The influence of porphyrins on iron-catalysed generation of hydroxyl radicals.

Uroporphyrin I, haematoporphyrin and haematoporphyrin derivative had no effect on O2-. generation during oxidation of hypoxanthine by xanthine oxidase and on the formation of hydroxyl radicals (OH.) in the hypoxanthine/xanthine oxidase/Fe3+-EDTA/deoxyribose system. On the other hand, these porphyrins strongly inhibited O2-. formation in a horseradish peroxidase/H2O2/NADPH mixture, whereas they augmented OH. generation in this system after addition of Fe3+-EDTA. Experimental evidence suggests that these observations should be ascribed to the formation of a porphyrin anion radical in the horseradish peroxidase/NADPH system. The formation of this anion radical was confirmed by e.s.r. spectroscopy. This radical is apparently unable to reduce cytochrome c, but it can replace O2-. in the OH.-generating Haber-Weiss reaction.     

The role of superoxide and hydroxyl radicals in the degradation of hyaluronic acid induced by metal ions and by ascorbic acid

Purified commercial hyaluronic acid contains significant amounts of iron. Addition of Fe2+ to solutions of it causes depolymerization, which is inhibited by catalase and scavengers of the hydroxyl radical (. OH) but not by superoxide dismutase. Fe3+ is ineffective. Ascorbic acid also depolymerizes hyaluronic acid, apparently because it can reduce Fe3+ in the reaction mixtures to Fe2+. Ascorbate-induced depolymerization is inhibited by the specific iron chelator desferrioxamine, by catalase, and by scavengers of the hydroxyl radical. The relevance of these observations to rheumatoid arthritis and inflammatory joint diseases is discussed.     

Ascorbic acid, metal ions and the superoxide radical.

1. No evidence could be found for production of the superoxide radical, O2-, during autoxidation of ascorbic acid at alkaline pH values. Indeed, ascorbate may be important in protection against O2- genat-d in vivo. 2. Oxidation of ascorbate at pH 10.2 was stimulated by metal ions. Stimulation by Fe2+ was abolished by superoxide dismutase, probably because of generation of O2-- during reduction of O2 by Fe2+, followed by reaction of O2-- with ascorbate. EDTA changed the mechanism of Fe2+-stimulated ascorbate oxidation. 3. Stimulation of ascorbate oxidation by Cu2+ was also decreased by superoxide dismutase, but this appears to be an artifact, since apoenzyme or bovine serum albumin showed similar effects.     

Induction of oxalate binding by lipid peroxidation in rat kidney mitochondria.

Enhanced oxalate binding (150-180% of control) was observed in kidney, liver, brain and heart, after subjecting them to lipid peroxidation in presence of iron. Kidney mitochondrial oxalate binding was stimulated by different promoters, and the order of stimulation was Fe2+ greater than t-BH greater than ascorbic acid greater than Fe3+ greater than H2O2. Oxalate binding was maximum when iron concentration was between 1-2 mM. The iron-induced oxalate binding was inhibited by reduced glutathione, beta-mercaptoethanol, alpha-tocopherol and hydroxyl ion scavengers, histidine and mannitol. Catalase inhibited both Fe(2+)-H2O2 induced oxalate binding and lipid peroxidation reactions, suggesting that the induced oxalate binding in mitochondria was mediated through the hydroxyl radical reaction mechanism.     

Selective oxidation of imidazole ring in histidine residues by the ascorbic acid — copper ion system

In connection with the physiological actions of active oxygen species on proteins, oxidative modification of histidine residues by the autoxidation of ascorbic acid was determined and the main oxidized compound was identified. Oxidation of imidazole ring by the ascorbic acid — copper ion system was considerably site-specific and assumed to be initiated by the addition of the hydroxyl radical (·OH) at C-2 position in the imidazole ring.     

Oxidative damage to fibronectin. II. The effect of H2O2 and the hydroxyl radical.

