PHOTOCHEMISTRY OF AQUEOUS ADRIAMYCIN AND DAUNOMYCIN. A SPIN TRAPPING STUDY WITH 17O ENRICHED OXYGEN AND WATER

Abstract— Photolyses at 485 or 490 nm of air saturated, H₂^I7O enriched, aqueous solutions of adriamycin (ADR) in the presence of 5,5-dimethyl-l-pyrroline-l-oxide (DMPO) produce a mixture of hydroxyl radical spin-adducts, i.e. DMPO-¹⁶OH- and DMPO-¹⁷OH-, as detected by electron spin resonance (ESR). DMPO-¹⁷OH- was also observed during the irradiation at these same wavelengths of aqueous solutions of ADR or daunomycin (DA) containing DMPO and ¹⁷O enriched oxygen. Therefore, the observed hydroxyl radical spin-adducts derive from both water and gaseous oxygen. It is concluded from the measured relative spin-adduct concentrations and from the enrichment fractions that most of the DMPO-OH adducts originate from water. However, if anaerobic diluted solutions of ADR (200 μ M ) or DA (600 μM) in the presence of DMPO (13 mM and 27 m M respectively) are irradiated at these same wavelengths no spin adduct is detected, indicating that oxygen is needed for the adduct formation, at these drug and spin trap concentrations. DMPO-OH- is always observed if relatively high concentrations of either the drug (1.1–2.9 mM) or the spin trap(100–150 m M ) are used in argon saturated solutions. A water photooxidation mediated mechanism is proposed in order to account for these results, analogous to previous observations in the photochemistry of other water soluble anthraquinone derivatives.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS—X. A SPIN-TRAPPING AND DIRECT ELECTRON SPIN RESONANCE STUDY OF THE PHOTOCHEMICAL PATHWAYS OF DAUNOMYCIN AND ADRIAMYCIN

Abstract— Irradiation of daunomycin (or adriamycin) and the spin trap 5,5-dimethyl-l-pyrroline-1-oxide (DMPO) at 490 nm in the presence or in the absence of air generated the hydroxyl radical adduct (DMPO-OH). The observed DMPO-OH signal was not affected by the addition of hydroxyl radical scavengers (ethanol, formate), suggesting that direct trapping of the hydroxyl radical was not involved. The DMPO-OH signal was insensitive to superoxide dismutase and catalase, which ruled out the possibility of superoxide or H₂O₂ involvement. These findings demonstrate that daunomycin (or adriamycin) does not generate hydroxyl radicals or superoxide radical anions when subjected to 490-nm excitation. However, when daunomycin (or adriamycin) was irradiated at 310 nm DMPO adducts derived from two carbon-centered radicals, superoxide and the hydroxyl radical were detected. The superoxide adduct of DMPO was abolished by the addition of SOD, providing unequivocal evidence for the generation of the superoxide anion radical. The daunomycin semiquinone radical, observed upon 310-nm irradiation of daunomycin in the absence of DMPO, appears to be the precursor of the superoxide radical anion. One of the carbon-centered radicals trapped by DMPO exhibited a unique set of hyperfine parameters and was identified as an acyl radical. This suggests that the known photochemical deacylation of daunomycin occurs via a homolytic cleavage mechanism. The free radicals generated photolytically from adriamycin and daunomycin may be involved in the etiology of the skin ulceration and inflammation caused by these drugs. A knowledge of the dependence of these photogenerated radicals on the wavelength of excitation may be important in the development of adriamycin and daunomycin for photodynamic therapy.     

QUENCHING OF THE EXCITED STATES OF 2-PHENYLBENZOXAZOLE BY AZIDE ANION. FLUORESCENCE AND ESR STUDY

Electron spin resonance, spin-trapping and fluorescence techniques demonstrate that 2-phenylbenzoxazole (P) participates in photo-induced reactions with alcohols and electron donors like the azide ion. Irradiation of Pat 300 nm in deaerated ethanol produces ethoxyl and hydroxyethyl radicals which can be detected with the spin trap, 5,5-dimethyl-l-pyrroline-l-oxide (DMPO). However, irradiation of P in the presence of N^-₃ leads to the appearance of the azide radical, N^-₃, which also reacts with DMPO. Studies with the nitroxyl radical, 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), suggest that electron transfer from the azide anion to an excited state of P yields the semi-reduced sensitizer, P^-, which in turn reacts with TEMPO. The effect of sodium azide upon the fluorescence intensity and lifetime of P in aqueous ethanol has also been studied.     

