Iron is the intracellular metal involved in the production of DNA damage by oxygen radicals.

The metal chelators 1,10-phenanthroline and 2,9-dimethyl-1,10-phenanthroline (neocuproine) showed distinct abilities to prevent hydroxyl radical formation from hydrogen peroxide and Cu+ or F2(2+) (Fenton reaction) as determined by electron spin resonance. o-Phenanthroline prevented both Fe- and Cu-mediated Fenton reactions whereas neocuproine only prevented the Cu-mediated Fenton reaction. Because only 1,10-phenanthroline but not neocuproine prevented DNA strand-break formation in hydrogen peroxide-treated mammalian fibroblasts it appears that the Fe-mediated, as compared to the Cu-mediated, intranuclear Fenton reaction is responsible for DNA damage.     

Influence of the physiologically widespread substances on the oxidative activity of copper(II) complexes with sinefungin, a nucleoside antibiotic

The oxidation-promoting reactivity of the Cu(II)-sinefungin complex in the presence of hydrogen peroxide was studied at pH 7.4, using N,N-dimethyl-p-nitrosoaniline (NDMA), as well as plasmid DNA as target molecules. Mixture of the complex with H(2)O(2) was found to be an efficient oxidant, bleaching NDMA solution, and generating single- and double-strand breaks in DNA. The oxidative DNA damage was investigated also in the presence of varying amounts of glutathione, histidine, Gly-Gly-His peptide, H2A histone, and ascorbic acid, showing diverse influence of those substances on the cleavage extension.     

Homocysteine potentiates copper- and amyloid beta peptide-mediated toxicity in primary neuronal cultures: possible risk factors in the Alzheimer's-type neurodegenerative pathways

Oxidative stress may have an important role in the progression of neurodegenerative disorders such as Alzheimer's disease (AD) and prion diseases. Oxidative damage could result from interactions between highly reactive transition metals such as copper (Cu) and endogenous reducing and/or oxidizing molecules in the brain. One such molecule, homocysteine, a thiol-containing amino acid, has previously been shown to modulate Cu toxicity in HeLa and endothelial cells in vitro. Due to a possible link between hyperhomocysteinemia and AD, we examined whether interaction between homocysteine and Cu could potentiate Cu neurotoxicity. Primary mouse neuronal cultures were treated with homocysteine and either Cu (II), Fe (II or III) or Zn (II). Homocysteine was shown to selectively potentiate toxicity from low micromolar concentrations of Cu. The toxicity of homocysteine/Cu coincubation was dependent on the ability of homocysteine to reduce Cu (II) as reflected by the inhibition of toxicity with the Cu (I)-specific chelator, bathocuproine disulphonate. This was supported by data showing that homocysteine reduced Cu (II) more effectively than cysteine or methionine but did not reduce Fe (III) to Fe (II). Homocysteine also generated high levels of hydrogen peroxide in the presence of Cu (II) and promoted Abeta/Cu-mediated hydrogen peroxide production and neurotoxicity. The potentiation of metal toxicity did not involve excitotoxicity as ionotropic glutamate receptor antagonists had no effect on neurotoxicity. Homocysteine alone also had no effect on neuronal glutathione levels. These studies suggest that increased copper and/or homocysteine levels in the elderly could promote significant oxidant damage to neurons and may represent additional risk factor pathways which conspire to produce AD or related neurodegenerative conditions.     

DNA-decorated nanoparticles as nanosensors for rapid detection of ascorbic acid

We designed an assay for rapid detection of ascorbic acid (AA) with a DNAzyme cleaving its DNA substrate in the presence of Cu(2+) and AA. The sensor consists of two DNA strands that form a complex between each other. The 5'-end of the DNAzyme binds the substrate DNA via Watson-Crick bonding and the 3'-end binds through formation of a DNA-triplex via Hoogsteen hydrogen bonding. The substrate DNA was prepared by two different methods. In the first case the nucleic acid was modified with fluorescein/dabcyl FRET pair across the cleavage site. In the second case the nucleic acid modified with fluorescein was immobilised on gold nanoparticles. DNAzyme contains a loop forming a complex with Cu(2+) ions. The oxidation of ascorbic acid (AA) with oxygen yields hydrogen peroxide. The latter interacts with Cu(2+) to give hydroxyl radicals. They break substrate DNA in close vicinity to the copper/DNA complex to separate fluorescein from gold nanoparticles leading to the increase in fluorescence intensity. Use of substrate DNA modified with the fluorescein/dabcyl couple allowed to measure AA concentration within 3 min with the detection limit of 2.5 μM. Employment of gold nanoparticles decorated with fluorescein-modified DNA allowed to improve the detection limit of AA quantification by two orders of magnitude due to enhanced cleavage of DNA catalysed by Au clusters. Fructose, sucrose, glucose, urea, and citric acid did not interfere with our assay even at concentration of 1mM. Good selectivity allowed us to apply our rapid and sensitive assays to detection of AA in vitamin C tablets, urine and orange juice.     