The effect of H2O2 and the hydroxyl radical (.OH) on fibronectin was investigated. .OH was generated in three ways: (i) by radiolysis with 60Co under N2O, or by the Fenton system using either (ii) equimolar Fe(2+)-EDTA and H2O2 or (iii) H2O2 and catalytic amounts of Fe(2+)-EDTA recycled with ascorbate. Each system had a different effect. H2O2 alone caused no changes, even at an 800-fold molar excess. Radiolytic .OH caused a rapid loss of tryptophan fluorescence, an increase in bityrosine fluorescence, and extensive crosslinking. The Fenton system using Fe-EDTA, H2O2, and ascorbate caused a loss in tryptophan fluorescence, a smaller increase in bityrosine than was seen with radiolytic .OH, and a threefold increase in carbonyl groups. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis fragmentation of fibronectin was seen. In contrast, when .OH was generated with equimolar Fe-EDTA and H2O2, the only change was a small increase in bityrosine fluorescence at the highest dose of oxidant. None of the systems used affected cysteine. All the changes except the loss of tryptophan by radiolytic .OH were completely inhibited with mannitol. The differences seen with radiolytic .OH and the Fe-EDTA, H2O2, ascorbate system were not solely due to O2 in the latter system since similar results were obtained under N2. The differences between radiolytic .OH and the Fenton systems could be partly due to the components of the latter systems reacting with .OH and thus competing with fibronectin. Our results demonstrate that the extent and type of fibronectin damage by .OH is dependent on the mode of radical generation.     

Copper + zinc and manganese superoxide dismutases inhibit deoxyribose degradation by the superoxide-driven Fenton reaction at two different stages. Implications for the redox states of copper and manganese.

When OH. radicals are formed in a superoxide-driven Fenton reaction, in which O2.- is generated enzymically, deoxyribose degradation is effectively inhibited by CuZn- and Mn-superoxide dismutases. The products of this reaction are H2O2 and a Fe3+-EDTA chelate. The mixing of H2O2 and a Fe3+-EDTA chelate also generates OH. radicals able to degrade deoxyribose with the release of thiobarbituric acid-reactive material. This reaction too is inhibited by CuZn- and Mn-superoxide dismutases, suggesting that most of the OH. is formed by a non-enzymic O2.--dependent reduction of the Fe3+-EDTA chelate. Since the reaction between the Fe3+-EDTA chelate and H2O2 leads to a superoxide dismutase-inhibitable formation of OH. radicals, it could suggest a much wider protective role for the superoxide dismutase enzymes in biological systems. Urate produced during the reaction of xanthine oxidase and hypoxanthine limits deoxyribose degradation as well as the effectiveness of the superoxide dismutase enzymes to inhibit damage to deoxyribose by H2O2 and the Fe3+-EDTA chelate. Some of this damage may result from an O2.--independent pathway to OH. formation in which urate reduces the ferric complex.     

Antioxidant effect of homogenetisic acid on hydrogen peroxide induced oxidative stress in human lung fibroblast cells

Homogentisic acid was found to scavenge intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and thus prevented lipid peroxidation in human fibroblast (WI 38) cells. The radical scavenging activity of homogentisic acid was found to protect WI 38 cells against hydrogen peroxide (H₂O₂) induced oxidative stress, via the activation of extracellular signal regulated kinase (ERK) protein. Homogentisic acid increased the activity of catalase. Hence, from the present study, it is suggested that homogentisic acid protects WI 38 cells against H₂O₂ damage by enhancing the intracellular antioxidative activity.     

