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 curcumin's 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.     

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, e_aq⁻, 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.     

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.     

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

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

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS. XVIII. INDOMETHACIN

Abstract— The photochemistry, photophysics, and photosensitization (Type I and II) of indomethacin (IN) (N-[p-chlorobenzoyl]-5-methoxy-2-methylindole-3-acetic acid) has been studied in a variety of solvents using NMR, high performance liquid chromatography-mass spectroscopy, transient spectroscopy, electron paramagnetic resonance in conjunction with the spin trapping technique, and the direct detection of singlet molecular oxygen (^l O₂) luminescence. Photodecomposition of IN (λ_ex > 330 nm) in degassed or air-saturated benzene proceeds rapidly to yield a major (2; N-[p-chlorobenzoyl]-5-methoxy-2-methyl-3-methylene-indoline) and a minor (3; N-[p-chlorobenzoyl]-5-methoxy-2, 3-dimethyl-indole) decarboxylated product and a minor indoline (5; 1-en-5-methoxy-2-methyl-3-methylene-in-doline), which is formed by loss of the p-chlorobenzoyl moiety. In air-saturated solvents two minor oxidized products 4 (N-[p-chlorobenzoyl]-5-methoxy-2-methylindol-3-aldehyde) and 6 (5-methoxy-2-methyl-indole-3-aldehyde) are also formed. When photolysis was carried out in ¹⁸O₂-saturated benzene, the oxidized products 4 and 6 contained ¹⁸O, indicating that oxidation was mediated by dissolved oxygen in the solvent. In more polar solvents such as acetonitrile or ethanol, photodecomposition is extremely slow and inefficient. Phosphorescence of IN at 77 K shows strong solvent dependence and its emission is greatly reduced as polarity of solvent is increased. Flash excitation of IN in degassed ethanol or acetonitrile produces no transients. A weak transient is observed at 375 nm in degassed benzene, which is not quenched by oxygen. Irradiation of IN (λ_ex > 325 nm) in N₂-gassed C₆H₆ in the presence of 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) results in the trapping of two carbon-centered radicals by DMPO. One adduct was identified as DMPO/^.COC₆H₄-p-CI, while the other was probably derived from a radical formed during IN decarboxylation. In air-saturated benzene, (hydro) peroxyl and alkoxyl radical adducts of DMPO are observed. A very weak luminescence signal from ¹O₂ at 1268 nm is observed initially upon irradiation (λ_ex= 325 nm) of IN in air-saturated benzene or chloroform. The intensity of this ¹O₂ signal increases as irradiation is continued suggesting that the enhancement in ¹O₂ yield is due to photoproduct(s). Accordingly, when 2 and 3 were tested directly, 2 was found to be a much better sensitizer of ¹O₂ than IN. In air-saturated ethanol or acetonitrile no IN ¹O₂ luminescence is detected even on continuous irradiation. The inability of IN to cause phototoxicity may be related to its photo stability in polar solvents, coupled with the low yield of active oxygen species (¹O₂, O₂^?_-) upon UV irradiation.     

MAGNETIC FIELD EFFECTS ON THE PHOTOHEMOLYSIS OF HUMAN ERYTHROCYTES BY KETOPROFEN AND PROTOPORPHYRIN IX

Abstract— Application of a static external magnetic field (3350 G) during UV-irradiation (>300 nm) reduced the time for 50% photohemolysis of human erythrocytes by the phototoxic drug ketoprofen (3-benzoyl-α-methylbenzoacetic acid) from 96 min to 78 min. This observation can be attributed to a magnetic field induced decrease in the rate of intersystem crossing (k_ISC) of the geminate triplet radical pair generated by the reduction of ketoprofen in its triplet excited state by erythrocyte membrane constituents, probably lipids. The decrease in k_JSC results in an increase in the concentration and/or lifetime of free radicals that escape from the triplet radical pair. Thus the critical radical concentration needed to cause membrane damage and cell lysis is reached sooner in the presence of the magnetic field. In contrast, the photohemolysis induced by the photodynamic agent protoporphyrin IX was not affected by the magnetic field. Protoporphyrin IX photohemolysis, which is initiated by singlet oxygen, does not involve the initial generation of a triplet radical pair and so is not influenced by the magnetic field. The enhancement of ketoprofen-induced photohemolysis by an externally applied magnetic field is the first example of a magnetic field effect on a toxicological process involving free radicals.     

