Photo-irradiated titanium dioxide catalyzes site specific DNA damage via generation of hydrogen peroxide

Titanium dioxide (TiO₂) is a potential photosensitizer for photodynamic therapy. In this study, the mechanism of DNA damage catalyzed by photo-irradiated TiO₂ was examined using [₃₂P]-5'-end-labeled DNA fragments obtained from human genes. Photo-irradiated TiO₂ (anatase and rutile) caused DNA cleavage frequently at the guanine residue in the presence of Cu(II) after E. coli formamidopyrimidine-DNA glycosylase treatment, and the thymine residue was also cleaved after piperidine treatment. Catalase, SOD and bathocuproine, a chelator of Cu(I), inhibited the DNA damage, suggesting the involvement of hydrogen peroxide, superoxide and Cu(I). The photocatalytic generation of Cu(I) from Cu(II) was decreased by the addition of SOD. These findings suggest that the inhibitory effect of SOD on DNA damage is due to the inhibition of the reduction of Cu(II) by superoxide. We also measured the formation of 8-oxo-7,8-dihydro-2' -deoxyguanosine, an indicator of oxidative DNA damage, and showed that anatase is more active than rutile. On the other hand, high concentration of anatase caused DNA damage in the absence of Cu(II). Typical free hydroxyl radical scavengers, such as ethanol, mannnitol, sodium formate and DMSO, inhibited the copper-independent DNA photodamage by anatase. In conclusion, photo-irradiated TiO₂ particles catalyze the copper-mediated site-specific DNA damage via the formation of hydrogen peroxide rather than that of a free hydroxyl radical. This DNA-damaging mechanism may participate in the phototoxicity of TiO₂.     

Nickel (II) Complexes of Histidyl-Peptides as Fenton-Reaction Catalysts

Addition of histidyl-peptides containing the glycyl-glycyl-L-histidyl sequence stimulated the catalysis of Ni(II) hydrogen peroxide reduction. Maximum bleaching of murexide or nitrosodimethylaniline was obtained with glycyl-glycyl-L-histidine. A decrease in the bleaching rates was observed upon addition of SOD or hydroxyl radical scavengers, showing that the hydrogen peroxide/Ni(II)/glycyl-glycyl-L-histidine system generated superoxide anions as well as hydroxyl radicals. In contrast, addition of glycyl-glycyl-L-histidine inhibited the Cu(II) hydrogen peroxide reduction.

When peptides or proteins were exposed to oxygen radicals produced by Ni(II)/glycyl-glycyl-L-histidine catalysis of hydrogen peroxide reduction, the observed effects were similar to those produced by oxygen radicals generated by water radiolysis or by Fe(II) or Cu(II) mediated Fenton-reactions: hydroxylation of phenylalanine, interchange of disulfides, destruction of tryptophans and dityrosine formation.     

Nickel (II) Complexes of Histidyl-Peptides as Fenton-Reaction Catalysts

Addition of histidyl-peptides containing the glycyl-glycyl-L-histidyl sequence stimulated the catalysis of Ni(II) hydrogen peroxide reduction. Maximum bleaching of murexide or nitrosodimethylaniline was obtained with glycyl-glycyl-L-histidine. A decrease in the bleaching rates was observed upon addition of SOD or hydroxyl radical scavengers, showing that the hydrogen peroxide/Ni(II)/glycyl-glycyl-L-histidine system generated superoxide anions as well as hydroxyl radicals. In contrast, addition of glycyl-glycyl-L-histidine inhibited the Cu(II) hydrogen peroxide reduction.

When peptides or proteins were exposed to oxygen radicals produced by Ni(II)/glycyl-glycyl-L-histidine catalysis of hydrogen peroxide reduction, the observed effects were similar to those produced by oxygen radicals generated by water radiolysis or by Fe(II) or Cu(II) mediated Fenton-reactions: hydroxylation of phenylalanine, interchange of disulfides, destruction of tryptophans and dityrosine formation.     

Prion-derived copper-binding peptide fragments catalyze the generation of superoxide anion in the presence of aromatic monoamines

OBJECTIVES: Studies have proposed two opposing roles for copper-bound forms of prion protein (PrP) as an anti-oxidant supporting the neuronal functions and as a pro-oxidant leading to neurodegenerative process involving the generation of reactive oxygen species. The aim of this study is to test the hypothesis in which putative copper-binding peptides derived from PrP function as possible catalysts for monoamine-dependent conversion of hydrogen peroxide to superoxide in vitro. MATERIALS AND METHODS: Four peptides corresponding to the copper (II)-binding motifs in PrP were synthesized and used for analysis of peptide-catalyzed generation of superoxide in the presence of Cu (II) and other factors naturally present in the neuronal tissues. RESULTS: Among the Cu-binding peptides tested, the amino acid sequence corresponding to the Cu-binding site in the helical region was shown to be the most active for superoxide generation in the presence of Cu(II), hydrogen peroxide and aromatic monoamines, known precursors or intermediates of neurotransmitters. Among monoamines tested, three compounds namely phenylethylamine, tyramine and benzylamine were shown to be good substrates for superoxide-generating reactions by the Cu-bound helical peptide. CONCLUSIONS: Possible roles for these reactions in development of prion disease were suggested.     

