Singlet oxygen trapping by DRD156 in micellar solutions

Recently, 4.4'-bis(1-p-carboxyphenyl-3-methyl-5-dydroxyl)-pyrazol (DRD156) has been developed as a new sensitive reagent that reacts specifically with singlet oxygen. The specificity of DRD156 for singlet oxygen in a biomimetic solution (micellar solution) and the effects of its coexistence with other reagent were examined with electron spin resonance (ESR). Singlet oxygen was generated using photosensitization reaction. The ESR spectrum of the radical derived from DRD156 after the reaction with singlet oxygen in phosphate buffered salines (PBS) was comprised of twenty-nine lines, whereas that in cetyltrimethylammonium bromide (CTAB) micelles was comprised of nine lines. Both 2,2,6,6-tetramethyl-4-piperidine (TMPD) and 1,3-diphenyl-isobenzofuran (DPBF) reduced the singlet oxygen-DRD156 signal intensity, and TMPD-mediated decrease in PBS (to 62%) was almost the same as that in CTAB micelle (to 65%). In contrast, DPBF reduced the DRD156 signal intensity more effectively in CTAB micelle (to 12%) than PBS (to 38%). These results indicate that the specificity of DRD156 for singlet oxygen is dependent on microenvironment.     

Diaziquone as a potential agent for photoirradiation therapy: formation of the semiquinone and hydroxyl radicals by visible light

When diaziquone was irradiated with 500 nm visible light, hydroxyl free radicals as well as the diaziquone semiquinone were produced. The diaziquone semiquinone is a stable free radical that exhibits a characteristic 5-line electron spin resonance (ESR) spectrum. Since hydroxyl free radicals are short lived, and not observable by conventional ESR, the nitrone spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) was used to convert hydroxyl radicals into longer lived ESR detectable spin adducts. The formation of hydroxyl radicals was further confirmed by investigating reactions in which hydroxyl radical scavangers, sodium formate and dimethylsulfoxide, compete with the spin traps DMPO or POBN (alpha-(4-Pyridyl-1-oxide)-N- tert-butylnitrone) for hydroxyl free radicals. The products of these scavenging reactions were also trapped with DMPO or POBN. If drug free radicals and hydroxyl free radicals are important in the activity of quinone-containing antitumor agents, AZQ may have a potential in photoirradiation therapy or photodynamic therapy.     

ESR and optical absorption evidence for free radical involvement in the photosensitizing action of furocoumarin derivatives and for their singlet oxygen production.

Frozen aqueous solutions of thymine and its derivatives were irradiated with visible light (lambda greater than 320 nm) in the presence of various furocoumarins. ESR analysis revealed the induction of hydrogen adduct free radicals at C-6 position of thymine, only with those furocoumarin derivatives which show a skin-photosensitizing ability. It has been shown, moreover, that the photocycloaddition of psoralen to thymine, which is responsible for the biological effects of this dye, is inhibited when the induction of free radicals in thymine moiety has been prevented by electron scavengers. It is suggested that the free radicals observed could be involved in the biological photosensitization. The mechanism of free radical generation and singlet oxygen production by furoccoumarins were also investigated.     

Generation of nitro radical anions of some 5-nitrofurans, 2- and 5-nitroimidazoles by norepinephrine, dopamine, and serotonin. A possible mechanism for neurotoxicity caused by nitroheterocyclic drugs

Catecholamine neurotransmitters such as norepinephrine, dopamine, and related catecholamine derivatives reduce nitroheterocyclic drugs such as nitrofurantoin, nifurtimox, nifuroxime, nitrofurazone, misonidazole, and metronidazole in slightly alkaline solutions. Drugs which contain 5-nitrofurans are reduced at lower pH than drugs which contain 2- and 5-nitroimidazoles. 5-Nitroimidazole derivatives such as metronidazole and ronidazole are known to be more difficult to reduce than 2-nitroimidazole derivatives, due to their lower redox potential. Catecholamines, when reducing nitro drugs, undergo concomitant oxidation to form semiquinone radicals. Both semiquinone radicals and nitro anion radicals formed in a reaction of nitro drug and catecholamine derivative were detected by electron spin resonance spectroscopy. Oxygen consumption studies in solutions containing nitro drug and catecholamine derivative showed that nitro anion radicals formed under aerobic conditions reduce oxygen to form the superoxide radical and hydrogen peroxide. Quinones formed in the reaction of catecholamine and nitro drug were detected by optical spectroscopy. Biosynthetic precursors and some metabolic products of catecholamines were also used in these studies, and they all exhibited reactions similar to catecholamines. Bovine chromaffin granules which synthesize and store catecholamines produced the nitrofurantoin anion radical when intact granules were treated with nitrofurantoin. These radicals formed inside the granules were observed by ESR spectroscopy. The formation of nitrofurantoin radical, semiquinone radicals of catecholamines, and oxygen-derived radicals by chromaffin granules is proposed to cause damage to adrenal medulla, and this process may lead to neurotoxicity.     

