Reactions of arylamine and aminophenol derivatives, and riboflavin with organic radicals

Based on product yield data on radiolysis of hexane, ethanol and 3 M aqueous ethylene glycol solutions, the ability of a number of arylamine, aminophenol and quinonimine derivatives to affect processes involving peroxyl, alkyl or alpha-hydroxyalkyl radicals was assessed. It has been shown that the introduction of a hydroxyl group into aromatic amine structure enhances its antioxidant performance and makes it significantly more reactive with respect to carbon-centered organic radicals. Replacement of the hydrogen atom of a hydroxyl group by a methyl group decreases the anti-radical activity of aminophenols drastically. Compounds containing (or capable of forming) a quinonimine moiety interact with alkyl or alpha-hydroxyalkyl radicals most effectively, suppressing recombination and fragmentation reactions of the latter. In the sequence: aromatic amines--aminophenols--quinonimines, a trend towards enhancement of the ability of the compounds studied to react with carbon-centered radicals was noted. Also, this study presents for the first time evidence of riboflavin reactivity with respect to organic radicals.     

Effects of Phenolic Compounds on Reactions Involving Various Organic Radicals

Investigation of effects produced by 26 various phenol and diphenol derivatives, including industrial and natural antioxidants (ionol, bis-phenol 2246, α-tocopherol), on final product yields of radiation-induced free-radical processes involving peroxyl, alkyl, α-hydroxyalkyl and α,β-dihydroxyalkyl radicals has been performed. Ionol and bis-phenol 2246 have been shown to be more effective than α-tocopherol or diphenol derivatives in suppressing hydrocarbon oxidation processes. At the same time, α-tocopherol and its water-soluble analogues, as well as diphenol-based substances, are more effective than phenol derivatives in regulating various homolytic processes involving carbon-centered radicals. This fact can be accounted for by taking into consideration the contribution to formation of the final product set and the respective yields made by semiquinone radicals and compounds with quinoid structure arising in the course of homolytic transformations in systems containing diphenol derivatives.     

Radical-regulating and antiviral properties of ascorbic acid and its derivatives

The ability of ascorbic acid and a number of its derivatives to suppress replication of Herpes simplex virus type I was investigated in human rhabdomyosarcoma cell line. In parallel, interaction of the test compounds with carbon- and oxygen-centered radicals formed on radiolysis of hydroxyl-containing organic compounds was studied using the steady state radiolysis method. It has been shown that 2-O-glycoside of ascorbic acid, displaying marked antiviral properties against Herpes simplex virus type I, is also capable of inhibiting fragmentation and recombination reactions of α-hydroxyl-containing carbon-centered radicals while not affecting processes involving oxygen-centered radicals.     

Chitosan gallate as a novel potential polysaccharide antioxidant: an EPR study

A novel biopolymer-based antioxidant, chitosan conjugated with gallic acid (chitosan galloylate, chitosan-GA), is proposed. Electron paramagnetic resonance (EPR) demonstrates a wide range of antioxidant activity for chitosan-GA as evidenced from its reactions with oxidizing free radicals, that is, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), horseradish peroxidase (HRP)-H₂O₂, carbon-centered alkyl radicals, and hydroxyl radicals. The EPR spectrum of the radical formed on chitosan-GA was attributed to the semiquinone radical of the gallate moiety. The stoichiometry and effective concentration (EC₅₀) of the DPPH free radical with chitosan-GA show that the radical scavenging capacity is maintained even after thermal treatment at 100 °C for an hour. Although the degree of substitution of GA on chitosan was about 15%, its antioxidant capacity, that is, the reaction with carbon-centered and hydroxyl radicals, is comparable to that of GA.     

Factors affecting the free radical scavenging behavior of chitosan sulfate

Scavenging activity of hydroxyethyl chitosan sulfate (HCS) against 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl and carbon-centered radical species were studied using electron spin resonance (ESR) spectroscopy. In addition, its antioxidant activity to retard lipid peroxidation was also evaluated in a linoleic acid model system. HCS could scavenge DPPH (33.78%, 2.5 mg/mL) and carbon-centered radicals (67.74%, 0.25 mg/mL) effectively. However, chitosan sulfate did not exhibit any scavenging activity against hydroxyl radicals, but increased its generation. This was different from the published literature and was presumed due to the loss of chelating ability on Fe2+. This assumption could further confirm from the results obtained for Fe2+-ferrozine method that upon sulfation chitooligosaccharides lost its chelation properties. Therefore, HCS can be identified as antioxidant that effectively scavenges carbon centered radicals to retard lipid peroxidation.     