Photochemistry of Water-soluble Quinones. Production of a Water-derived Spin Adduct

Abstract— The photolyses of phosphate-buffered (pH 7) air- and nitrogen-saturated solutions containing the water-soluble quinones, 1,4-benzoquinone (BQ), 2-methyl-l,4-ben-zoquinone (MBQ), sodium 1,4-naphthoquinones-sulfonate (NQ2S), 9,10-anthraquinone-2-sulfonate (AQ2S) or 9,10-anthraquinone-l,5-disulfonate (AQDS), and the spin trap 5,5-dimethylpyrroline-l-oxide (DMPO) produce a DMPO-OH adduct. Electron paramagnetic resonance spectroscopy of the photolyzed samples in ₁₇0-enriched water demonstrates that this adduct derives almost exclusively from water. With the exception of BQ, quantum yields for the formation of DMPO-OH are larger in air than in nitrogen-saturated samples, thus supporting the idea of the formation of air-oxidized intermediates that enhance the DMPO hydroxylation reaction rate. Evidence has been obtained which suggests that BQ and MBQ, but not AQDS, are able to photoox-idize water, with the consequent production of the free OH radical.     

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.     

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.     

ULTRAVIOLET LIGHT-INDUCED FREE RADICAL FORMATION IN SKIN: AN ELECTRON PARAMAGNETIC RESONANCE STUDY

It has been suggested that ultraviolet light induces free radical formation in skin, leading to photoaging and cancer. We have demonstrated by electron paramagnetic resonance that the ascorbate free radical is naturally present in unexposed skin at a very low steady state level. When a section of SKH-1 hairless mouse skin in an EPR cavity is exposed to UV light (4,500 J m⁻²−1, Xe lamp, 305 nm cutoff and IR filters), the ascorbate free radical signal intensity increases. These results indicate that UV light increases free radical oxidative stress, consistent with ascorbate's role as the terminal, small-molecule antioxidant. The initial radicals produced by UV light would have very short lifetimes at room temperature; thus, we have applied EPR spin trapping techniques to detect these radicals. Using α-[4-pyridyl 1-oxide]-N- tert -butyl nitrone (POBN), we have for the first time spin trapped a UV light-produced carbon-centered free radical from intact skin. The EPR spectra exhibited hyperfine splittings that are characteristic of POBN/alkyl radicals, a^N= 15.56 G and a^H= 2.70 G, possibly generated from membrane lipids as a result of β-scission of lipid alkoxyl radicals. Iron can act as a catalyst for free radical oxidative reactions; chronic exposure of skin to UV radiation causes increased iron deposition. Using our spin trapping system, we have shown that topical application of the iron-chelator, Desferal, to a section of skin reduces the UV light-induced POBN adduct radical signal. These results provide direct evidence for free radical generation and a role for iron in UV light-induced dermatopathology. We suggest that iron chelators can serve as photoprotective agents by preventing these oxidations.     

Characteristics of the spin-trapping reaction of a free radical derived from AAPH: further development of the ORAC-ESR assay

The characteristics of the spin-trapping reaction in the oxygen radical absorbance capacity (ORAC)-electron spin resonance (ESR) assay were examined, focusing on the kind of spin traps. 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH) was used as a free radical initiator. The spin adducts of the AAPH-derived free radical were assigned as those of the alkoxyl radical, RO· (R=H(2)N(HN)C-C(CH(3))(2)). Among the spin traps tested, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5,5-dimethyl-4-phenyl-1-pyrroline N-oxide (4PDMPO), 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO), and 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) were applicable to the ORAC-ESR assay. Optimal formation of spin-trapped radical adduct was observed with 1 mM AAPH, 10 mM spin trap, and 5 s UV irradiation. The calibration curve (the Stern-Volmer's plot) for each spin trap showed good linearity, and their slopes, k (SB)/k (ST), were estimated to be 87.7±2.3, 267±15, 228±9, and 213±16 for DMPO, 4PDMPO, CYPMPO, and DEPMPO, respectively. Though the k (SB)/k (ST) values for selected biosubstances varied with various spin traps, their ratios to Trolox (the relative ORAC values) were almost the same for all spin traps tested. The ORAC-ESR assay also had a very good reproducibility. The ORAC-ESR assay was conducted under stoichiometric experimental conditions. The present results demonstrate the superiority of the ORAC-ESR assay.     