Protective effect of metallothionein to ras DNA damage induced by hydrogen peroxide and ferric ion-nitrilotriacetic acid

Metallothionein (MT) is a strong antioxidant, due to a large number of thiol groups in the MT molecule and MT has been found in the nucleus. To investigate whether MT can directly protect DNA from damage induced by hydroxyl radical, the effects of MTs on DNA strand scission due to incubation with ferric ion-nitrilotriacetic acid and H2O2 (Fe3+ -NTA/H2O2) were studied. The Fe3+-NTA/H2O2 resulted in a higher rate of deoxyribose degradation, compared to incubation of Fe3+/H2O2, presumably mediated by the formation of hydroxyl radicals (*OH). This degradation was inhibited by either Zn-MT or Cd-MT, but not by Zn2+ or Cd2+ at similar concentrations. The Fe3+ -NTA/H2O2 resulted in a concentration dependent of increase in DNA strand scission. Damage to the sugar-phosphodiester chain was predominant over chemical modifications of the base moieties. Incubation with either Zn-MT or Cd-MT inhibited DNA damage by approximately 50%. Preincubation of MT with EDTA and N-ethylmaleimide, to alkylate sulfhydryl groups of MT, resulted in MT that was no longer able to inhibit DNA damage. These results indicates that MT can protect DNA from hydroxyl radical attack and that the cysteine thiol groups of MT may be involved in its nuclear antioxidant properties.     

Generation of *OH initiated by interaction of Fe2+ and Cu+ with dioxygen; comparison with the Fenton chemistry

Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (*OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating *OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce *OH. The addition of Fe2+ and Cu+ (0-20 microM) to KH resulted in a concentration-dependent increase in *OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 microM) did not result in *OH formation, these ions mediated significant *OH production in the presence of a number of reducing agents. The *OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 microM, 5 microM or 10 microM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in *OH formation. For each Fe2+ concentration tested, the *OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one. In conclusion: (i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting *OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the *OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate *OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.     

库克诺你果汁提取物体外清除自由基及抗氧化活性研究

本文对诺你果汁多糖、乙醇溶出物和乙酸乙酯萃取物体外对超氧阴离子(O2·)、羟自由基(·OH)、DPPH和脂质过氧化(LPO)的抑制作用进行了研究。超氧阴离子(O2·)由邻苯三酚自氧化产生;羟自由基(·OH)由Fenton反应产生;利用Fe2 诱发卵黄脂蛋白产生丙二醛(MDA),TBA法测定。所有测定均为分光光度法。结果表明,与已知抗氧化剂L抗坏血酸相比,乙醇溶出物和乙酸乙酯萃取物均有明显的捕捉自由基和抗氧化能力,而多糖捕捉自由基和抗氧化能力很低,且对O2·没有抑制作用,反而会增加其生成速度。     

The reaction of superoxide radical with iron complexes of EDTA studied by pulse radiolysis.

The reactions of Fe3+-EDTA and Fe2+-EDTA with O2- and CO2- were investigated in the pH range 3.8--11.8. Around neutral pH O2- reduces Fe3+-EDTA with a rate constant which is pH dependent kpH 5.8--8.1 = 2 - 10(6)--5 - 10(5) M-1 - s-1. At higher pH values this reaction becomes much slower. The CO2- radical reduces Fe3+-EDTA with kpH 3.8--1- = 5 +/- 1 - 10(7) M-1 - s-1 independent of pH. At pH 9--11.8, Fe2+-EDTA forms a complex with O2- with kFe2+-EDTA + O2 = 2 - 10(6)--4 - 10(6) M-1 - s-1 which is pH dependent. We measured the spectrum of Fe2+-EDTA-O2- and calculated epsilon 290 over max = 6400 +/- 800 M-1 - cm-1 in air-saturated solutions. In O2-saturated solutions another species is formed with a rate constant of 7 +/- 2 s-1. This intermediate absorbs around 300 nm but we were not able to identify it.     

Lactoferrin-catalysed hydroxyl radical production. Additional requirement for a chelating agent.

The ability of lactoferrin to catalyse hydroxyl radical production was determined by measuring ethylene production from methional (2-amino-4-methylthiobutyraldehyde) or 4-methylthio-2-oxobutyrate. Lactoferrin, isolated from human milk and saturated by adding the exact equivalents of Fe3+-nitrilotriacetic acid and dialysing, give little if any catalysis of the reaction between H2O2 and either O2-. or ascorbic acid at either pH 7.4 or pH 5.0. However, in the presence of chelating agents such as EDTA or nitrilotriacetic acid that can complex with lactoferrin, hydroxyl radical production by both mechanisms was observed.     