Enzymatic transformations of morphinane alkaloids

Horseradish peroxidase transforms morphinane alkaloids into N-oxides and morphine to pseudomorphine in the presence of hydrogen peroxide. The crude poppy enzyme fraction shows the same activities. The rates of reactions were influenced by phenolic compounds and their relation controlled by the concentration of hydrogen peroxide and the presence of ascorbic acid.     

Carcinogenic semicarbazide induces sequence-specific DNA damage through the generation of reactive oxygen species and the derived organic radicals

Semicarbazide, a hydrazine derivative, is carcinogenic to mice but shows no or little mutagenicity in the Salmonella-microsome test. To clarify whether or not the genotoxic mechanism contributes to the non-mutagenic carcinogenicity of semicarbazide, we investigated DNA damage induced by semicarbazide using 32P-5'-end-labeled DNA fragments obtained from the c-Ha-ras-1 protooncogene and the p53 tumor suppressor gene. Semicarbazide caused DNA damage frequently at the thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine partially inhibited DNA damage, suggesting that hydrogen peroxide plus Cu(I) participates in DNA damage. When a high concentration of semicarbazide was used in the presence of catalase, DNA damage was induced, especially at G in 5'-AG and slightly at 5'-G in GG and GGG sequences. An electron paramagnetic resonance (EPR) spectroscopic study has confirmed that the reaction of semicarbazide with Cu(II) produces carbamoyl radicals (z.rad;CONH(2)), possibly generated via the nitrogen-centered radicals of semicarbazide. Azodicarbonamide also produced carbamoyl radicals and induced DNA damage frequently at 5'-G in GG and GGG sequences, suggesting that carbamoyl radicals participate in this sequence-specific DNA damage by semicarbazide. On the basis of our previous reports, we consider that the sequence-specific DNA damage at G in 5'-AG in the present study is due to the nitrogen-centered radicals. This study has shown that semicarbazide induces DNA damage in the presence of Cu(II) through the formation of hydrogen peroxide and Cu(I). In addition, semicarbazide-derived free radicals participate in DNA damage. DNA damage induced by these reactive species may be relevant to the carcinogenicity of semicarbazide.     

Oxalic acid is available as a natural antioxidant in some systems

Oxalic acid is found in a wide variety of plants. This study showed that oxalic acid suppressed in vitro lipid peroxidation in a concentration-dependent manner. Furthermore, oxalic acid reduced the rate of ascorbic acid oxidation in the presence of hydrogen peroxide and Cu(2+). These results suggest that oxalic acid is available as a natural antioxidant.     

Is the lens canned?

The ocular lens somehow remains pellucid despite bombardment by ultraviolet radiation and endogenous hydrogen peroxide (present in the humoral fluids which bathe this tissue). The lens and adjacent aqueous and vitreous humors contain exceptionally high concentrations of reducing substances, particularly ascorbic acid, thought to be important in lenticular oxidant defense. However, in the presence of traces of transition metals, or when exposed to ultraviolet radiation, ascorbic acid readily reacts with oxygen, yielding hydrogen peroxide, and damaging lens crystallins. We propose the alternative hypothesis that the real antioxidant function of ascorbic acid, particularly that in the aqueous and vitreous humors, may be effecting the conversion of oxygen to water. Because the lens lacks a blood supply, coupled reactions of ascorbic acid with oxygen in the humoral fluid spaces should produce a metabolically sustained anaerobiosis. If so, nature may have preinvented the process of canning, wherein food (or in this case, the lens) is preserved by a combination of sterility and anoxia.     