脂氧合酶催化双高-γ-亚麻酸脂质过氧化产生的碳自由基的结构确证

采用液相色谱-电子自旋共振波谱(LC/ESR)联用技术、液相色谱-质谱(LC/MS)联用技术结合自旋捕集技术,研究了脂氧合酶(LOX)催化双高-γ-亚麻酸(DGLA)脂质过氧化过程中产生的碳自由基.以α-[4-吡啶基-1-氧]-N-叔丁基氮酮(POBN)为自旋捕集剂,在LOX-DGLA反应混合物中与碳自由基形成自旋加合物后,根据各加合物在LC/UV/ESR和LC/MS中对应的保留时间,确定加合物的分子量,进一步根据加合物质谱裂解方式确定其结构.结果表明,在LOX催化DGLA脂质过氧化过程中产生的碳自由基主要包括~·C_7H_(13)O_2,~·C_(10)H_(17)O_2和~·C_5H_(11),分别来自DGLA脂氧自由基(8-,11-,15-LO~·)的β-裂解.此结果有利于进一步研究DGLA在体内的脂质过氧化过程及该过程中产生的碳自由基的生理作用.     

Direct ESR detection and spin trapping of radicals generated by reaction of oxygen radicals with sulfonated poly(ether ether ketone) (SPEEK) membranes

The stability of membranes under the strong oxidizing conditions in fuel cells is one of the major challenges in the development of fuel cells based on proton exchange membranes (PEMs). This study is centered on the determination of the susceptibility to degradation of SPEEK membranes exposed to OH radicals, using both direct ESR and spin trapping with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). In order to achieve a complete picture on SPEEK degradation, two types of experiments were performed: 1. UV irradiation at 77 K of SPEEK membranes swollen by aqueous solutions of H₂O₂; 2. UV irradiation of SPEEK membranes swollen by aqueous solutions of H₂O₂ in the presence of DMPO as a spin trap. UV irradiation without oxygen of SPEEK at 77 K in acid or basic form in the presence of H₂O₂/H₂O produced phenoxyl radicals as the predominant radicals detected by direct ESR or spin trapping methods. At pH 4, the oxygen radicals produced phenyl radicals as the predominant species detected by spin trapping methods. The hydroperoxyl radical, as DMPO/OOH adduct, was detected only when the DMPO/OH adduct was absent. The appearance of phenyl and phenoxyl radicals provides the evidence that OH radicals react with the aromatic ring of SPEEK or leading to the scission of its ether bridge.     

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.     

Spin trapping by 5,5-dimethylpyrroline-N-oxide in Fenton media in the presence of Nafion perfluorinated membranes: limitations and potential

Spin trapping by 5,5-dimethylpyrroline-N-oxide (DMPO) was used for the detection of radicals in Fenton media in the presence and absence of Nafion perfluorinated ionomers. For ethanol as solvent, the same types of spin adducts were detected in the presence or absence of Nafion. Solvent-derived adducts, DMPO/*OC2H5 and DMPO/*CH(OH)CH3, were identified, and their presence was rationalized by Fe(III)-catalyzed nucleophilic addition of ethanol to the spin trap and hydrogen abstraction by *OH radicals; oxygen radical adducts, DMPO/*O2(-) and DMPO/*OOH, were also detected. In Fenton media with methanol as solvent (and no Nafion), the DMPO/*O2(-) adduct dominated immediately after sample preparation, and a mixture consisting of DMPO/*OCH3, DMPO/*CH3, DMPO/*O2(-), and DMPO/*OOH adducts was detected after 30 min. In the presence of Nafion, only the adduct DMPO/*OH was detected. For water as solvent, only the DMPO/*OH adduct was detected, in both the absence and the presence of Nafion. The full hyperfine tensor components of this adduct were determined in Fenton media in the presence of Nafion with water and methanol as solvents. In Nafion/water exposed to the Fenton reagent at 358 K for 3 h, a DMPO adduct of a carbon-centered radical was also identified and assigned to a Nafion-derived fragment; its exact nature is under investigation. Variations of the 14N and Hbeta hyperfine splittings of a given adduct with the local polarity were key to the identification of some DMPO adducts, in particular DMPO/*O2(-). Both *OOH and O2*- adducts, with different 14N and Hbeta splittings, were detected simultaneously in some samples, for the first time in the spin trapping literature. Comparison with the results of a direct electron spin resonance study of Nafion exposed to the Fenton reagent indicated that spin trapping by DMPO can provide complementary information on the type of radicals present during Nafion degradation. The spin trapping approach described in this paper is limited, however, to systems that do not contain organic solvents.     