Hydralazine stimulates production of oxygen free radicals in Eagle's medium and cultured fibroblasts.

The effect of hydralazine on the oxygen free radical production was studied in whole cultured murine liver fibroblasts and mitochondrial and microsomal fractions of the cells by ESR spin trapping with DMPO and measurement of Tiron semiquinone formation. Hydralazine itself was found to generate free radicals in phosphate buffer and especially in Eagle's Minimal Essential Medium. Most of the adduct of the spin trap DMPO was due to its reaction with hydralazine-induced hydroxyl radical. Moreover, this compound stimulated free radical formation in fibroblasts. These data suggest that hydralazine alters the cellular free radical metabolism which may have implications for the biological activity of this drug.     

Reaction of beta-alkannin (shikonin) with reactive oxygen species: detection of beta-alkannin free radicals

beta-Alkannin (shikonin), a compound isolated from the root of Lithospermum erythrorhizon Siebold Zucc., has been used as a purple dye in ancient Japan and is known to exert an anti-inflammatory activity. This study aimed to understand the biological activity in terms of physico-chemical characteristics of beta-alkannin. Several physico-chemical properties including proton dissociation constants, half-wave potentials and molecular orbital energy of beta-alkannin were elucidated. This compound shows highly efficient antioxidative activities against several types of reactive oxygen species (ROS), such as singlet oxygen ((1)O2). superoxide anion radical (.O2), hydroxyl radical (.OH) and tert-butyl peroxyl radical (BuOO.) as well as iron-dependent microsomal lipid peroxidation. During the reactions of beta-alkannin with 1O2, .O2- and BuOO., intermediate organic radicals due to beta-alkannin were detectable by ESR spectrometry. Compared with the radicals due to naphthazarin, the structural skeleton of beta-alkannin, the beta-alkannin radical observed as an intermediate in the reactions with (1)O2, and .O2- was concluded to be a semiquinone radical. On the other hand, during the reactions of beta-alkannin and naphthazarin with BuOO., ESR spectra different from the semiquinone radical were observed, and proposed to result from the abstraction of hydrogen atoms from phenolic hydroxyl groups of beta-alkannin by BuOO.. Based on the ROS-scavenging abilities of beta-alkannin, the compound was concluded to react directly with ROS and exhibits antioxidative activity, which in turn exerts anti-inflammatory activity.     

The enzymatic one-electron reduction of porphyrins to their anion free radicals

The anaerobic enzymatic one-electron reduction of uroporphyrin I (in the absence of light) by the ferredoxin/ferredoxin:NADP+ oxidoreductase system was investigated using NADPH as the source of reducing equivalents. The porphyrin anion free radical metabolite formed by one-electron reduction of the parent molecule was detected with ESR spectroscopy. The ESR spectrum exhibited a singlet (g = 2.0021) with a 5.4-G peak-to-peak linewidth. The reduction process was also investigated under aerobic conditions. The reduction of molecular oxygen to superoxide anion radical by the porphyrin anion radical was demonstrated by using the ESR technique of spin trapping. The ESR spectra of the spin-trapped oxygen-derived radicals were superoxide dismutase-sensitive and catalase-insensitive, supporting the assignment of the trapped radical to the superoxide anion radical. These aerobic experiments demonstrate electron transfer from the porphyrin anion radical to molecular oxygen. The anaerobic reduction of Photofrin II by hepatic microsomes and the ferredoxin/ferredoxin:NADP+ oxidoreductase system to a porphyrin anion radical was also investigated. Free radical formation by ferredoxin: NADP+ oxidoreductase is totally dependent upon ferredoxin. The ESR spectrum of this porphyrin free radical also exhibited a singlet (g = 2.0026) with a 15-G peak-to-peak linewidth.     