Damage to the cytoplasmic membrane of Escherichia coli by catechin-copper (II) complexes

In the presence of a nonlethal concentration of Cu(II), washed Escherichia coli ATCC11775 cells were killed by (-)-epigallocatechin (EGC) and (-)-epicatechin (EC). Cell killing was accompanied by a depletion in both the ATP and potassium pools of the cells, but the DNA double strand was not broken, indicating that the bactericidal activity of catechins in the presence of Cu(II) results from damage to the cytoplasmic membrane. Induction of endogenous catalase in E. coli cells increased their resistance to being killed by the combination of catechins and Cu(II). In all cases studied, EGC and EC with Cu(II) were found to generate hydrogen peroxide, but its concentration was too low to account for the bactericidal activity. The bactericidal activity of EGC in the presence of Cu(II) was completely suppressed by ethylenediaminetetraacetate, bathocuproine, catalase, superoxide disumutase (SOD), heated catalase, and heated SOD, but not by dimethyl sulfoxide. When catalase, either heated or unheated, was added to the cells incubated with EGC in the presence of Cu(II), it completely inhibited further killing of the cells. These findings suggest that recycling redox reactions between Cu(II) and Cu(I), involving catechins and hydrogen peroxide on the cell surface, must be important in the mechanism of the killing.     

Metal-mediated oxidative damage to cellular and isolated DNA by gallic acid, a metabolite of antioxidant propyl gallate

Propyl gallate (PG), widely used as an antioxidant in foods, is carcinogenic to mice and rats. PG increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, but not in HP100, which is hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. Although PG induced no or little damage to 32P-5'-end-labeled DNA fragments obtained from genes that are relevant to human cancer, DNA damage was observed with treatment of esterase. HPLC analysis of the products generated from PG incubated with esterase revealed that PG converted into gallic acid (GA). GA induced DNA damage in a dose-dependent manner in the presence of Fe(III)EDTA or Cu(II). In the presence of Fe(III) complex such as Fe(III)EDTA or Fe(III)ADP, GA caused DNA damage at every nucleotide. Fe(III) complex-mediated DNA damage by GA was inhibited by free hydroxy radical (*OH) scavengers, catalase and an iron chelating agent. These results suggested that the Fe(III) complex-mediated DNA damage caused by GA is mainly due to *OH generated via the Fenton reaction. In the presence of Cu(II), DNA damage induced by GA occurred at thymine and cytosine. Although *OH scavengers did not prevent the DNA damage, methional inhibited the DNA damage. Cu(II)-mediated DNA damage was inhibited by catalase and a Cu(I) chelator. These results indicated that reactive oxygen species formed by the interaction of Cu(I) and H2O2 participates in the DNA damage. GA increased 8-oxodG content in calf thymus DNA in the presence of Cu(II), Fe(III)EDTA or Fe(III)ADP. This study suggested that metal-mediated DNA damage caused by GA plays an important role in the carcinogenicity of PG.     

Preparation of dehydro-L-ascorbic acid dimer by air oxidation of L-ascorbic acid in the presence of catalytic amounts of copper(II) acetate and pyridine

The catalytic system Cu(AcO)2-pyridine 1:4 mol% in methanol, slowly catalyses the air oxidation of ascorbic acid to the 2-methyl hemi-ketal of dehydroascorbic acid 5, and hydrogen peroxide. However, with Cu(AcO)2-pyridine 3:4 mol% the air oxidation is quite fast and no hydrogen peroxide is present at the end of the reaction. Removal of the catalyst and refluxing the foamy 5 in MeCN gives the oxidized, dimeric, dehydroascorbic acid in very good yields (approximately 70%) contaminated by approximately 1-2% MeCN.     

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.     

Damage to the cytoplasmic membrane of Escherichia Coli by catechin-copper (II) complexes

In the presence of a nonlethal concentration of Cu(II), washed Escherichia coli ATCC11775 cells were killed by (-)-epigallocatechin (EGC) and (-)-epicatechin (EC). Cell killing was accompanied by a depletion in both the ATP and potassium pools of the cells, but the DNA double strand was not broken, indicating that the bactericidal activity of catechins in the presence of Cu(II) results from damage to the cytoplasmic membrane. Induction of endogenous catalase in E. coli cells increased their resistance to being killed by the combination of catechins and Cu(II). In all cases studied, EGC and EC with Cu(II) were found to generate hydrogen peroxide, but its concentration was too low to account for the bactericidal activity. The bactericidal activity of EGC in the presence of Cu(II) was completely suppressed by ethylenediaminetetraacetate, bathocuproine, catalase, superoxide disumutase (SOD), heated catalase, and heated SOD, but not by dimethyl sulfoxide. When catalase, either heated or unheated, was added to the cells incubated with EGC in the presence of Cu(II), it completely inhibited further killing of the cells. These findings suggest that recycling redox reactions between Cu(II) and Cu(I), involving catechins and hydrogen peroxide on the cell surface, must be important in the mechanism of the killing.     