NADPH- and adriamycin-dependent microsomal release of iron and lipid peroxidation

In a previous study (Minotti, G., 1989, Arch. Biochem. Biophys. 268, 398-403) NADPH-supplemented microsomes were found to reduce adriamycin (ADR) to semiquinone free radical (ADR-.), which in turn autoxidized at the expense of oxygen to regenerate ADR and form O2-. Redox cycling of ADR was paralleled by reductive release of membrane-bound nonheme iron, as evidenced by mobilization of bathophenanthroline-chelatable Fe2+. In the present study, iron release was found to increase with concentration of ADR in a superoxide dismutase- and catalase-insensitive manner. This suggested that membrane-bound iron was reduced by ADR-. with negligible contribution by O2-. or interference by its dismutation product H2O2. Following release from microsomes, Fe2+ was reconverted to Fe3+ via two distinct mechanisms: (i) catalase-inhibitable oxidation by H2O2 and (ii) catalase-insensitive autoxidation at the expense of oxygen, which occurred upon chelation by ADR and increased with the ADR:Fe2+ molar ratio. Malondialdehyde formation, indicative of membrane lipid peroxidation, was observed when approximately 50% of Fe2+ was converted to Fe3+. This occurred in presence of catalase and low concentrations of ADR, which prevented Fe2+ oxidation and favored only partial Fe2+ autoxidation, respectively. Lipid peroxidation was inhibited by superoxide dismutase via increased formation of H2O2 from O2-. and excessive Fe2+ oxidation. Lipid peroxidation was also inhibited by high concentrations of ADR, which favored maximum Fe2+ release but also caused excessive Fe2+ autoxidation via formation of very high ADR:Fe2+ molar ratios. These results highlighted multiple and diverging effects of ADR, O2-., and H2O2 on iron release, iron (auto-)oxidation and lipid peroxidation. Stimulation of malondialdehyde formation by catalase suggested that lipid peroxidation was not promoted by reaction of Fe2+ with H2O2 and formation of hydroxyl radical. The requirement for both Fe2+ and Fe3+ was indicative of initiation by some type of Fe2+/Fe3+ complex.     

Iron(II) and hydrogen peroxide detoxification by human H-chain ferritin. An EPR spin-trapping study

Overexpression of human H-chain ferritin (HuHF) is known to impart a degree of protection to cells against oxidative stress and the associated damage to DNA and other cellular components. However, whether this protective activity resides in the protein's ability to inhibit Fenton chemistry as found for Dps proteins has never been established. Such inhibition does not occur with the related mitochondrial ferritin which displays much of the same iron chemistry as HuHF, including an Fe(II)/H(2)O(2) oxidation stoichiometry of approximately 2:1. In the present study, the ability of HuHF to attenuate hydroxyl radical production by the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + OH(-) + *OH) was examined by electron paramagnetic resonance (EPR) spin-trapping methods. The data demonstrate that the presence of wild-type HuHF during Fe(2+) oxidation by H(2)O(2) greatly decreases the amount of .OH radical produced from Fenton chemistry whereas the ferroxidase site mutant 222 (H62K + H65G) and human L-chain ferritin (HuLF) lack this activity. HuHF catalyzes the pairwise oxidation of Fe(2+) by the detoxification reaction [2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH(core) + 4H(+)] that occurs at the ferroxidase site of the protein, thereby preventing the production of hydroxyl radical. The small amount of *OH radical that is produced in the presence of ferritin (相似文献   