Synthesis and EPR investigations of new aminated hypocrellin derivatives

Hypocrellins are novel photodynamic agents. A recent advance in the synthesis of hypocrellin congeners results in the production of two amino-substituted hypocrellin B derivatives in high yield via photochemical reaction. Both compounds exhibit similar photodynamic activity as hypocrellin B in terms of type I and type II mechanisms. In anaerobic media, semiquinone anion radicals can be detected by electron paramagnetic resonance (EPR) under irradiation; while superoxide anion radical, hydroxyl radical and singlet oxygen are photoproduced when oxygen was present. The quantum yields of singlet oxygen by these two new compounds are determined to be 0.72 and 0.64, respectively, similar to that of hypocrellin B. The comparison of the photosensitization chemistry of compounds 1 and 2 in liposomes with that in homogeneous solution has also been made. In liposomes, the type II photoprocess was favored and predominant over the type I photoprocess due to the decreased interactions between dye molecules. Both compounds exhibit much stronger red light absorption than the parent hypocrellin and therefore, merit investigation as photosensitizers.     

An electron spin resonance study on the free radicals produced from aclacinomycin a and its derivatives: analysis of hyperfine structure of the spectra by means of molecular orbital method

Quinone-containing carcinostatics, aclacinomycin A and its derivatives were investigated on the convertibility to free radical under a mild reducing condition. The hyperfine structures of electron spin resonance (ESR) spectra were satisfactorily reproduced by computer simulations, using the hyperfine coupling constants calculated by the Intermediate Neglect of Differential Overlap Molecular Orbital (INDO MO) method. This verifies the reliability of molecular orbital calculations and opens a way to analyze theoretically the correlation between chemical structures and carcinostatic activities. By analyzing hyperfine structures of ESR spectra, the free radical produced from aclacinomycin was identified as a neutral form of semiquinone radical of intact aclacinomycin. Taking into account the previous finding that 7-deoxyaklavinone (C1) is formed reductively by cytochrome P-450 reductase (EC 1.6.2.4; Komiyama et al., 1979), it is postulated that two types of semi-quinone radicals exist in vivo.     

Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps

Abstract

Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-β-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated β-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.     

ESR study of electron transfer reactions between gamma-irradiated pyrimidines, adriamycin and oxygen

Solid pyrimidine nucleic acid bases (cytosine, thymine, and uracil) were gamma-irradiated (50 KGy) and dissolved in deaerated solutions of adriamycin in water and dimethylsulfoxide (DMSO). Analogous experiments using unirradiated pyrimidines as controls were also performed. In water only gamma-irradiated cytosine showed a reaction with the adriamycin yielding a single ESR peak (g = 2.0033) consistent with the adriamycin semiquinone radical. Since the unirradiated cytosine gave no reaction, the result suggests an electron transfer from cytosine radicals (generated by gamma-radiolysis) to adriamycin. In DMSO the three gamma-irradiated and unirradiated pyrimidines reacted with adriamycin yielding the adriamycin semiquinone radical observed by ESR. These results suggest that in DMSO an electron is transferred to adriamycin from the pyrimidine radicals and from the parent pyrimidine molecules. However, the process is on the order of 10(5) times more efficient for the pyrimidine radicals. Superoxide radicals (O2-.) were formed following addition of oxygen to the deaerated DMSO solutions containing adriamycin semiquinone radicals. O2-. was spin trapped using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results show a possible reaction sequence in which an electron transferred to adriamycin, by pyrimidine radicals and parent pyrimidine molecules, is subsequently transferred to dissolved oxygen.     

