no singlet oxygen phosphorescence could be observed. Curcumin photogenerates superoxide in toluene and ethanol, which was detected using the electron paramagnetic resonance/spin-trapping technique with 5,5-dimethyl-pyrroline-N-oxide as a trapping agent. Unidentified carbon-centered radicals were also detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Photosensitization by anticancer agents. 11. Mechanisms of photosensitization of human leukemic cells by diaminoanthraquinones: singlet oxygen and radical reactions.

The synthesis of several aminoanthraquinone derivatives (AAQs), designed to suppress the dark toxicity and to promote more efficient cancer cell photosensitization for potential use in photodynamic therapy (PDT), is described. The following AAQs were synthesized: 1-NH2-4,5-(MeO)2-AQ (1), 1,5-(NH2)2-4,8-(MeO)2-AQ (2), 1,8-(NH2)2-4,5-(MeO)2-AQ (3), and 1,5-(NHPhMe)2-4,8-(MeO)2-AQ (8). The agents exhibit strong absorption in the region 480-620 nm. Possible mechanisms of photosensitization were studied by measuring 1O2 phosphorescence at 1270 nm, detecting superoxide radicals employing an electron paramagnetic resonance (EPR)-spin trapping technique, and measuring oxygen consumption during the photo-oxidation of a representative biological electron donor, NADH. Strong phosphorescence from 1O2 was observed upon illumination of 2 and 3 in C6H6 (quantum yield of 0.25 and 0.5 respectively), and in EtOH (quantum yield of 0.23 and 0.34). The 1-amino-AQ (1) was the weakest 1O2 sensitizer, with quantum yield of 0.13 in benzene. No phosphorescence was observed in EtOH. A superoxide radical was detected as a spin adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in irradiated benzene solutions of 1, 2 or 3 and DMPO. AAQs 2 and 3 sensitized photo-oxidation of NADH in H2O/EtOH mixture with the intermediacy of singlet oxygen as judged by the effect of sodium azide on the photostimulated oxygen consumption. Evolution of O2 upon addition of catalase to the illuminated solution confirmed the ultimate formation of hydrogen peroxide. These findings suggested that the (di)amino-dimethoxyanthraquinones might exert photosensitization via both Type I and Type II mechanisms. The AAQs were tested for their ability to photosensitize K562 human chronic myeloid leukemic cells in culture. Viability was measured using the 3,4,5-diethylthiazol-2,5-diphenyl tetrazolium blue assay, and DNA and possible membrane damage were assessed. The results from illuminating cells with light > 475 nm show that for the 1,5-compounds, the presence of methoxy substituents at 4,8 positions reduces the dark toxicity from ID50 of 23 to 250 microM and for the 1,8-compounds correspondingly from ID50 of 53 to > 300 microM. In the 1,5-series this decrease of the dark toxicity is accompanied by an increase in light-induced dose modification from 8.85 to 14.4. Differences exist in the mechanisms of cytotoxicity between the prototype phenolic AAQs and their methoxy counterparts. It appears that the cytotoxic action of the latter causes cell damage by the formation of a high proportion of alkali labile sites in addition to frank strand breaks.(ABSTRACT TRUNCATED AT 400 WORDS)

Spectroscopic studies of cutaneous photosensitizing agents--XVI. Disperse blue 35.

The photochemistry (Type I and II) of the phototoxic textile dye Disperse Blue (DB-35) and its purified components has been studied using electron spin resonance in conjunction with spin trapping technique and the direct detection of singlet oxygen (1O2) luminescence. The main components of DB-35 (which is synthesized by the successive nitration, reduction and methylation of 1,8-dihydroxy-anthraquinone) were separated by HPLC and identified by mass spectrometry and 2-D NMR as 4,5-diamino-1,8-dihydroxyanthraquinone (4,5-DDHAQ; 62% of total dye) and 2,7-diamino-1,8-dihydroxyanthraquinone (2,7-DDHAQ; 31% of total dye). Minor components included 2,5-diamino-1,8-dihydroxyanthraquinone (2,5-DDHAQ) and a monomethylated derivative of either 4,5-DDHAQ or 2,7-DDHAQ. Irradiation (624 nm) of 4,5-DDHAQ and 2,7-DDHAQ in dimethylsulfoxide resulted in the generation of superoxide which was trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Visible light irradiation of the components in ethanol generated 1O2 with the yields decreasing in the following order: 4,5-DDHAQ greater than 2,5-DDHAQ greater than 2,7-DDHAQ. These findings indicate that upon irradiation by visible light DB-35 can generate active oxygen species which may be responsible for the photocontact dermatitis caused by this dye.