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.     

Double-channel contact recombination of radical pairs subjected to spin conversion via the Delta g mechanism

The contact recombination from both singlet and triplet states of a radical pair is studied assuming that the spin conversion is carried out by the fast transversal relaxation and Delta g mechanism. The alternative HFI mechanism is neglected as being much weaker in rather large magnetic fields. The magnetic-field-dependent quantum yields of the singlet and triplet recombination products, as well as of the free radical production, are calculated for any initial spin state and arbitrary separation of radicals in a pair. The magnetic field effect is traced and its diffusional (viscosity) dependence is specified.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID BILAYER VESICLE SYSTEMS: SECONDARY OXIDATION OF REDUCED VIOLOGEN ACCEPTORS BY Ru(NH3)63+

Abstract— The kinetics of the oxidation of a homologous series of 4,4'-di(n-alkyl)-bipyridinium (viologen) radicals by Ru(NH₃)₆³⁺ in vesicle suspensions was studied using laser flash photolysis. The viologen radicals were produced photochemically in the bilayer membrane phase of the vesicles by electron transfer from the triplet state of chlorophyll-α. At high concentrations of Ru(NH₃)₆³⁺, the rate of oxidation of the viologen radicals in the aqueous phase was limited by the rate at which the radicals diffused from the membrane to the aqueous phase. The exit rate constant decreased from 2 × 10⁵ s⁻¹ for the methyl viologen radical to 4 × 10³ s⁻¹ for the pentyl viologen radical. Both the exit rate constants and the calculated values for the equilibrium association constants of the viologen radicals were unexpectedly insensitive to the length of their alkyl substituents. This, as well as other data, suggests that the radicals that diffused into the aqueous phase tended to remain associated with the membrane-water interface.     

Effect of Magnetite Particles on Photoinduced and Nonphotoinduced Free Radical Processes in Human Erythrocytes

Magnetite (Fe₃0₄) encapsulated in polystyrene microspheres dramatically decreased the time for 50% hemolysis (t_1/2) of human erythrocytes irradiated (λ300 nm) in the presence of ketoprofen (0.1 mM). The magnetic microspheres were present at a very low concentration (0.002%) such that on average there was only one particle per four erythrocytes. No such effect was seen when nonmagnetic microspheres were employed or when the equivalent concentration of soluble iron (FeCl₃) was present. A decrease in t_1/2 was also observed when the magnetic microspheres were added after UVA/ketoprofen treatment or when they were present during hemolysis initiated by thermolysis of 2,2′-azobis(2-amidinopro-pane). These findings may be attributed to an increase in the membrane concentration of lipid radicals as a result of a magnetic field-induced increase in radicals escaping from triplet radical pairs.     

AN EPR STUDY OF THE SPECIES PRODUCED DURING THE UV PHOTOLYSIS OF HETEROCYCLIC COMPOUNDS IN METHYLTETRAHYDROFURAN AT 77 K

Abstract —The ultraviolet irradiation (290 nm ≤Λ≤ 390 nm) of indole, purine, indazole, acridine and quinoline in 2-methyltetrahydrofuran glass at 77 K produces trapped radicals. Two electron-paramagnetic-resonance (EPR) signals are found at 77 K during illumination, one at high magnetic field (3–25 times 10^-1 T) assigned to the matrix radical and the other at low field (1.3 times 10^-1 to 1–5 times 10^-1 T) attributed to the lowest triplet state of the heterocyclic molecule. Quantum yields for triplet production at 77 K are 0–34 for indole, 0.51 for purine, 0.55 for indazole, 0.15 for acridine, and 0.94 for quinoline. The rate of formation of matrix radicals varies as the n _R^th power of the incident light intensity, I ₀^nR, where 1.6 ≤ n _R=≤ 2. Solvent radical yields, which depend on the light intensity, have been determined. Under the experimental conditions, no signals attributable to trapped electrons or cations have been observed. The dependence of the reciprocal value of the rise lifetime of the low field EPR signal as a function of the intensity of exposure is in accordance with a biphotonic mechanism.     