Prevention of oxidative DNA degradation by copper-binding peptides

Free divalent ions of copper (Cu) are capable of generating radical species such as hydroxyl radicals in the presence of hydrogen peroxide or ascorbic acid through Harbor-Weiss-like reactions under physiological conditions. It has been reported that radical-mediated damage to DNA molecules in animal cells leads to programmed cell death. Hence it is important to seek for methods to prevent Cu-mediated DNA damage. In this study we identified on effect of Cu binding of short peptides (chiefly Gly-Gly-His tripeptide) in the prevention of DNA degradation caused by Cu-mediated reactions in the presence of hydrogen peroxide and of ascorbic acid.     

Cupric-amyloid beta peptide complex stimulates oxidation of ascorbate and generation of hydroxyl radical

A growing body of evidence supports an important role for oxidative stress in the pathogenesis of Alzheimer's disease. Recently, a number of papers have shown a synergistic neurotoxicity of amyloid beta peptide and cupric ions. We hypothesized that complexes of cupric ions with neurotoxic amyloid beta peptides (Abeta) can stimulate copper-mediated free radical formation. We found that neurotoxic Abeta (1-42), Abeta (1-40), and Abeta (25-35) stimulated copper-mediated oxidation of ascorbate, whereas nontoxic Abeta (40-1) did not. Formation of ascorbate free radical was significantly increased by Abeta (1-42) in the presence of ceruloplasmin. Once cupric ion is reduced to cuprous ion, it can be oxidized by oxygen to generate superoxide radical or it can react with hydrogen peroxide to form hydroxyl radical. Hydrogen peroxide greatly increased the oxidation of cyclic hydroxylamines and ascorbate by cupric-amyloid beta peptide complexes, implying redox cycling of copper ions. Using the spin-trapping technique, we have shown that toxic amyloid beta peptides led to a 4-fold increase in copper-mediated hydroxyl radical formation. We conclude that toxic Abeta peptides do indeed stimulate copper-mediated oxidation of ascorbate and generation of hydroxyl radicals. Therefore, cupric-amyloid beta peptide-stimulated free radical generation may be involved in the pathogenesis of Alzheimer's disease.     

A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals

The metal-mediated site-specific mechanism for free radical-induced biological damage is reviewed. According to this mechanism, cooper- or iron-binding sites on macromolecules serve as centers for repeated production of hydroxyl radicals that are generated via the Fenton reaction. The aberrations induced by superoxide, ascorbate, isouramil, and paraquat are summarized. An illustrative example is the enhancement of double-strand breaks by ascorbate/copper. Prevention of the site-specific free radical damage can be accomplished by using selective chelators for iron and copper, by displacing these redox-active metals with other redox-inactive metals such as zinc, by introducing high concentrations of hydroxyl radicals scavengers and spin trapping agents, and by applying protective enzymes that remove superoxide or hydrogen peroxide. Histidine is a special agent that can intervene in free radical reactions in variety of modes. In biological systems, there are traces of copper and iron that are at high enough levels to catalyze free-radical reactions, and account for such deleterious processes. In the human body Fe/Cu = 80/1 (w/w). Nevertheless, both (free) copper and iron are soluble enough, and the rate constants of their reduced forms with hydrogen peroxide are sufficiently high to suggest that they might be important mediators of free radical toxicity.     