Tannic acid inhibits in vitro iron-dependent free radical formation

The antioxidant activity of tannic acid (TA), a plant polyphenol claimed to possess antimutagenic and anticarcinogenic activities, was studied by monitoring (i) 2-deoxyribose degradation (a technique for OH detection), (ii) ascorbate oxidation, (iii) ascorbate radical formation (determined by EPR analysis) and (iv) oxygen uptake induced by the system, which comprised Fe(III) complexes (EDTA, nitrilotriacetic acid (NTA) or citrate as co-chelators), ascorbate and oxygen. TA removes Fe(III) from the co-chelators (in the case of EDTA, this removal is slower than with NTA or citrate), forming an iron-TA complex less capable of oxidizing ascorbate into ascorbate radical or mediating 2-deoxyribose degradation. The effectiveness of TA against 2-deoxyribose degradation, ascorbate oxidation and ascorbate radical formation was substantially higher in the presence of iron-NTA (or iron-citrate) than with iron-EDTA, which is consistent with the known formation constants of the iron complexes with the co-chelators. Oxygen uptake and 2-deoxyribose degradation induced by Fe(II) autoxidation were also inhibited by TA. These results indicate that TA inhibits OH formation induced by Fe(III)/ascorbate/O(2) mainly by arresting Fe(III)-induced ascorbate oxidation and Fe(II) autoxidation (which generates Fe(II) and H(2)O(2), respectively), thus limiting the production of Fenton reagents and OH formation. We also hypothesize that the Fe(II) complex with TA exhibits an OH trapping activity, which explains the effect of TA on the Fenton reaction.     

DNA sequence context as a determinant of the quantity and chemistry of guanine oxidation produced by hydroxyl radicals and one-electron oxidants

DNA sequence context has emerged as a critical determinant of the location and quantity of nucleobase damage caused by many oxidizing agents. However, the complexity of nucleobase and 2-deoxyribose damage caused by strong oxidants such as ionizing radiation and the Fenton chemistry of Fe2+-EDTA/H2O2 poses a challenge to defining the location of nucleobase damage and the effects of sequence context on damage chemistry in DNA. To address this problem, we developed a gel-based method that allows quantification of nucleobase damage in oxidized DNA by exploiting Escherichia coli exonuclease III to remove fragments containing direct strand breaks and abasic sites. The rigor of the method was verified in studies of guanine oxidation by photooxidized riboflavin and nitrosoperoxycarbonate, for which different effects of sequence context have been demonstrated by other approaches (Margolin, Y., Cloutier, J. F., Shafirovich, V., Geacintov, N. E., and Dedon, P. C. (2006) Nat. Chem. Biol. 2, 365-366). Using duplex oligodeoxynucleotides containing all possible three-nucleotide sequence contexts for guanine, the method was used to assess the role of DNA sequence context in hydroxyl radical-induced guanine oxidation associated with gamma-radiation and Fe2+-EDTA/H2O2. The results revealed both differences and similarities for G oxidation by hydroxyl radicals and by one-electron oxidation by riboflavin-mediated photooxidation, which is consistent with the predominance of oxidation pathways for hydroxyl radicals other than one-electron oxidation to form guanine radical cations. Although the relative quantities of G oxidation produced by hydroxyl radicals were more weakly correlated with sequence-specific ionization potential than G oxidation produced by riboflavin, damage produced by both hydroxyl radical generators and riboflavin within two- and three-base runs of G showed biases in location that are consistent with a role for electron transfer in defining the location of the damage products. Furthermore, both gamma-radiation and Fe2+-EDTA/H2O2 showed relatively modest effects of sequence context on the proportions of different damage products sensitive to E. coli formamidopyrimidine DNA glycosylase and hot piperidine, although GT-containing sequence contexts displayed subtle biases in damage chemistry (formamidopyrimidine DNA glycosylase/piperidine ratio). Overall, the results are consistent with the known chemistry of guanine oxidation by hydroxyl radical and demonstrate that charge migration plays a relatively minor role in determining the location and chemistry of hydroxyl radical-mediated oxidative damage to guanine in DNA.     