Biological Reduction of Aromatic Nitroso Compounds: Evidence for the Involvement of Superoxide Anions

The in vitro formation of phenylhydronitroxide and 2-methylphenylhydronitroxide free radicals from nitrosobenzene (NB) and 2-nitrosotoluene (NT), respectively, in either red blood cells (RBC) or RBC hemolysates, was confirmed by electron spin resonance spectroscopy (ESR). Free radicals were generated nonenzymatically from reaction of the respective nitroso compounds with a number of biological reducing agents as corroborated by model studies of NB or NT with NAD(P)H. Under aerobic conditions, phenylhydronitroxide and 2-methylphenylhydronitroxide underwent a subsequent one-electron transfer to oxygen, which then resulted in the formation of superoxide anion (O₂^-). The latter product was confirmed by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (cyt c). Apparently, oxygen is needed for continuous formation of the hydronitroxide radical derivatives. On the other hand, under anaerobic conditions, no phenylhydronitroxide radical was generated from NB in the presence of NADH, but the formation of phenylhydroxylamine from NB was detected by the absorption spectrometry. These results suggest that oxygen is a preferential electron acceptor for hydronitroxide radical derivatives.     

Kinetic analysis-based quantitation of free radical generation in EPR spin trapping

Because short-lived reactive oxygen radicals such as superoxide have been implicated in a variety of disease processes, methods to measure their production quantitatively in biological systems are critical for understanding disease pathophysiology. Electron paramagnetic resonance (EPR) spin trapping is a direct and sensitive technique that has been used to study radical formation in biological systems. Short-lived oxygen free radicals react with the spin trap and produce paramagnetic adducts with much higher stability than that of the free radicals. In many cases, the quantity of the measured adduct is considered to be an adequate measure of the amount of the free radical generated. Although the intensity of the EPR signal reflects the magnitude of free radical generation, the actual quantity of radicals produced may be different due to modulation of the spin adduct kinetics caused by a variety of factors. Because the kinetics of spin trapping in biochemical and cellular systems is a complex process that is altered by the biochemical and cellular environment, it is not always possible to define all of the reactions that occur and the related kinetic parameters of the spin-trapping process. We present a method based on a combination of measured kinetic data for the formation and decay of the spin adduct alone with the parameters that control the kinetics of spin trapping and radical generation. The method is applied to quantitate superoxide trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). In principle, this method is broadly applicable to enable spin trapping-based quantitative determination of free radical generation in complex biological systems.     

The reduction of anti-tumour diaziridinyl benzoquinones

The properties of the semiquinone radicals produced for 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ) and 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), have been investigated. AZQ semiquinone radicals can be produced from the reduction of AZQ by superoxide radicals, whereas BZQ semiquinone radicals are unstable in the presence of oxygen. The one-electron reduction potentials of the couples Q/Q-. at pH 7.0 were determined as -70 +/- 10 mV for AZQ and -376 +/- 15 mV for BZQ. The difference in these potentials is explained. As a consequence of ESR studies on the enzymatically produced radicals, we have considered the factors which determine the detection of ESR signals for reduced quinones produced in a biological system.     

Stable free radicals as ubiquitous components of red wines.

A stable ESR signal, centred at g = 2.0037 +/- 0.0002, characterised by a single resonance and assignable to a free radical, was found in all the bottled red wines, both commercial and experimental, that we have examined. The radical concentration was calculated to be in the range of 5-82 nM. After exposure of the wines to air for a few minutes a two fold increase of the ESR signal, followed by a slow decrease with time, was observed. The intensity of ESR signal in experimental red wines, was found to increase with the ageing of the wines and was strictly correlated to the total content of polyphenols. The formation of semiquinone radicals of polyphenols is suggested as one possible mechanism leading to the presence of stable free radicals in red wines.     

Pulse radiolysis studies of antitumor quinones: Radical lifetimes,reactivity with oxygen,and one-electron reduction potentials