Antioxidant responses to enhanced generation of superoxide anion radical and hydrogen peroxide in the copper-stressed mulberry plants

The aim of the study was to implicate the generation of reactive oxygen species (ROS) and altered cellular redox environment with the effects of Cu-deficiency or Cu-excess in mulberry (Morus alba L.) cv. Kanva 2 plants. A study of antioxidative responses, indicators of oxidative damage and cellular redox environment in Cu-deficient or Cu-excess mulberry plants was undertaken. While the young leaves of plants supplied with nil Cu showed chlorosis and necrotic scorching of laminae, the older and middle leaves of plants supplied with nil or 0.1 μM Cu showed purplish-brown pigmented interveinal areas that later turned necrotic along the apices and margins of leaves. The Cu-excess plants showed accelerated senescence of the older leaves. The Cu-deficient plants showed accumulation of hydrogen peroxide and superoxide anion radical. The accumulation of hydrogen peroxide was strikingly intense in the middle portion of trichomes on Cu-deficient leaves. Though the concentration of total ascorbate increased with the increasing supply of Cu, the ratio of the redox couple (DHA/ascorbic acid) increased in Cu-deficient or Cu-excess plants. The activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) increased in both Cu-deficient and Cu-excess plants. The results suggest that deficiency of Cu aggravates oxidative stress through enhanced generation of ROS and disturbed redox couple. Excess of Cu damaged roots, accelerated the rate of senescence in the older leaves, induced antioxidant responses and disturbed the cellular redox environment in the young leaves of mulberry plants.     

Detoxification of SO2 derivatives in chloroplasts ofHardwickia binata

Intact chloroplasts isolated from sulphur dioxide fumigatedHardwickia binata leaves showed inhibition of PS II electron transport activity without any significant effect on photosystem I. Sulphur dioxide exposed leaves accumulated more hydrogen peroxide than those from non-fumigated plants and this was caused by increase in superoxide radical production. Hydrogen peroxide formation was inhibited by addition of cytochrome C and superoxide disrnutase. In sulphur dioxide fumigated leaves, increase in superoxide dismutase activity showed resistance to sulphite toxicity. The localization of ascorbate peroxidase, glutathione reductase and dehydroascorbate reductase activities in chloroplasts provide evidence for the photogeneration of ascorbate. The scavenging of hydrogen peroxide in chloroplast due to ascorbate regenerated from DHA by the system: PS I → Fd → NADP → glutathione. The system can be considered as a means for preliminary detoxification of sulphur dioxide by chloroplasts     

Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates

Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).     

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

An analog of the human albumin N-terminus (Asp-Ala-His-Lys) prevents formation of copper-induced reactive oxygen species

Copper mobilization and redox activity form damaging reactive oxygen species (ROS) and are implicated in the pathogenesis of ischemia-reperfusion injury, chronic inflammation, Alzheimer's disease, aging, and cancer. Protein sequestration of Cu(II) ions has been shown to prevent ROS-generating reactions. The first four amino acids of the N-terminus of human albumin, Asp-Ala-His-Lys (DAHK), form a tight binding site for Cu(II) ions. We synthesized several analogs, including the enantiomer d-DAHK, to study their effects on copper-induced hydroxyl radical and superoxide formation in the presence of ascorbate. d-DAHK prevented thiobarbituric acid-reactive species (TBARS) formation within physiological and acidic pH ranges (7.5-6.5) and inhibited low-density lipoprotein lipid peroxidation. A d-DAHK/Cu complex exhibited superoxide dismutase-like activity by significantly inhibiting superoxide formation. These in vitro results suggest that d-DAHK may shift the Cu(II)-binding equilibrium from the exchangeable Cu(II) pool to the tightly-bound, nonexchangeable pool, prevent ROS formation, and potentially provide therapeutic benefit for ROS-related diseases.     

Oxidation of NADH by vanadium: kinetics, effects of ligands and role of H2O2 or O2.

The mechanism of oxidation of NADH by either vanadium(V) or vanadium(IV) was examined in the presence of reducing agents, complexing agents, and hydrogen peroxide. Reducing agents that stimulate the oxidation of NADH by V(V) include: a variety of cysteine analogues, glutathione, beta-mercaptoethanol, dithiothreitol, and ascorbate. Complexing agents which stimulate NADH oxidation by V(V) include cystine, glutathione disulfide, and dehydroascorbate. Vanadium(IV)-dependent systems which oxidize NADH include combinations of V(IV) with cysteine or air alone. Combination of either V(V) or V(IV) with hydrogen peroxide leads to NADH oxidation. Based on kinetic analysis and the use of the diagnostic inhibitors--superoxide dismutase, catalase, albumin, mannitol, ethanol, and anaerobic conditions--we have assigned two major mechanisms of NADH oxidation. One is the previously reported mechanism which involves V(V)-superoxide as the NADH oxidant. This reaction is inhibited by superoxide dismutase and anaerobic conditions but not by catalase or ethanol. This reaction is observed for V(V) in the presence of reducing agents and complexing agents. The second reaction mechanism operates when V(IV) comes in contact with hydrogen peroxide and involves V(III)-superoxide as the NADH oxidant. This reaction is inhibited by catalase (if unligated hydrogen peroxide is an intermediate) and superoxide dismutase but not anaerobic conditions or ethanol. This mechanism is observed for reactions of V(IV) with air or hydrogen peroxide.     