PHOTOCHEMISTRY OF FLAVINS—I. CONVENTIONAL AND LASER FLASH PHOTOLYSIS STUDY OF ALLOXAZINE

Abstract— The photobleaching of alloxazine in buffered aqueous solution has been studied by means of flash photolysis using conventional and laser excitation sources. Several transient species have been characterized. The alloxazine triplet state (Λ_max 420 nm and 550 nm, times; = 9 μs) was identified with the aid of low-temperature comparison experiments in ethanol. Transient absorption with Λ_max 440 nm, which appears after decay of the triplet state, and whose second-order decay is pH-dependent, is postulated to be due to the semiquinone radical (AH₂*) and a radical derived from alloxazine by addition of water and loss of a hydrogen atom (HAOH*), which are in equilibrium with their conjugate cation radicals. The results of experiments in the presence of oxygen indicate that these species are not primarily formed from the triplet state. The enhanced second-order decay of the flavin radicals in oxygen-containing solutions is interpreted in terms of their reaction with the peroxy radicals. The proposed mechanisms account for the production of hydroxylated alloxazines.     

Dramatic Effect of Magnetite Particles on the Dynamics of Photogenerated Free Radicals

Abstract— Polymeric particles of about 1 u.m in diameter and containing around 40% magnetite have a dramatic influence on the dynamics of triplet radical pair reactions occurring in micelles in their vicinity. The effect has been monitored with laser flash photolysis techniques and results in a decrease of the number of radicals that separate from their geminate partner and an acceleration of radical pair geminate reactions. The effect involves remote interactions because the solution contains ca 10¹¹micelles for each polymeric particle. Magnetite also perturbs the way in which the radicals interact with an external magnetic field.     

ON THE PHOTOCHEMISTRY OF X-RAY PRODUCED RADICALS TRAPPED IN FROZEN ETHANOL MATRICES

Abstract— Ethanol and ethanol-water matrices were exposed to X-rays at 77K and the photochemistry and paths of radical conversion were investigated by EPR methods. The main X-ray induced radical, CH3ĊHOH, is probably photoionized by 254 nm light. The following radicals are produced during prolonged UV-irradiation of CH₃ĊHOH radicals: ĊH₃, ĊHO, H and 2 types of radicals giving singlet EPR spectra. One of these radicals (d) is bleachable with 580 nm light, ĊH₃ and ĊH₃ĊHOH being formed during the bleaching, the other one (e) is unbleachable and the most stable radical in the matrix during annealing. The CH₃ radicals decay at 77 K (τ∽ 10 min) and produce CH₃-CHOH radicals and the unbleachable radical (e). Stable H-atom signals were seen in X-irradiated ethanol-water mixtures (volume ratio 2:1) at 77 K. The H-atom signals increased during photobleaching of the trapped electrons in the matrix and during UV-photolysis of CH₃CHOH radicals.     

Photochemistry and Photobiological Properties of Dicloran, a Postharvest Fungicide with Photosensitizing Side Effects

Photochemical and laser flash photolysis studies on dicloran have shown that this fungicide undergoes photo-reactions such as photoreduction of the nitro group and homolytic rupture of the C-NH₂ bond. Dramatic changes in the dicloran photoreactivity by the influence of the solvents have been observed. More efficient photodegradation of this fungicide was observed in diethyl ether and chloroform than in methanol or acetonitrile. Photoreduction of the nitro group from the dicloran triplet state seems to be the most important photodegradation path-way in solvents of low polarity. Hydrogen abstraction by the triplet state or the intermediate radicals appears to be in the origin of linoleic acid peroxidations photosensitized by dicloran. The photohemolysis assay has been used, as an in vitro phototoxicity test, to demonstrate the involvement of radical-mediated cellular membrane damage in dicloran photosensitization.     

Singlet and triplet products of the geminate recombination of a radical pair with a single magnetic nucleus (I = 1/2)

The double-channel recombination and separation of the photochemically created singlet radical pair is investigated, taking into account the spin conversion in a zero magnetic field and the arbitrary initial distance between the radicals. The quantum yields of the singlet and triplet products and the free radicals production are found analytically, assuming that the recombination of the diffusing radicals occurs at contact. All the yields are related to the singlet and triplet populations of the recombining radical pair, subjected to spin conversion and contact exchange interaction. The general analytical expressions for the quantum yields are specified for the particular limits of the weak and strong exchange. They are greatly simplified in the case of polar solvents, especially at the contact start. A close similarity is obtained with the results of a previously developed incoherent model of spin conversion, provided that the conversion rate is appropriately related to the hyperfine coupling constant.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS—II. SPIN TRAPPING OF PHOTOLYSIS PRODUCTS FROM SULFANILAMIDE AND 4-AMINOBENZOIC ACID USING 5,5-DIMETHYL-1-PYRROLINE-1-OXIDE