Antioxidant properties of EPC-K1: a study on mechanisms.

Scavenging effects of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8- tetramethyl-2-(4,8,12-trimethytridecyl)-2H-1-benzopyran- 6-yl-hydrogen phosphate] potassium salt (EPC-K1) on hydroxyl radicals, alkyl radicals and lipid radicals were studied with ESR spin trapping techniques. The inhibition effects of EPC-K1 on lipid peroxidation were assessed by TBA assay. The kinetics of EPC-K1 reacting with hydroxyl radicals and linoleic acid radicals were studied by pulse radiolysis. The active site of EPC-K1 and the structure-antioxidative activity relationships were discussed. The superoxide radicals scavenging capacity of the brain homogenate of EPC-K1-treated rats was measured. The results revealed that in comparison with Trolox and vitamin C, EPC-K1 showed better overall antioxidative capacity in vitro and in vivo. EPC-K1 was a moderate scavenger on hydroxyl radicals and alkyl radicals, a potent scavenger on lipid radicals, and an effective inhibitor on lipid peroxidation. EPC-K1 could react with hydroxyl radicals with a rate constant of 7.1 x 10(8) dm3 mol-1 s-1 and react with linoleic acid radicals with a rate constant of 2.8 x 10(6) dm3 mol-1 s-1. The active site of EPC-K1 was the enolic hydroxyl group. After administration of EPC-K1, the ability of rat brain to scavenge superoxide radicals was significantly increased. The potent scavenging effects of EPC-K1 on both hydrophilic and hydrophobic radicals were relevant with its molecular structure, which consisted of both hydrophilic and hydrophobic groups.     

The oxidation of N-substituted aromatic amines by horseradish peroxidase

The mechanism of N-dealkylation by peroxidases of the Ca2+ indicator quin2 and analogs was investigated and compared with the mechanism of N-dealkylation of some N-methyl-substituted aromatic amines. Nitrogen-centered cation radicals were detected by ESR spectroscopy for all the compounds studied. Further oxidation of the nitrogen-centered cation radicals, however, was dependent upon the structure of the radical formed. In the case of quin2 and analogs, a carbon-centered radical could be detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. By using the spin trap 2-methyl-2-nitrosopropane (tert-nitrosobutane), it was determined that the carbon-centered radical was formed due to loss of a carboxylic acid group. This indicated that bond breakage most likely occurred through a rearrangement reaction. Furthermore, extensive oxygen consumption was detected, which was in agreement with the formation of carbon-centered radicals, as they avidly react with molecular oxygen. Thus, reaction of the carbon-centered radical with oxygen most likely led to the formation of a peroxyl radical. The peroxyl radical decomposed into superoxide that was spin trapped by 5,5-dimethyl-1-pyrroline N-oxide and an unstable iminium cation. The iminium cation would subsequently hydrolyze to the monomethyl amine and formaldehyde. In the case of N-methyl-substituted aromatic amines, carbon-centered radicals were not detected during the peroxidase-catalyzed oxidation of these compounds. Thus, rearrangement of the nitrogen-centered radical did not occur. Furthermore, little or no oxygen consumption was detected, whereas formaldehyde was formed in all cases. These results indicated that the N-methyl-substituted amines were oxidized by a mechanism different from the mechanism found for quin2 and analogs.     

Free radicals in iron-containing systems

All oxidative damage in biological systems arises ultimately from molecular oxygen. Molecular oxygen can scavenge carbon-centered free radicals to form organic peroxyl radicals and hence organic hydroperoxides. Molecular oxygen can also be reduced in two one-electron steps to hydrogen peroxide in which case superoxide anion is an intermediate; or it can be reduced enzymatically so that no superoxide is released. Organic hydroperoxides or hydrogen peroxide can diffuse through membranes whereas hydroxyl radicals or superoxide anion cannot. Chain reactions, initiated by chelated iron and peroxides, can cause tremendous damage. Chain carriers are chelated ferrous ion; hydroxyl radical .OH, or alkoxyl radical .OR, and superoxide anion O2-. or organic peroxyl radical RO2.. Of these free radicals .OH and RO2. appear to be most harmful. All of the biological molecules containing iron are potential donors of iron as a chain initiator and propagator. An attacking role for superoxide dismutase is proposed in the phagocytic process in which it may serve as an intermediate enzyme between NADPH oxidase and myeloperoxidase. The sequence of reactants is O2----O2-.----H2O2----HOCl.     

