Ann G. Motten

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.

Spectroscopic studies of cutaneous photosensitizing agents. VIII: A spin-trapping study of light induced free radicals from chlorpromazine and promazine

Abstract— The clinically important phenothiazine drugs, particularly chlorpromazine, often elicit phototoxic and photoallergic reactions. We have used the spin traps 2‐methyl‐2‐nitrosopropane (MNP) and 5,5‐dimethyl‐pyrroline‐N‐oxide (DMPO) to define the radical photolysis pathways of chlorpromazine and promazine. In the absence of oxygen the dechlorination product of chlorpromazine is trapped by MNP. The reactivity of the dechlorination product is similar to that of the phenyl radical as shown by its ability to extract hydrogen atoms from donors. Our results suggest that the dechlorination product is sufficiently reactive to account for the observation that chlorpromazine is more phototoxic than its parent promazine. In the presence of oxygen both chlorpromazine and promazine form a superoxide‐dismutase‐insensitive oxygen‐centered intermediate which, when trapped by DMPO, rapidly decays to DMPO‐OOH and subsequently to DMPO‐OH. In addition, chlorpromazine readily undergoes photoelectron ejection only when it is excited into the second excited singlet state (Δ < 280 nra). This previously unknown wavelength dependence of photoionization should be considered in establishing the mechanism of chlorpromazine photosensitization.

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.

Oxidative metabolism of hydralazine. Evidence for nitrogen centered radicals formation

Abstract The oxidative metabolism of hydralazine, a hydrazine-containing hypotensive drug, has been studied using a spin-trapping technique. In the presence of Cu 2+ , Fe 2+ and Fe 3+ , hydralazine rapidly forms a nitrogen-centered-DMPO adduct with a N = 15.0G, a β H = 16.7G and a β N = 2.55G. While catalase has a very small inhibitory effect, superoxide dismutase completely inhibits the formation of the DMPO adduct. Mass spectral analysis of the adduct indicates that the hydralazyl radical is trapped with DMPO. Human red blood cells also catalyze the formation of a nitrogen-centered-DMPO adduct, a N = 15.9G, a β H = 19.4G and a β N = 1.7G, which is different than that obtained with metal ions. DMPO-H adduct is also formed in the red cells from hydralazine.

Generation of radical anions of nitrofurantoin, misonidazole, and metronidazole by ascorbate

Nitrofurantoin, misonidazole, and metronidazole were reduced to their corresponding nitro anion radicals by ascorbate in anaerobic solutions at high pH. The nitrofurantoin anion radical could be detected at neutral pH. In neutral solutions, the nitro anion radicals of misonidazole and metronidazole were too unstable to be observed by electron spin resonance spectroscopy. At neutral pH, solutions containing ascorbate, nitrofurantoin, or misonidazole consumed oxygen. The addition of superoxide dismutase, catalase, or both superoxide dismutase and catalase decreased the rate of oxygen consumption. These results show that nitro anion radicals are formed by reduction with ascorbate, and superoxide anion radical and hydrogen peroxide are produced by reactions of these radicals with oxygen.

ESR DETECTION OF ENDOGENOUS ASCORBATE FREE RADICAL IN MOUSE SKIN: ENHANCEMENT OF RADICAL PRODUCTION DURING UV IRRADIATION FOLLOWING TOPICAL APPLICATION OF CHLORPROMAZINE

Using electron spin resonance spectroscopy, we observed that UV radiation (330 nm) increased the endogenous ascorbate free radical concentration in hairless mouse (HRS/J) skin. When the skin was topically treated with a chlorpromazine solution prior to illumination, UV irradiation caused the ascorbate free radical concentration to increase even more. This observation suggests that there is an increased UV‐induced oxidative stress in the presence of chlorpromazine, probably caused by the production of free radicals from chlorpromazine.

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.

Free radical production by chlorpromazine sulfoxide, an ESR spin-trapping and flash photolysis study.

Abstract— Using the spin‐trapping technique we have investigated the photolysis of chlorpromazine sulfoxide and promazine sulfoxide. Photolysis of these sulfoxides in aqueous solution resulted in a species which is capable of oxidizing ascorbate, cysteine, glutathione, NADH, and azide by one electron, in addition to extracting hydrogen atoms from ethyl alcohol and dimethyl sulfoxide. These oxidations were not dependent on the presence of dissolved oxygen. The oxidizing species is proposed to be the hydroxyl free radical arising from the homolytic cleavage of the S‐O bond of the sulfoxide. Flash photolysis of the chlorpromazine and promazine sulfoxides demonstrated the formation of cation radicals consistent with the loss of the hydroxyl radical from the sulfoxides. In addition we present a simple direct method for the quantitative synthesis of promazine and chlorpromazine sulfoxides from the parent promazine derivatives.

The photooxidation of diethylhydroxylamine by rose bengal in micellar and nonmicellar aqueous solutions.

The photooxidation of N,N‐diethylhydroxylamine (DEHA) by Rose Bengal (RB) has been investigated in micellar and nonmicellar aqueous solutions. We measured the quantum yield of oxygen consumption forming H2O2 and monitored two intermediates, the superoxide and diethylnitroxide radicals. When the pH was vaned, the quantum yield of oxidation remained constant for 6 < pH < 10.5, decreased in acidic pH, and increased considerably in NaOH solution; these changes could be attributed to the protonation and dissociation processes of the >N‐OH moiety of DEHA. The formation of diethylnitroxide radical was enhanced by superoxide dismutase or strong alkaline solution. Around neutral pH, the oxidation proceeded mainly via electron transfer from DEHA to the RB triplet (kq= 107M‐1 s‐1) with little 1O2 participation (kq < 105M‐1 s‐1). However, when RB was incorporated into micelles in alkaline solution, the contribution of the singlet oxygen pathway increased at the expense of electron transfer, which was inhibited by the less polar micellar environment. Dark autoxidation of DEHA was accelerated by heavy metal impurities and increased very strongly in NaOH solution.

Correlation analysis of ESR spectra on a small computer

Abstract A correlation approach to computer solutions of electron spin resonance spectra has been adapted for use on a small computer. The correlation analysis is capable of finding solutions for complex, well-resolved spectra, including those containing spin-1 species such as nitrogen. Even for spectra that can be solved readily by hand, a tuning procedure using correlation as a criterion for goodness of fit saves much time and effort. A fast procedure for adjusting hyperfine constants to within approximately 0.05% of the scan range is presented. Fast methods of calculation for other parts of the correlation analysis are described and their application to the aminophenoxy and the tetramethylaminophenoxy radicals is shown.