Abstract— The photodecomposition of sulfanilamide, 4-aminobenzoic acid and related analogs in aqueous solution has been studied with the aid of spin traps 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and CH₃NO₂ as well as by direct electron spin resonance techniques. The NH₂ radical was trapped by DMPO during the photolysis of aqueous solutions of sulfanilamide with a Xe arc lamp. Studies with [¹⁵N¹]-sulfanilamide indicated that the NH₂ radical was generated by homolytic fission of the sulfur-nitrogen bond. Under the same conditions DMPO trapped the H and SO⁻₃ radicals during photolysis of sulfanic acid. Direct photolysis of sulfanilamide, sulfanilic acid and Na₂SO₃ in the absence of any spin trap yielded the SO⁻³ radical. Photolysis of 4-aminobenzoic acid at pH 7 gave the H radical which was trapped by DMPO. At low pH values OH and C₆H₄COOH radicals were generated during the photolysis of 4-aminobenzoic acid. No e⁻_aq were trapped by CH₃NO₂ when acid (pH 4) and neutral aqueous solutions of sulfanilamide or 4-aminobenzoic acid were photoirradiated. The mechanism of formation of known photoproducts from the free radicals generated by sulfanilamide and 4-aminobenzoic acid during irradiation are discussed. The free radicals generated by these agents may play an important role in their phototoxic and photoallergic effects.     

REACTIVITY OF ACRIDINE DYE TRIPLET STATES IN ELECTRON TRANSFER REACTIONS

Abstract—Rate constants, k _q , for the reaction of cationic and neutral acridine orange and 10-methylacridine orange triplet states (³AOH ⁺, ³AO, ³MAO⁺) with a series of electron donors have been measured. Two different protolytic forms of the semireduced dye radical are produced by acridine orange triplet quenching at various pH^M values in methanolic solution.
It is found that k ₄ decreases with increasing oxidation potential of the reducing agent for all triplet states in a manner which is expected for electron transfer reactions. The different reactivities of the cationic and neutral triplet forms can, therefore, be attributed to the difference in reduction potentials of these species. The difference in reduction potentials is related to the p K ^M values of triplet state, p K _T^M , and semireduced dye radical, p K ^M_S , by thermodynamic consideration. p K _T^M (³AOH⁺/³AO) is determined to be 11.2. From thisp K _S^M (AOH./AO;) is estimated to be 17–18. This is in striking contrast to the protolytic equilibrium of the semireduced dye radicals found to be pK^F= 4.1. We conclude that the last value represents the second protonation equilibrium (AOH⁺₂./AOH). This conclusion is confirmed by spectroscopic data.     

Nanosecond time-resolved magnetic field effect on radical recombination in solution

The time dependence of the magnetic field effect on radical recombination in solution has been analyzed experimentally and theoretically. For the geminate recombination of anthracene anions and dimethylaniline cations in a polar solvent, the effect originates from a magnetic field dependent production of triplet states in an initially singlet phased radical pair, induced by hyperfine interaction of the unpaired electrons with the nuclei. The magnetic field dependence of the triplet yield shows a lifetime broadening of the energy levels of the radical pair if a short delay-time between radical production and triplet observation is chosen. The agreement of this delay-time dependent broadening effect with the theoretical results proves directly the coherence of the spin motion in the radical pairs.     