PORPHYRIN-SENSITIZED PHOTOREACTIONS IN THE PRESENCE OF ASCORBATE: OXIDATION OF CELL MEMBRANE LIPIDS AND HYDROXYL RADICAL TRAPS

Abstract— Photooxidation reactions in ascorbate (AH)-containing erythrocyte membrane suspensions have been studied in broad perspective by simultaneously monitoring lipid peroxidation in the membrane compartment and formation of hydrogen peroxide (H₂O₂) and hydroxyl radical (OH) in the aqueous compartment. Non-bound uroporphyrin (UP) and membrane-bound protoporphyrin (PP) were used as sensitizers. Photoreduction of UP to the radical anion (UP^-) was detected by electron spin resonance when UP/AH/membrane mixtures were irradiated anaerobically. Aerobic irradiation resulted in a strong AH^--stimulation of lipid peroxidation, H₂O₂ formation, and OH^- generation (detected with 2-deoxyribose (DOR) and the spin trap 5,5-dimethyl-l-pyrroline-N-oxide). Use of diagnostic agents (e.g. catalase, desferrioxamine, mannitol) revealed that OH^- is involved in light-stimulated DOR oxidation, but not in lipid peroxidation. Similar irradiation in the presence of PP resulted in far greater lipid peroxidation than observed with UP, but less DOR oxidation, and insignificant accumulation of H₂O₂. This suggests that photoreduction of membrane-bound PP is less efficient, possibly due to hindered access of AH^-.     

PHOTOSENSITIZED FORMATION OF ASCORBATE RADICALS BY RIBOFLAVIN: AN ESR STUDY

Abstract— The riboflavin-sensitized photooxidation of ascorbate ion (HA^-) to ascorbate radical (A^-) was followed by electron spin resonance (ESR) spectroscopy in conjunction with oxygen depletion measurements. In air-saturated aqueous media, steady-state amounts of A^- are rapidly established upon irradiation. The ESR signal disappears within a few seconds after the light is extinguished–more slowly under constant irradiation as oxygen is depleted. No photooxidation was observed in deaerated media. Similar results were obtained with other flavins and when ascorbyl palmitate was substituted for HA^-. The effect of added superoxide dismutase, catalase, desferrioxamine, and singlet oxygen scavengers (NaN₃ and tryptophan) was studied, as was replacement of water by D₂O and saturation with O₂. The results are indicative of ascorbate free radical production via direct reaction between ascorbate ion and triplet riboflavin in the presence of O₂. While the presence of superoxide ion tends to reduce the steady-state concentration of A^-, competition from the reaction of HA^- with singlet oxygen is less apparent in this system (at HA^-≥ 1 m M ) than in the previously studied aluminum phthalocyanine tetrasulfonate-photosensitized reaction.     