Activities of antioxidant enzymes in mitochondria of growing and dormant sugar beet roots

In mitochondria isolated from growing (70–85 days) and dormant (stored for 8–12 weeks) sugar beet (Beta vulgaris L.) roots, activities of superoxide dismutase (SOD) and enzymes of the ascorbate-glutathione cycle were determined. The activity of SOD, the enzyme involved in superoxide detoxification, was much higher in mitochondria of the growing root, whereas activities of ascorbate peroxidase (APO) and glutathione reductase (GR), key enzymes of the ascorbate-glutathione cycle involved in the hydrogen peroxide degradation, increased substantially in mitochondria of dormant storage roots. Catalase (CAT) activity was detected in the fraction of root mitochondria purified in the sucrose density gradient, which activity was inhibited by cyanide by 85–90% and much weaker, by aminotriazol (by 30–35%). Submitochondrial localization of APO and CAT was analyzed using proteinase K. It was established that a substrate-binding APO center is localized on the external side of the inner membrane, whereas CAT is localized in the mitochondrial matrix. A possible role of mitochondria as ROS (hydrogen peroxide) acceptors in the cells of storage parenchyma of the stored root is discussed.     

The antimicrobial peptide histatin-5 causes a spatially restricted disruption on the Candida albicans surface, allowing rapid entry of the peptide into the cytoplasm

Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.     

胸腺相关肽及其自旋标记类似物的抗氧化活性研究

胸腺相关肽及其自旋标记类似物的抗氧化活性研究杨国玲,文永均（兰州大学生物系,７３００００）胡晓愚,佘世望（南昌中德联合研究院,３３００４７）关键词胸腺五肽（ＴＰ－５）;脂质过氧化物;超氧阴离子自由基;羟自由基;自旋标记类似物胸腺素水平随着人的年龄的增...     

Damage to the oxygen-evolving complex by superoxide anion, hydrogen peroxide, and hydroxyl radical in photoinhibition of photosystem II

Under strong illumination of a photosystem II (PSII) membrane, endogenous superoxide anion, hydrogen peroxide, and hydroxyl radical were successively produced. These compounds then cooperatively resulted in a release of manganese from the oxygen-evolving complex (OEC) and an inhibition of oxygen evolution activity. The OEC inactivation was initiated by an acceptor-side generated superoxide anion, and hydrogen peroxide was most probably responsible for the transportation of reactive oxygen species (ROS) across the PSII membrane from the acceptor-side to the donor-side. Besides ROS being generated in the acceptor-side induced manganese loss; there may also be a ROS-independent manganese loss in the OEC of PSII. Both superoxide anion and hydroxyl radical located inside the PSII membrane were directly identified by a spin trapping-electron spin resonance (ESR) method in combination with a lipophilic spin trap, 5-(diethoxyphosphoryl)-5-phenethyl-1-pyrroline N-oxide (DEPPEPO). The endogenous hydrogen peroxide production was examined by oxidation of thiobenzamide.     

Redox cycling of potential antitumor aziridinyl quinones

The formation of reactive oxygen intermediates (ROI) during redox cycling of newly synthesized potential antitumor 2,5-bis (1-aziridinyl)-1,4-benzoquinone (BABQ) derivatives has been studied by assaying the production of ROI (superoxide, hydroxyl radical, and hydrogen peroxide) by xanthine oxidase in the presence of BABQ derivatives. At low concentrations (< 10 microM) some BABQ derivatives turned out to inhibit the production of superoxide and hydroxyl radicals by xanthine oxidase, while the effect on the xanthine-oxidase-induced production of hydrogen peroxide was much less pronounced. Induction of DNA strand breaks by reactive oxygen species generated by xanthine oxidase was also inhibited by BABQ derivatives. The DNA damage was comparable to the amount of hydroxyl radicals produced. The inhibiting effect on hydroxyl radical production can be explained as a consequence of the lowered level of superoxide, which disrupts the Haber-Weiss reaction sequence. The inhibitory effect of BABQ derivatives on superoxide formation correlated with their one-electron reduction potentials: BABQ derivatives with a high reduction potential scavenge superoxide anion radicals produced by xanthine oxidase, leading to reduced BABQ species and production of hydrogen peroxide from reoxidation of reduced BABQ. This study, using a unique series of BABQ derivatives with an extended range of reduction potentials, demonstrates that the formation of superoxide and hydroxyl radicals by bioreductively activated antitumor quinones can in principle be uncoupled from alkylating activity.     

Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.