PHOTO ACTIVATION OF ISOFLAVONOID PHYTOALEXINS: INVOLVEMENT OF FREE RADICALS

Abstract— Upon irradiation with ultraviolet light the isoflavonoid phytoalexins phaseollin, 3,6a, 9-trihydroxypterocarpan, glyceollin, tuberosin and pisatin, but not medicarpin, brought about inactivation ofglucose–6-phosphate dehydrogenase in an in vitro assay system. Photoinactivation of the enzyme by photoactivated pisatin in air-saturated solutions was hardly affected by singlet oxygen quenchers such as NaN₃, bovine serum albumin, histidine or methionine. Neither addition of the hydroxyl radical scavengers mannitol, Na-benzoate and ethanol nor the presence of catalase or supcroxide dismutase protected the enzyme against photoinactivation, suggesting that OH, H₂O₂ and O₂ are not the reactive oxygen species involved. However, the free radical scavenger S-(2-amino-ethyl)isothiouronium bromide hydrobromide (AET) protected the enzyme against inactivation by photoactivated pisatin. Direct evidence for the generation of free radicals was obtained by ESR measurements of solutions of phaseollin, pisatin and medicarpin in hexane irradiated with ultraviolet light in the presence or absence of O₂. Phaseollin produced the most stable free radicals, whereas medicarpin hardly gave rise to free radical formation; pisatin took a somewhat intermediate position by producing a strong ESR signal which, however, decayed rather quickly. Photodegradation of all phytoalexins, except for medicarpin, was accompanied with loss of fungitoxicity, as shown in thin-layer chromatographic bioassays, and formation of new products.
These results indicate free radical formation as the causative process for photoinactivation of enzymes by photoactivated isoflavonoid phytoalexins.     

AN EPR STUDY ON THE TRIPLET STATE OF PURINE FREE BASE IN AQUEOUS GLASSES AT 77 K

Abstract— Since purine free base in aqueous solution exists in different ionic forms at different pHs and most of its photoreactions are likely to involve the triplet excited states of these different ionic forms, electron paramagnetic resonance studies (EPR) have been performed in order to determine intersystem crossing quantum yields and other characteristic parameters of the excited triplet state of these forms. Intersystem crossing yields decrease with a decrease in pH, being 0.62, 0.37 and 0.10 in 8 M NaOH, 8 M NaCIO₄ and 6M H₃P0₄ glasses, respectively. Differences in triplet decay lifetimes 3.4, 2.5 and 3.1 s, as well as in root mean-square zero-field splitting (ZFS) parameter, D_*, (0,1304, 0.1512 and 0.1353 cm_-1) are also observed for the anionic, neutral and cationic species of purine free base. The EPR signals of the triplet state of the neutral and anionic forms have been observed simultaneously in the pH range of7–10.     

ELECTRON TRANSFER FROM THE EXCITED STATE OF TYROSINE TO COMPOUNDS CONTAINING DISULFIDE LINKAGES

Abstract— The flash photolysis of aqueous solutions of tyrosine has been studied in the presence of various concentrations of the cyclic disulfide sodium lipoate (thioctic acid, Na⁺ salt). In addition to the formation of phenoxyl radicals and hydrated electrons (and possibly H atoms) from the photoionization of tyrosine, the characteristic spectrum of the radical anion RSSR^- of lipoate was also observed in neutral as well as in alkaline solutions. From the dependence of these yields upon the concentration of lipoate, it was found that a long–lived triplet excited state of tyrosine, rather than the singlet excited state, is involved in these reactions. The negative radical ions RSSR^- are formed by two distinct pathways: (a) Na⁺–lipoate reacts with the solvated electrons which are ejected from the tyrosine triplets ³Tyr → RO.+ e ^-_aq+ H⁺ followed by e ^-_aq+ RSSR → RSSR^-, and (b) by direct interaction of lipoate with triplet excited tyrosine, resulting in the transfer of a negative charge from tyrosine to the disulfide linkage. At high lipoate concentrations, the singlet excited state of lipoate is quenched, k ₄= 1.6 × 10¹⁰ M ^-1 sec^-1, but this reaction does not lead to the formation of RSSR^- radical ions.     

STUDIES ON THE UV PHOTODESTRUCTION OF PURINE FREE BASE IN AQUEOUS SOLUTIONS AT 77 and 298K

Abstract— The photodestruction of purine free base used as a model for the purine bases in DNA has been studied in order to better understand the effect of UV light on these molecules. Photodestruction yields have been determined in glassy aqueous solutions at 77 K and at room temperature at different pH's. The yield decreases in the order of 0.04, 0.01, 0.001 in 8 M NaOH, 8 M NaCIO₄ and 6 M H₃PO₄, respectively, while at room temperature the highest destruction yield is 0.005 for the unbuffered neutral solution. These yields have been determined by measuring the initial decrease in the purine absorption maximum as a function of irradiation time. During the illumination stable photoproducts, as well as reactive intermediates, such as trapped electrons, radical anions and cations, are formed and have been characterized from their absorption spectra. The addition of triplet quenchers and an electron scavenger resulted in a decrease in the yield, implying the participation of the purine triplet state and a radical anion in the reactions leading to the photodestruction of purine.     