Identification of isomeric spin adducts of Leu-Tyr and Tyr-Leu free radicals using liquid chromatography-tandem mass spectrometry

Free radical species are generally short-lived due to their high reactivity and thus direct measurement and identification are often impossible. In this study we used a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), to trap radical intermediates formed during the oxidation of isomeric dipeptides tyrosine-leucine (Tyr-Leu) and leucine-tyrosine (Leu-Tyr), induced by the hydroxyl radical. To investigate the influence of the amino acid position in the peptide chain on the oxidation and free radical generation, the spin adducts were characterized using LC-MS and MS(n) . We detected carbon and oxygen DMPO adducts and adducts bearing two DMPO, which were analyzed by MS(n) . Both alkoxyl and peroxyl radicals were identified. Radical intermediates were localized in Tyr during oxidation of Tyr-Leu, while radicals were identified in Leu and Tyr during oxidation of Leu-Tyr. DMPO adducts of acyl radical species formed from cleavage of the peptide backbone, promoted by the alkoxyl radical in α carbon of the N-terminal amino acid were observed. The results show that the amino acid position has an influence in the oxidation process, at least on small peptides, and that the α carbon of the N-terminal amino acid is more vulnerable to the attack of the electrophilic hydroxyl radical.     

Identification of free radicals induced by UV irradiation in collagen water solutions

Electron paramagnetic resonance (EPR) method has shown that hydrogen atoms and acetic acid free radicals appear in surrounding acetic acid-water solution of collagen under ultraviolet (UV) irradiation. These free radicals interact with the collagen molecule; consequently, seven superfine components of EPR spectrum with the split of aH = 11.3G and g-factor 2.001 appear. It is assumed that this spectrum is related to the free radical occurred on the proline residue in collagen molecule. In order to discover .OH hydroxyl radicals even in minor concentration, spin trap 5.5-dimethyl-1-pyrroline N-oxide (DMPO) has been applied. During the irradiation of collagen water solution in the presence of spin trap, EPR spectrum of the DMPO/.OH adduct has not been identified, while the above mentioned spectrum has been observed once the hydrogen peroxide H2O2 and FeSO4 were added to the sample. That means that water photolysis does not take place in collagen water-solution due to UV irradiation. It was suggested that occurrence of hydrogen radical is connected with the electron transmission to the hydrogen ion. The possible source of free electrons can be aromatic residues, photo ionization of which takes place in collagen molecule due to UV irradiation.     

The fidelity of spin trapping with DMPO in biological systems

Unlike direct ESR, spin trap methodology depends on the absolute fidelity of the spin trap reaction. Two alternative reactions of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) leading to radical adduct artifacts have been discovered and investigated: inverted spin trapping and the Forrester-Hepburn nucleophilic mechanism. These two alternate pathways to radical adducts are a combination of one-electron oxidation and nucleophilic addition, in either order. In biological systems, serious artifacts have been reported due to the Forrester-Hepburn mechanism, which is initiated by the addition of a nucleophile to DMPO. It has recently been demonstrated that (bi)sulfite (hydrated sulfur dioxide) can react with DMPO via a nonradical, nucleophilic reaction, and it has been further proposed that DMPO/(?)SO(3)(-) formation in biological systems is an artifact and not the result of spin trapping of sulfur trioxide anion radical ((?)SO(3)(-)). The one-electron oxidation of (bi)sulfite catalyzed by horseradish peroxidase (HRP)/hydrogen peroxide (H(2)O(2)) has been reinvestigated by ESR spin trapping with DMPO and oxygen uptake studies to obtain further evidence for the radical reaction mechanism. In the absence of DMPO, the initial rate of (bi)sulfite-dependent oxygen and H(2)O(2) consumption was determined to be half of the initial rate of DMPO/(?)SO(3)(-) radical adduct formation as determined by ESR, demonstrating that, under our experimental conditions, DMPO exclusively forms the radical adduct by trapping the (?)SO(3)(-).     

PHOTOCHEMISTRY OF 2-MERCAPTOPYRIDINES. PART 3. EPR STUDY OF PHOTOPRODUCTION OF HYDROXYL RADICALS BY N-HYDROXYPYRIDINE-2-THIONE USING 5,5-DIMETHYL-1-PYRROLINE N-OXIDE IN AQUEOUS SOLUTIONS*

