Krzysztof J. Reszka

Reaction of Melatonin and Related Indoles With Hydroxyl Radicals: EPR and Spin Trapping Investigations

It has been suggested that the indole hormone melatonin (N-acetyl-5-methoxytryptamine, MLT) is an important natural antioxidant and free radical scavenger [J. Pineal Res., 14:51; 1993]. In the present work we determined the rate constants, k(r), for scavenging .OH radicals by melatonin, 5-methoxytryptamine (5-MeO-T), 5-hydroxytryptamine (serotonin, 5-OH-T), 6-chloromelatonin (6-Cl-MLT), 6-hydroxymelatonin (6-OH-MLT), and kynurenine (KN) in aqueous solutions. Hydroxyl radicals were generated using a Fenton reaction in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which competed with the indoles for the radicals. It was found that MLT reacts with .OH with k(r) = 2.7 x 10(10) M(-1) s(-1). Other indoles and KN reacted with .OH radicals with similarly high rates (k(r) > 10(10) M(-1) s(-1)). In contrast to nonhydroxylated indoles (MLT, 6-Cl-MLT, and 5-MeO-T), hydroxylated indoles (5-OH-T and 6-OH-MLT) may function both as .OH promoters and .OH scavengers. The melatonin precursor serotonin promoted the generation of .OH radicals in the presence of ferric iron and H2O2, and the melatonin metabolite 6-hydroxymelatonin generated large quantities of .OH radicals in aerated solutions containing Fe3+ ion, even in the absence of externally added hydrogen peroxide. These reactions may be relevant to the biological action of these physiologically important indolic compounds.

SPECTRAL AND PHOTOCHEMICAL PROPERTIES OF CURCUMIN

Curcumin, bis(4‐hydroxy‐3‐methoxyphenyl)‐l,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 (λmax= 524 nm), ethanol (λmax= 549 nm) or micellar solution (λmax= 557 nm) but has some structure in toluene (λmax= 460, 488 nm). The fluorescence quantum yield of curcumin is low in sodium dodecyl sulfate (SDS) solution (φ= 0.011) but higher in acetonitrile (φ= 0.104). Curcumin produced singlet oxygen upon irradiation (φ > 400 nm) in toluene or acetonitrile (Φ= 0.11 for 50 μM curcumin); in acetonitrile curcumin also quenched 1O2 (kq, = 7 × 106 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. These findings indicate that the spectral and photochemical properties of curcumin are strongly influenced by solvent. In biological systems, singlet oxygen, superoxide and products of photodegradation may all participate in curcumin phototoxicity depending on the environment of the dye.

PHOTOCYTOTOXICITY OF CURCUMIN

Curcumin, bis(4‐hydroxy‐3‐methoxyphenyl)‐l,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 curcumins phototoxicity since singlet oxygen and reduced oxygen species alone could not explain the biological results, such as the relatively long lifetime (t12= 27 s) of the toxicant responsible for decreased cell viability.

Surface catalyzed electron transfer from polycyclic aromatic hydrocarbons (PAH) to methyl viologen dication: evidence for ground-state charge transfer complex formation on silica gel

Abstract Porous silica surfaces are shown to slowly catalyze the oxidation of adsorbed polycyclic aromatic hydrocarbons (PAH) to the corresponding radical cation via Lewis acid sites present on the surface. When a good electron acceptor such as methyl viologen dication (MV ++ ) is co-adsorbed on silica surface, a red-shifted structureless absorption band characteristic of a ground-state charge transfer (CT) complex formed between the PAH and MV ++ is observed. Oxygen efficiently competes with MV ++ for the trapped electrons on the active sites of silica surface causing a significant decrease in the concentration of ground-state CT complex. The rate of this electron transfer process is enhanced dramatically at the solid/liquid interface when solution of PAH in a non-polar solvent is added to dry silica containing adsorbed MV ++ . Room temperature electron paramagnetic resonance (EPR) spectra of PAHs adsorbed on silica show a broad unresolved signal ( g =2.0031–2.0045) due to PAH ⋅+ radical cation which disappears in the presence of air but can be restored upon evacuation of the sample. The EPR measurements of mixed samples containing PAH and MV ++ co-adsorbed on silica show a composite signal with hyperfine structure that may be due to presence of two paramagnetic species corresponding to MV ⋅+ and possibly PAH radical cation.

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.