PHOTODEGRADATION AND in vitro PHOTOTOXICITY OF FENOFIBRATE, A PHOTOSENSITIZING ANTI-HYPEIUIPOPROTEINEMIC DRUG

The phototoxic anti-hyperlipoproteinemic drug fenofibrate was found to be photolabile under aerobic and anaerobic conditions. Irradiation under argon of a methanol solution of this drug produced the photoproducts isopropyl 4-(1-[4-chlorophenyl]-1,2-dihydroxy)ethylphenoxyisobutyrate, 1,2- bis (4-chlorophenyl)-1,2- bis (4-[isopro-poxycarbonylisopropoxy]phenyl)ethane-1,2-diol and 4-(4-chlorobenzoyl)phenol, while under oxygen the photoproducts were 4-chloroperbenzoic acid, methyl 4-chlorobenzoate, 4-chlorobenzoic acid and singlet oxygen, as evidenced by trapping with 2,5-dimethylfuran. These results can be rationalized through hydrogen abstraction by excited fenofibrate, to afford a free radical as key intermediate. Biologically active antioxidants such as glutathione and cysteine efficiently reduced 4-chloroperbenzoic acid to 4-chlorobenzoic acid. The involvement of an electron transfer mechanism is suggested by detection (UV-vis spectrophotometry) of the radical cation TMP⁺ during the oxidation of tetramethylphenylenediamine (TMP) with 4-chloroperbenzoic acid. Fenofibrate was phototoxic in vitro when examined by the photohemolysis test, both under oxygen and argon atmosphere, although the photohemolysis rate was markedly lower under anaerobic conditions. The photoproducts 4-(1-[4-chlorophenyl]-1,2-dihy-droxy)ethylphenoxyisobutyrate and 4-chloroperbenzoic acid induced hemolysis in the dark however, this effect was quantitatively less important than photohemolysis by fenofibrate. On the other hand, fenofibrate photosensitized peroxidation of linoleic acid, monitored by the UV detection of dienic hydroperoxides. Based on the inhibition of this process upon addition of butylated hydroxyanisole, a radical chain (type I) mechanism appears to operate. In summary, fenofibrate is phototoxic in vitro . This behavior can be explained through the involvement of free radicals, singlet oxygen and stable photoproducts.     

LUMIFLAVIN-SENSITIZED PHOTOOXYGENATION OF INDOLE

Abstract— The lumiflavin-sensitized photooxygenation of indole in aqueous solutions has been investigated by means of steady light photolysis and flash photolysis. The semiquinone of lumiflavin and the half-oxidized radical of indole were formed by the reaction between triplet lumiflavin and indole (3.7 times 10⁹ M ^-1 s^-1). The semiquinone anion radical of lumiflavin reacted with oxygen to form superoxide radical. The triplet state of lumiflavin also reacted with oxygen forming singlet oxygen, ¹O₂. But the reaction between ¹O₂ and indole (7 times 10⁷ M^_l s^_1; estimated from steady light photolysis using Rose Bengal as a sensitizer) was far less efficient than the reaction between indole and triplet lumiflavin. The quantum yield of the lumiflavin-sensitized photooxygenation of dilute indole via radical processes was much higher than that via ¹O₂ processes, though appreciable ¹O₂ was formed.     

ESR STUDY OF ORIENTED TRIPLET MOLECULES GENERATED BY EXCITATION WITH POLARIZED LIGHT

Abstract— The lowest-lying triplet states of a variety of aromatic molecules and complexes have been generated by the irradiation of these compounds in solvent glasses with plane-polarized light. Measurements of the allowed (Δ M=± 1) ESR transitions clearly demonstrate that the triplets so formed are oriented with respect to the external magnetic field. By this method the triplet zero-field splitting parameters, D and E , can be evaluated simply and reliably. Intramolecular energy transfer is postulated to explain the triplet spectra of Zn( o -phen)₂(NO₃)₂ and Zn( o -phen)₃(NO₃)₂. It was observed that in triplet-triplet energy transfer from benzophenone to naphthalene there is no apparent orientation requirement between the donor and acceptor molecules. Further areas of significance and application of this technique are suggested.