Copper- and Arene-Catalyzed Cleavage of DNA

DNA was found to be cleaved by arenes and copper(II) salts in neutral solutions. The efficiency of this reaction is comparable with the DNA cleavage by such systems as Cu(II)–phenanthroline and Cu(II)–ascorbic acid in efficiency, but, unlike them, it does not require the presence of an exogenous reducing agent or hydrogen peroxide. The Cu²⁺–arene system does not cleave DNA under anaerobic conditions. Catalase, sodium azide as well as bathocuproine, a specific chelator of Cu(I), completely inhibit the reaction. Our results suggest that Cu(I) ions, superoxide radical and singlet oxygen participate in this reaction. It was shown by EPR and spin traps that the reaction proceeds with the formation of alkoxyl radicals capable of inducing breaks in DNA molecules. An efficient cleavage of DNA in the Cu(II)–o-bromobenzoic acid system requires the generation of radicals under the conditions of formation of a specific copper–DNA–o-bromobenzoic acid complex, in which copper ions are likely to be coordinated with oxygen atoms of the DNA phosphate groups.     

Protective effect of seminal plasma proteins on the degradation of ascorbic acid

The objectives of this study were to determine ascorbic acid stability and its effect on antiproteinase activity of seminal plasma in the presence of an oxidant. Effect of seminal plasma, and additives: glutathione, albumin, hydrogen peroxide and Tris buffer, on ascorbic acid degradation was investigated by UV absorbance. Antiproteinase against trypsin amidase activity was measured spectrophotometrically using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Ascorbic acid was destroyed much more rapidly with the addition of hydrogen peroxide than in Tris buffer at pH 8.2 alone. Seminal plasma protected ascorbic acid more efficiently than glutathione and albumin alone. The protective effect of seminal plasma on ascorbic acid degradation may closely relate to the function of ascorbic acid in reproductive system of scurvy-prone animals including teleost fish. Within the range of 1–8 mM concentrations, ascorbic acid had a pro-oxidant action on seminal plasma antiproteinase activityin vitro when they were incubated with hydrogen peroxide.Abbreviations AA Ascorbic acid - BAPNA N-benzoyl-DL-arginine-p-nitroanilide - DMSO dimethyl sulfoxide - GSH glutathione - H₂O₂ hydrogen peroxide     

Real-time detection of L-ascorbic acid and hydrogen peroxide in crude food samples employing a reversed sequential differential measuring technique of the SIRE-technology based biosensor.

Detection of the common electrochemical interferents, ascorbic acid and hydrogen peroxide, using a SIRE (Sensors based on Injection of the Recognition Element) technology based biosensor in reverse mode operation is reported. The differential measuring principle employed in the SIRE biosensor during operation in reverse mode is such that the sample is measured first in the presence of enzyme (yielding matrix signal only), and then measured again in the absence of enzyme (yielding signal from matrix+analyte). Subtraction of the signal obtained in the presence of enzyme from the signal obtained in the absence of enzyme gives a specific signal for the analyte only and correlates directly to its concentration in solution. The linear range for the determination of ascorbic acid and hydrogen peroxide was 0-3 mM and 0-2 mM, respectively, with an enzyme concentration of 25 U ascorbate oxidase/ml and 1000 U catalase/ml. The reproducibility was 5% for ascorbic acid (R.S.D. n=15) and 10% for hydrogen peroxide (R.S.D. n=18). The cost per measurement was 0.28 USD for ascorbic acid analysis and 0.0008 USD for hydrogen peroxide analysis. The degradation of ascorbic acid in cereal was followed in real-time, as was the stabilization of low pH on the degradation process.     