Extracellular superoxide dismutase (ecSOD) in vascular biology: an update on exogenous gene transfer and endogenous regulators of ecSOD

Extracellular superoxide dismutase (ecSOD) is the major extracellular scavenger of superoxide (O(2)(.-)) and a main regulator of nitric oxide (NO) bioactivity in the blood vessel wall, heart, lungs, kidney, and placenta. Involvement of O(2)(.-) has been implicated in many pathological processes, and removal of extracellular O(2)(.-) by ecSOD gene transfer has emerged as a promising experimental technique to treat vascular disorders associated with increased oxidant stress. In addition, recent studies have clarified mechanisms that regulate ecSOD expression, tissue binding, and activity, and they have provided new insight into how ecSOD interacts with other factors that regulate vascular function. Finally, studies of a common gene variant in humans associated with disruption of ecSOD tissue binding suggest that displacement of the enzyme from the blood vessel wall may contribute to vascular diseases. The purpose of this review is to summarize recent research findings related to ecSOD function and gene transfer and to stimulate other investigations into the role of this unique antioxidant enzyme in vascular pathophysiology and therapeutics.

PHOTOPHYSICAL STUDIES ON ANTIMALARIAL DRUGS

Abstract— Most drugs used in the treatment of malaria produce phototoxic side effects in both the skin and the eye. Cutaneous and ocular effects that may be caused by light include changes in skin pigmentation, corneal opacity, cataract formation and other visual disturbances including irreversible retinal damage (retinopathy) leading to blindness. The mechanism for these reactions in humans is unknown. We irradiated a number of antimalarial drugs (amodiaquine, chloroquine, hydroxychloroquine, mefloquine, primaquine and quinacrine) with light (Λ > 300 nm) and conducted electron paramagnetic resonance (EPR) and laser flash photolysis studies to determine the possible active intermediates produced. Each antimalarial drug produced at least one EPR adduct with the spin‐trap 5,5‐dimethyl‐l‐pyrroline N‐oxide in benzene: superoxide/hydroperoxyl adducts (chloroquine, mefloquine, quinacrine, amodiaquine and quinine), carbon‐centered radical adducts (all but primaquine), or a nitrogen‐centered radical adduct only (primaquine). In ethanol all drugs except primaquine produced some superoxide/hydroperoxyl adduct, with quinine, quinacrine, and hydroxychloroquine also producing the ethoxyl adduct. As detected with flash photolysis and steady‐state techniques, mefloquine, quinine, amodiquine and a photoproduct of quinacrine produced singlet oxygen (τ= 0.38; τ= 0.36; τ= 0.011; τ= 0.013 in D2O, pD7), but only primaquine quenched singlet oxygen efficiently (2.6 × 108M−1 s1 in D2O, pD7). Because malaria is a disease most prevalent in regions of high light intensity, protective measures (clothing, sunblock, sunglasses or eye wraps) should be recommended when administering antimalarial drugs.

A SPIN TRAPPING STUDY OF THE PHOTOCHEMISTRY OF 5,5-DIMETHYL-1-PYRROLINE N-OXIDE (DMPO)

The photochemistry of 5,5‐dimethyl‐l‐pyrroline N‐oxide (DMPO) has been studied in benzene, cyclohexane and aqueous buffer solutions (pH 7.4) by means of electron paramagnetic resonance (EPR) and the spin trapping technique. Ultraviolet irradiation of DMPO in aqueous buffer with unfiltered UV radiation from a Xe arc lamp results in photoionization of the spin trap and the generation of the DMPO cation radical, DMPO+. The aqueous electron, eaq−, was trapped by DMPO and detected as the DMPO/H adduct. The DMPO+‐ reacted with the water to yield the DMPO/OH adduct. Ultraviolet irradiation of DMPO in nitrogen‐saturated benzene gave an unidentified carbon‐centered DMPO adduct that was replaced by hydroperoxyl and alkoxyl adducts of DMPO when oxygen was present. Experiments employing 17O2 gas indicated that the oxygen in the DMPO alkoxyl adduct was derived from molecular oxygen. However, UV irradiation of DMPO in cyclohexane yielded the cyclohexyl and cyclohexyloxyl adducts of DMPO in nitrogen‐saturated and air‐saturated solutions, respectively. These observations suggest that in aprotic solvents UV irradiation of DMPO generates a carbon‐centered radical (R−), derived from the trap itself, which in benzene reacts with oxygen to yield an alkoxyl radical (RO−), possibly via a peroxyl radical (ROO−) intermediate. In cyclohexane R− abstracts a hydrogen atom from the solvent to yield the cyclohexyl radical in the absence of oxygen and the cyclohexyloxyl radical in the presence of oxygen. These findings indicate that when DMPO is used as a spin trap in studies employing short‐wavelength UV radiation (λ < 300 nm) the photochemistry of DMPO cannot be ignored.

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 × 107M–1 s–1 in D2O (pD 7), 6.0 × 107M–1 s–1 in acetonitrile, and 6.1 × 107M–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.

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.