Abstract— N-Hydroxypyridine-2-thione, 2-S-PyrNOH, a potent antimicrobial, antifungal and anticancer agent, is photochemically active and upon UV irradiation generates free radicals. We have employed EPR and the spin-trap 5,5-dimethyl-l-pyrroline TV-oxide (DMPO) to investigate the photochemistry in aqueous solutions of 2-S-PyrNOH (used here in the form of a sodium salt, 2-S-PyrNONa). We found that upon photoactivation 2-S-PyrNONa can follow two different pathways: it can produce hydroxyl radicals and/or it can act as a photoreducing agent. The capacity of 2-S-PyrNONa to produce “OH” radicals has been demonstrated by: (1) EPR detection of the DMPO/OH adduct in UV-irradiated samples; (2) inhibition of the DMPO/OH formation by OH scavengers such as methyl alcohol, formate and DMSO and (3) by detection of EPR signals of DMPO adducts with radicals derived from reaction of OH with these inhibitors. The photoreductive capacity of 2-S-PyrNONa was deduced from the observation that the amplitude of the EPR signal of the spin adduct DMPO/OH decreased on UV irradiation in air-free pH 7.0 buffers and that the signal recovered in the dark and after aeration. The ability to generate free radicals upon UV irradiation suggests that 2-S-PyrNONa can be regarded as a potential photocytotoxic agent. This feature may be relevant to the biological action of this compound. Our findings also emphasize that caution should be used when 2-S-PyrNOH is employed as a source of OH radicals in biological or chemical systems.     

Photochemistry of naphthalene diimides: EPR study of free radical formation via photoredox process

Earlier studies have shown that on exposure to UVA, hydroperoxynaphthalene diimide (IA) generates hydroxyl radicals, induces DNA strand scission, and kills cells.Here we employed electron paramagnetic resonance (EPR) and spin trapping to investigate the free radical photochemistry of IA and that of related naphthalene diimides, which are devoid of the hydroperoxyl moiety (N,N'-bis[2-methyl]-1,4,5,8-naphthaldiimide [IB], N,N'-bis[2-thiomethyl-2-methoxyethyl]-1,4,5,8-naphthaldiimide [IC]) and therefore are unable to generate hydroxyl radicals. It is shown that on UV irradiation (>300 nm) in air-free methanol or ethanol solutions all these naphthalene diimides undergo one-electron reduction to corresponding anion radicals, positively identified by EPR. With EPR and a spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), we found that the photogeneration of the naphthalene diimide radicals is concomitant with the formation of radicals from the solvents, presumably through electron/hydrogen atom abstraction by photoactivated diimides. Irradiation of IA, IB or IC in the presence of oxygen generates superoxide, which was detected as a DMPO adduct. The high photoreactivity of IB and IC supports the notion that hydroperoxide IA can induce oxidative damage via photoprocesses that are independent of *OH generation. These observations could be pertinent to the application of naphthalene diimides as selective photonucleases, PDT anticancer agents or both.     

PHYSIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHTON DICTYOSTELIUM DISCOIDEUM SPORE GERMINATION

Abstract— The spore germination in Dictyostelium discoideum consists of four stages: activation, postactivation lag, swelling and emergence. Ultraviolet irradiation (total fluence of 250 J/m²) of spores at any time prior to late spore swelling allows full swelling, but inhibits the emergence of myxamoebae. In the case of freshly activated spores, a UV exposure time of 30 s (total fluence of 50 J/m²) is sufficient to reduce emergence to about 6% when measured after 24 h of incubation. This same fluence results in about 10% viability as measured by plaque forming ability. Experiments utilizing 'fractionated exposures' result in the same percentage inhibition of emergence as that found for 'single exposures' provided the total fluence is equivalent. The higher fluences (250 J/m²) which completely prevent emergence, do not affect the endogenous oxygen uptake of spores during swelling. Ultraviolet light irradiated spores respond to the same activation and deactivation treatments as control unirradiated spores. Ultraviolet irradiation after late spore swelling allows emergence to occur in only a small fraction of the population. This fraction of cells which can emerge after UV treatment is said to have passed a 'competence point', which is believed to be the time when all the events necessary for emergence have been completed. Though the sites of UV inactivation in spores can only be postulated at present, it is apparent that the initial stages of germination (activation, postactivation lag and spore swelling) occur independently of the UV sensitive sites. The final stage of germination (emergence), however, is dependent on UV sensitive functions.