Hydroxyl radical and hypochlorous acid scavenging activity of small Centaury (Centaurium erythraea) infusion. A comparative study with green tea (Camellia sinensis)

Small centaury (Centaurium erythraea Rafin.) is a herbal species with a long use in traditional medicine due to its digestive, stomachic, tonic, depurative, sedative and antipyretic properties. This species is reported to contain considerable amounts of polyphenolic compounds, namely xanthones and phenolic acids as the main constituents. Although the antiradicalar activity of some pure polyphenolic compounds is already known, it remains unclear how a complex mixture obtained from plant extracts functions against reactive oxygen species. Thus, the ability of small centaury infusion to act as a scavenger of the reactive oxygen species hydroxyl radical and hypochlorous acid was studied and compared with that of green tea (Camellia sinensis L.). Hydroxyl radical was generated in the presence of Fe3+-EDTA, ascorbate and H2O2 (Fenton system) and monitored by evaluating hydroxyl radical-induced deoxyribose degradation. The reactivity towards hypochlorous acid was determined by measuring the inhibition of hypochlorous acid-induced 5-thio-2-nitrobenzoic acid oxidation to 5,5'-dithiobis(2-nitrobenzoic acid). The obtained results demonstrate that small centaury infusion exhibits interesting antioxidant properties, expressed both by its capacity to effectively scavenge hydroxyl radical and hypochlorous acid, although with a lower activity against the second than that observed for green tea. Green tea exhibited a dual effect at the hydroxyl radical scavenging assay, stimulating deoxyribose degradation at lower dosages.     

Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators.

The pH of the solution along with chelation and consequently coordination of iron regulate its reactivity. In this study we confirmed that, in general, the rate of Fe(II) autoxidation increases as the pH of the solution is increased, but chelators that provide oxygen ligands for the iron can override the affect of pH. Additionally, the stoichiometry of the Fe(II) autoxidation reaction varied from 2:1 to 4:1, dependent upon the rate of Fe(II) autoxidation, which is dependent upon the chelator. No partially reduced oxygen species were detected during the autoxidation of Fe(II) by ESR using DMPO as the spin trap. However, upon the addition of ethanol to the assay, the DMPO:hydroxyethyl radical adduct was detected. Additionally, the hydroxylation of terephthalic acid by various iron-chelator complexes during the autoxidation of Fe(II) was assessed by fluorometric techniques. The oxidant formed during the autoxidation of EDTA:Fe(II) was shown to have different reactivity than the hydroxyl radical, suggesting that some type of hypervalent iron complex was formed. Ferrous iron was shown to be able to directly reduce some quinones without the reduction of oxygen. In conclusion, this study demonstrates the complexity of iron chemistry, especially the chelation of iron and its subsequent reactivity.     

Fenton chemistry. Amino acid oxidation

The oxidation of amino acids by Fenton reagent (H2O2 + Fe(II] leads mainly to the formation of NH+4, alpha-ketoacids, CO2, oximes, and aldehydes or carboxylic acids containing one less carbon atom. Oxidation is almost completely dependent on the presence of bicarbonate ion and is stimulated by iron chelators at levels which are substoichiometric with respect to the iron concentration but is inhibited at higher concentrations. The stimulatory effect of chelators is not due merely to solubilization of catalytically inactive polymeric forms of Fe(OH)3 nor to the conversion of Fe(II) to complexes incapable of scavenging hydroxyl radicals. The results suggest that an iron chelate and another as yet unidentified form of iron are both required for maximal rates of amino acid oxidation. The metal ion-catalyzed oxidation of amino acids is likely a "caged" process, since the oxidation is not inhibited by hydroxyl radical scavengers, and the relative rates of oxidation of various amino acids by the Fenton system as well as the distribution of products formed (especially products of aromatic amino acids) are significantly different from those reported for amino acid oxidation by ionizing radiation. Several iron-binding proteins, peptides, and hemoglobin degradation products can replace Fe(II) or Fe(III) in the bicarbonate-dependent oxidation of amino acids. In view of their ability to sequester metal ions and their susceptibility to oxidation by H2O2 in the presence of physiological concentrations of bicarbonate, amino acids may serve an important role in antioxidant defense against tissue damage.