The formation of semiquinone free radicals from antitumor drugs has been studied by pulse radiolysis. The semiquinone free radicals are reactive and have short half-lives in aqueous media under anaerobic conditions. The half-lives of the radicals formed from adriamycin, mitomycin C, and 2,5-diaziridinyl-3,6-bis(carboethoxy)amine-1,4-benzoquinone (AZQ) are 50,100, and 200 μs, respectively. The mean diffusion distance of the semiquinone free radical is less than 0.6 μm. In the presence of molecular oxygen the half-life of the semiquinone free radical is shortened. Adriamycin semiquinone reacts rapidly with oxygen, k = 4.4 × 10⁷m^?1s^?1. In air-saturated buffer the half-life of adriamycin semiquinone radical can be calculated to be 8 μs with a mean diffusion distance of less than 0.1 μm. If the half-lives in buffer are comparable to those within a cell, semiquinone free radicals must be generated close to the site at which they produce a biological effect. One-electron reduction potentials (E₇¹) were determined and were AZQ, ?168 mV, adrenochrome, ?253 mV, mitomycin C, ?271 mV, adriamycin, ?292 mV, daunomycin, ?305 mV, and anthracenedione, ?348 mV. Enzymatic one-electron reduction of these antitumor quinones by NADPH-cytochrome P-450 reductase increased at more positive values of quinone E₇¹.     

Biological effects of cigarette smoke, wood smoke, and the smoke from plastics: The use of electron spin resonance

This review compares and contrasts the chemistry of cigarette smoke, wood smoke, and the smoke from plastics and building materials that is inhaled by persons trapped in fires. Cigarette smoke produces cancer, emphysema, and other diseases after a delay of years. Acute exposure to smoke in a fire can produce a loss of lung function and death after a delay of days or weeks. Tobacco smoke and the smoke inhaled in a burning building have some similarities from a chemical viewpoint. For example, both contain high concentrations of CO and other combustion products. In addition, both contain high concentrations of free radicals, and our laboratory has studied these free radicals, largely by electron spin resonance (ESR) methods, for about 15 years. This article reviews what is known about the radicals present in these different types of smokes and soots and tars and summarizes the evidence that suggests these radicals could be involved in cigarette-induced pathology and smoke-inhalation deaths. The combustion of all organic materials produces radicals, but (with the exception of the smoke from perfluoropolymers) the radicals that are detected by ESR methods (and thus the radicals that would reach the lungs) are not those that arise in the combustion process. Rather they arise from chemical reactions that occur in the smoke itself. Thus, a knowledge of the chemistry of the smoke is necessary to understand the nature of the radicals formed. Even materials as similar as cigarettes and wood (cellulose) produce smoke that contains radicals with very different lifetimes and chemical characteristics, and mechanistic rationales for this are discussed. Cigarette tar contains a semiquinone radical that is infinitely stable and can be directly observed by ESR. Aqueous extracts of cigarette tar, which contain this radical, reduce oxygen to superoxide and thus produce both hydrogen peroxide and the hydroxyl radical. These solutions both oxidize alpha-1-proteinase inhibitor (a1PI) and nick DNA. Because of the potential role of radicals in smoke-inhalation injury, we suggest that antioxidant therapy (such as use of an inhaler for persons brought out of a burning building) might prove efficacious.     

Spontaneous generation of adriamycin semiquinone radicals at physiologic pH.

Adriamycin semiquinone radicals are spontaneously generated by adriamycin solutions at physiologic pH. Rate of radical formation and equilibrium-state radical yield increase with increasing pH from 7.4 to 8.85. The radicals are oxygen sensitive, but the mechanism of radical formation is oxygen independent and associated with proton removal from the dihydroquinone of adriamycin. The less cardiotoxic and non-mutagenic (Ames test) anthracycline 5-iminodaunorubicin does not form semiquinone radicals spontaneously at physiologic pH.     

Stable free radicals as ubiquitous components of red wines

A stable ESR signal, centred at g = 2.0037 ± 0.0002, characterised by a single resonance and assignable to a free radical, was found in all the bottled red wines, both commercial and experimental, that we have examined. The radical concentration was calculated to be in the range of 5–82 nM. After exposure of the wines to air for a few minutes a two fold increase of the ESR signal, followed by a slow decrease with time, was observed. The intensity of ESR signal in experimental red wines, was found to increase with the ageing of the wines and was strictly correlated to the total content of polyphenols. The formation of semiquinone radicals of polyphenols is suggested as one possible mechanism leading to the presence of stable free radicals in red wines.     

Photoreduction of the fluorescent dye 2′-7′-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements

The photoreduction of 2′-7′-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (λ > 300 nm) 2′-7′-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/^·OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.