Peroxyl, alkoxyl, and carbon-centered radical formation from organic hydroperoxides by chloroperoxidase

The decomposition of organic hydroperoxides as catalyzed by chloroperoxidase was investigated with electron spin resonance (ESR) spectroscopy. Tertiary peroxyl radicals were directly detected by ESR from incubations of tert-butyl hydroperoxide or cumene hydroperoxide with chloroperoxidase at pH 6.4. Peroxyl, alkoxyl, and carbon-centered free radicals from tertiary hydroperoxide/chloroperoxidase systems were successfully trapped by the spin trap 5,5-dimethyl-1-pyrroline N-oxide, whereas alkoxyl radicals were not detected in the ethyl hydroperoxide/chloroperoxidase system. The carbon-centered free radicals were further characterized by spin-trapping studies with tert-nitrosobutane. Oxygen evolution measured by a Clark oxygen electrode was detected for all the hydroperoxide/chloroperoxidase systems. The classical peroxidase mechanism is proposed to describe the formation of peroxyl radicals. In the case of tertiary peroxyl radicals, their subsequent self-reactions result in the formation of alkoxyl free radicals and molecular oxygen. beta-Scission and internal hydrogen atom transfer reactions of the alkoxyl free radicals lead to the formation of various carbon-centered free radicals. In the case of the primary ethyl peroxyl radicals, decay through the Russell pathway forms molecular oxygen.     

Why is the hydroxyl radical the only radical that commonly adds to DNA? Hypothesis: it has a rare combination of high electrophilicity, high thermochemical reactivity, and a mode of production that can occur near DNA

Free radicals do not commonly add to nucleotides in DNA, despite the fact that radicals are produced in all aerobically metabolizing cells. Why is this? For oxy-radicals, the ratio of the rate constant for addition to double bonds divided by that for H-abstraction from good H-donors parallels the electrophilicity of the radical, and among oxy-radicals the hydroxyl radical is the most electrophilic, with an unusually high ratio of Kad/kH. The hydroxyl radical also is very reactive in H-atom abstraction reactions, with a large absolute value of kH. However, the hydroxyl radical's high reactivity makes it unselective and relatively nondiscriminating between H-abstraction from a sugar moiety in DNA and penetration to, and reaction with, a base. Oxy-radicals such as alkoxyl and peroxyl radicals do not have as high electrophilicity or as high reactivity. Interestingly, carbon-centered radicals (such as the methyl radical) also can both add to double bonds and abstract H-atoms, but carbon-centered radicals are not commonly observed to add to DNA bases. However, they cannot be generated near DNA in vivo. In contrast, hydroxyl radical generating systems appear to complex with DNA and produce the hydroxyl radical in the immediate vicinity of the DNA, producing a type of DNA damage that is called site specific. Thus, addition of a radical to a DNA base may require all three features possessed by the hydroxyl radical: high electrophilicity, high thermokinetic reactivity, and a mechanism for production near DNA.     

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.     

Nonenzymatic reduction of Met-Hb under the action of NAD radicals generated in the presence of HAD.H and Fe(II) salts. Antioxidant properties of Met-Hb]

It has been found that superoxide radicals formed at autooxidation of Fe(II) ions or of their complexes with EDTA in the presence of NAD.H cause reduction of met-Hb into deoxy or oxy-form. Under the effect of hydroxyl radicals generated in the ultrasonic field in the presence of NADH reduction of met-Hb into the ferro-form (carboxy-Hb) was observed in Co atmosphere and not in O2 atmosphere. It was induced by a high oxidation rate of oxy-Hb by hydroxyl radicals into met-Hb as compared to carboxy-Hb. Reduction of met-Hb was shown to be accompanied with the formation of NAD+. The role of NAD. radicals in the reduction of met-Hb which acts as an antioxidant in respect to organic free radicals was discussed.     

Spin-trapping studies of hepatic free radicals formed following the acute administration of ethanol to rats: in vivo detection of 1-hydroxyethyl radicals with PBN.