The Effect of Static Magnetic Fields on the Photohemolysis of Human Erythrocytes by Ketoprofen

Ultraviolet irradiation (λ > 300 nm) of the nonsteroidal anti-inflammatory agent ketoprofen (KP, 3-benzoyl-α-methylbenzoacetic acid) in aqueous solution, pH 7.4, results in heterolytic decarboxylation of the drug to give 3-ethylbenzophenone (EtBP). Ketoprofen caused the photohemolysis of human erythrocytes probably as a result of lipid peroxidation. Application of a static magnetic field (250–1500 G) during UV (>300 nm) irradiation of KP and erythrocytes significantly decreased the time required for photohemolysis. This observation suggests that KP-induced photohemolysis involves the initial generation of a triplet radical pair derived from the reaction of triplet state KP (or 3-EtBP) with erythrocyte component(s) probably lipids. The magnetic field increases the concentration and/or lifetime of free radicals that escape from the radical pair so that the critical radical concentration needed to initiate membrane damage and cause cell lysis is reached sooner. Spin-trapping studies with 2,6-dibromo-1-nitrosobenzene-4-sulfonate confirmed that the application of an external static magnetic field increased the concentration of radicals released during the photolysis of either KP or 3-EtBP dissolved in organized media such as sodium dodecylsulfate micelles.     

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.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS–XII. SPIN TRAPPING STUDY OF THE FREE RADICALS GENERATED DURING THE PHOTOLYSIS OF PHOTO ALLERGENS BITHIONOL and FENTICHLOR

Free radicals were trapped and observed by ESR when photoallergens bithionol and fentichlor were irradiated in the presence of spin traps N- t -butyl-α-phenylnitrone (PBN) and 5,5-dimethyl-pyrroline-N-oxide (DMPO). In the absence of air, both PBN and DMPO trapped a carbon-centered radical. The carbon-centered radical, which was capable of abstracting a hydrogen atom from cysteine, glutathione, ethanol and formate, was identified as an aryl radical derived from the homolytic cleavage of the carbon-chlorine bond. In the presence of air, both carbon-centered radicals and hydroxyl radicals were trapped by DMPO. Under similar conditions, the yield of the hydroxyl radicals was greater from bithionol than from fentichlor. The presence of the hydroxyl radical was confirmed by kinetic experiments employing hydroxyl radical scavengers (ethanol, formate). Superoxide and H₂O₂ were not involved. Experiments with oxygen-¹⁷O indicated that the hydroxyl radicals came exclusively from dissolved oxygen. The precursor of the hydroxyl radical is postulated to be a peroxy intermediate (ArOO*) derived from the reaction of an aryl radical (Ar*) with molecular oxygen. Both bithionol and fentichlor photoionized only when excited in the UVC (<270 nm) region. Free radicals have long been postulated in the photodechlorination of bithionol and fentichlor and the present study provides supporting evidence for such a mechanism. Aryl and hydroxyl radicals are reactive chemical species which may trigger a series of events that culminate in photoallergy.     

Comparison of A2E Cytotoxicity and Phototoxicity with all-trans-Retinal in Human Retinal Pigment Epithelial Cells†

All-trans-retinal is the precursor of A2E, a fluorophore within lipofuscin, which accumulates in human retinal pigment epithelial (hRPE) cells and contributes to age-related macular degeneration. Here we have compared the in vitro dark cytotoxicity and visible-light-mediated photoreactivity of all-trans-retinal and A2E in hRPE cells. All-trans-retinal caused distinct cytotoxicity in hRPE cells measured with cell metabolic activity (MTS) and lactate dehydrogenase release assays. Significant increases in intracellular oxidized glutathione (GSSG), extracellular GSH and GSSG levels and lipid hydroperoxide production were observed in cells incubated in the dark with 25 and 50 μm all-trans-retinal. Light modified all-trans-retinal’s harmful action and decreased extracellular glutathione and hydroperoxide levels. A2E (<25 μm ) did not affect cell metabolism or cytoplasmic membrane integrity in the dark or when irradiated. 25 μm A2E raised the intracellular GSSG level in hRPE cells to a much smaller extent than 25 μm all-trans-retinal. A2E did not induce glutathione efflux or hydroperoxide generation in the dark or after irradiation. These studies support our previous conclusions that although A2E may be harmful at high concentrations or when oxidized, its phototoxic properties are insignificant compared to those of all-trans-retinal. The endogenous production of A2E may serve as a protective mechanism to prevent damage to the retina by free all-trans-retinal.