The generation of free radicals in rat liver following the acute oral administration of ethanol was studied with the spin-trapping method, using a deuterated derivative of phenyl-N-tert-butylnitrone (PBN-d14) as the spin-trapping agent. After administration of ethanol and PBN-d14 to rats, organic extracts of the liver were prepared and subjected to ESR spectroscopy. In the case of ethanol-treated rats, the ESR spectra indicated that mixtures of radicals had been trapped, while spectra from control rats were essentially negative. The predominant spin adduct detected after ethanol treatment is proposed to be from a carbon-centered, primary alkyl radical, based on gamma-hydrogen hyperfine splitting patterns observed with PBN-d14. Oxygen-centered radicals also contributed to the ESR spectra. Liver extracts also contained low concentrations of the 1-hydroxyethyl radical spin adduct, which was indicated by weak spectral lines corresponding to those of the 1-13C-ethanol adduct. These data confirm previous suggestions that ethanol is metabolized to a free radical metabolite in rat liver. In addition, some information on types of lipid radicals generated during alcohol intoxication has been obtained.     

The production of oxygen-centered radicals by Bacillus-Calmette-Guerin-activated macrophages: An electron paramagnetic resonance study of the response to phorbol myristate acetate

The spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide of free radicals formed from Bacillus-Calmette-Guerin elicited peritoneal macrophages stimulated with phorbol myristate acetate resulted in the formation of a superoxide and hydroxyl spin adducts. The formation of both spin adducts was inhibited by copper/zinc superoxide dismutase. Only 70% of the hydroxyl spin adduct could be inhibited by catalase or the scavenger dimethyl sulfoxide. This suggests that the production of hydroxyl radicals involves prior formation of both superoxide radicals and hydrogen peroxide, implicating a Fenton catalysed Haber-Weiss reaction. The metal scavenger desferrioxamine also reduced the hydroxyl radical signal by 70%. The unaccounted 30% hydroxyl radical-like signals are probably due to carbon-centered free radicals formed by the lipoxygenase reaction. Spin trapping in the presence of the lipid-soluble spin trap, 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide, resulted in a spectrum consistent with the presence of an oxaziridine nitroxide. This results from the free radical-induced cyclisation of a nitrone with an unsaturated fatty acid.     

Spin Trapping Using 2,2-Dimethyl-2H-Imidazole-1-Oxides

The ability of novel cyclic nitrones, 4-substituted 2,2-dimethyl-2H-imidazole-1-oxides (IMO's) to trap a variety of short-lived free radicals has been investigated using ESR spectroscopy. IMO's scavenge oxygen-, carbon- and sulfur-derived free radicals to give persistent nitroxides. Compared to the spin trap 5,5-dimethyl-pyrroline-1-oxide, a higher lifetime of hydroxyl radical adducts and a higher selectivity related to the trapping of carbon-centered radicals was found. A reaction between IMO's and superoxide was not observed. ESR parameters of 4-carboxyl-2,2-dimethyl-2H-imidazole-1-oxide (CIMO) spin adducts are highly sensitive to the structure of the trapped radical, e.g., different spectra were detected with radicals derived from Na₂SO₃ and NaHSO₃. From the data obtained, a successful application of these new spin traps in biological systems can be expected.     

NADPH-cytochrome-P-450 reductase promoted hydroxyl radical production by the iron(III)-ochratoxin A complex

The Fe3+ complex of ochratoxin A has been shown to produce hydroxyl radicals in the presence of NADPH and NADPH-cytochrome-P-450 reductase. ESR spin-trapping experiments carried out in the presence of the hydroxyl radical scavenger ethanol and the spin trap DMPO (5,5-dimethyl-1-pyrroline-1-oxide) produced ESR spectra characteristic of the hydroxyl radial-derived carbon-centered DMPO-alkoxyl radical adduct. Thus hydroxyl radicals produced by the Fe3(+)-ochratoxin A complex in the presence of an enzymatic reductase may be be partly responsible for ochratoxin A toxicity.     

Protection by estrogens of biological damage by 2,2'-azobis(2-amidinopropane) dihydrochloride

We examined by using 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) as a radical generator the ability of estrogens to scavenge carbon-centered and peroxyl radicals. Electron spin resonance signals of carbon-centered radicals from AAPH were diminished by catecholestrogens but not by phenolic estrogens, showing that catecholestrogens efficiently scavenged carbon-centered radicals. However, fluorescent decomposition of R-phycoerythrin by AAPH-derived peroxyl radicals was inhibited by catecholestrogens and phenolic estrogens. Evidently, peroxyl radicals were scavenged by catecholestrogens and by phenolic estrogens. However, the scavenging ability of 4-hydroxyestradiol was less than 2-hydroxyestradiol. Strand break of DNA induced by AAPH was inhibited by catecholestrogens, but not by phenolic estrogens under aerobic and anaerobic conditions. Inactivation of lysozyme induced by AAPH was completely blocked by 2-hydroxyestradiol under aerobic and anaerobic conditions, and by 4-hyroxyestradiol only under anaerobic conditions. Peroxidation of arachidonic acid by AAPH was strongly inhibited by catecholestrogens at low concentrations. Only large amounts of phenolic estrogens markedly inhibited lipid peroxidation. These results show that catecholestrogens were antioxidant against AAPH-induced damage to biological molecules through scavenging both carbon-centered and peroxyl radicals, but phenolic estrogens partially inhibited AAPH-induced damage because they scavenged only peroxyl radicals.     

Reactivity of an indolinonic aminoxyl with superoxide anion and hydroxyl radicals

The increasing knowledge on the participation of free radicals in many diverse clinical and pathological conditions, has consequently expanded the search for new and versatile antioxidants aimed at combating oxidative stress. Our interest in this field concerns aromatic indolinonic aminoxyls (nitroxides) which efficiently react with alkoxyl, peroxyl, aminyl, arylthiyl and alkyl radicals to give non-paramagnetic species. This prompted us to test their antioxidant activity on different biological systems exposed to free radical-induced oxidative stress and the results obtained so far have been very promising. However little is known about their behaviour towards superoxide and hydroxyl radicals.

Here, we report on the reactivity of an indolinonic aminoxyl, with the two above mentioned radicals using hypoxanthine/xanthine oxidase and potassium superoxide for generating the former and the Fenton reagent for the latter. Besides performing the deoxyribose assay for studying the reaction of the aminoxyl with hydroxyl radical and monitoring spectral changes of the aminoxyl in the presence of superoxide radical, macroscale reactions were performed in both cases and the products of the reactions isolated and identified. The EPR technique was used in this study to help elucidate the data obtained. The results show that this compound efficiently reacts with both hydroxyl and superoxide radicals and furthermore, it is capable of maintaining iron ions in its oxidized form. The results thus contribute to increasing the knowledge on the reactivity of indolinonic aminoxyls towards free radical species and as a consequence, these compounds and/or other aminoxyl derivatives, may be considered as complementary, and sometimes alternative sources for combating oxidative damage.     

不依赖于过渡金属离子的卤代醌介导的新型有机类Fenton反应机理

卤代醌是许多卤芳香持久有机污染物的致癌代谢产物和饮用水消毒副产物。羟基自由基(.OH)被公认为生物系统中最具活性的活性氧物种,能导致生物体内DNA等生物大分子的氧化损伤。目前,最被广泛接受的.OH产生机理是过渡金属离子催化的Fenton反应。综合采用电子自旋共振二级自旋捕获和其他分析方法,发现四氯苯醌和其它卤代醌皆可通过不依赖于过渡金属离子的途径,显著促进氢过氧化物(H2O2或有机氢过氧化物)的分解而产生.OH或烷氧自由基,并首次检测到一种新型的、以碳为中心的醌自由基。基于以上研究,提出一类不依赖于过渡金属离子的卤代醌介导的新型有机类Fenton反应机理。     

Significant enhancement in radical-scavenging activity of curcuminoids conferred by acetoxy substituent at the central methylene carbon

For a compound to be a radical-trapping antioxidant, the antioxidant-derived radical must be sufficiently inert to molecular oxygen as this would generate harmful chain-propagating peroxyl radicals. Curcumin has a unique structure with phenolic hydroxyl group as well as β-diketone moiety in the same molecule, both of which are able to donate electrons to free radicals. However, due to the reactivity toward molecular oxygen, the carbon-centered radical derived from β-diketone moiety do not serve as radical-trapping antioxidants. In this study, we reasoned that stabilization of the carbon-centered radical through substitution with an electron-withdrawing group would enhance the radical-scavenging antioxidative activity of the resulting curcuminoids. Thus, various substituents (methyl, allyl, methoxy, xanthate, and acetoxy) covering broad spectrum of the polar substituent effect were introduced to the central methylene position of both phenolic and non-phenolic curcuminoids. With the free phenolic hydroxyl groups present, the methylene-substituent did not exert significant effect on the antioxidant activity of the curcuminoids (EC(50)=23.2-30.3 μM) with the exception of the acetoxy-substituted derivative (EC(50)=8.7 μM) which showed more potent activity than curcumin (EC(50)=22.6 μM). When substituted to the non-phenolic curcumin scaffold, however, the methylene-substituent enhanced antioxidant activity of the otherwise inactive curcuminoids in the increasing order of methyl相似文献