The oxidative damage of plasmid DNA by ascorbic acid derivatives in vitro: the first research on the relationship between the structure of ascorbic acid and the oxidative damage of plasmid DNA

To study the structure-function relationship of the oxidative-damage effect of ascorbic acid, we have focused on the interaction between plasmid DNA pUC19 and a series of ascorbic acid derivatives modified on different OH groups in the presence of transition metal ions. Some ascorbic acid derivatives can selectively cleave plasmid DNA from Form I to Form II in the presence of low concentration of Cu2+ just like ascorbic acid itself, while other derivatives oxidatively damage plasmid DNA slightly. We found that those derivatives with unattached 2-OH and 3-OH groups retain the ability to cleave the plasmid DNA. The derivatives that have been methylated on 2-OH or 3-OH can only cleave plasmid DNA softly, and those derivatives that have been protected on both 2-OH and 3-OH can hardly exert an oxidative damage on plasmid DNA under the same condition. Form these results, we can draw the conclusion that 2-OH and 3-OH groups of the ascorbic acid molecule contribute most to this biological activity.     

Studies on the mutagenic activity of ascorbic acid in vitro and in vivo

In vitro data are presented to show that ascorbic acid does not have intrinsic mutagenicity towards strain TA100 of S. typhimurium if deionized water is used to prepare the incubation medium. The addition of Cu2+ ions to the bacterial medium that contains ascorbic acid, or the use of tap water and ascorbic acid alone, causes a mutagenic and cytotoxic response that is blocked by EDTA. Additional in vitro data demonstrate that hydrogen peroxide is mutagenic to S. typhimurium strain TA100 and it is suggested that ascorbic acid may be mutagenic and cytotoxic through the generation of hydrogen peroxide. In vivo studies using a sensitive intrahepatic host-mediated mutagenicity assay indicate that ascorbic acid is not genotoxic in guinea pigs even when the dietary intake of vitamin C is above the level required for tissue saturation (5000 mg/kg body weight/day).     

Damage to the DNA bases in mammalian chromatin by hydrogen peroxide in the presence of ferric and cupric ions.

Modification of DNA bases in mammalian chromatin upon treatment with hydrogen peroxide in the presence of ferric and cupric ions was studied. Ten DNA base products in mammalian chromatin were identified and quantitated by the use of gas chromatography-mass spectrometry with selected-ion monitoring after hydrolysis of chromatin and trimethylsilylation of hydrolysates. This technique permitted the analysis of modified DNA bases in chromatin without the necessity of isolation of DNA from chromatin first. Modified bases identified were typical hydroxyl radical-induced products of DNA, indicating the involvement of hydroxyl radical in their formation. This was also confirmed by inhibition of product formation by typical scavengers of hydroxyl radical. The inhibition of product formation was much more prominent in the presence of chelated ions than unchelated ions, indicating a possible site-specific formation of hydroxyl radical when metal ions are bound to chromatin. Hydrogen peroxide in the presence of cupric ions caused more DNA damage than in the presence of ferric ions. Chelation of cupric ions caused a marked inhibition in product formation. By contrast, DNA was damaged more extensively in the presence of chelated ferric ions than in the presence of unchelated ferric ions. The presence of ascorbic acid generally increased the yields of the products, indicating increased production of hydroxyl radical by reduction of metal ions by ascorbic acid. Superoxide dismutase afforded partial inhibition of product formation only in the case of chelated iron ions. The yields of the modified bases in chromatin were lower than those observed with calf thymus DNA under the same conditions.     

Redox reactions of copper complexes formed with different beta-amyloid peptides and their neuropathological [correction of neuropathalogical] relevance

The binding stoichiometry between Cu(II) and the full-length beta-amyloid Abeta(1-42) and the oxidation state of copper in the resultant complex were determined by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) and cyclic voltammetry. The same approach was extended to the copper complexes of Abeta(1-16) and Abeta(1-28). A stoichiometric ratio of 1:1 was directly observed, and the oxidation state of copper was deduced to be 2+ for all of the complexes, and residues tyrosine-10 and methionine-35 are not oxidized in the Abeta(1-42)-Cu(II) complex. The stoichiometric ratio remains the same in the presence of more than a 10-fold excess of Cu(II). Redox potentials of the sole tyrosine residue and the Cu(II) center were determined to be ca. 0.75 and 0.08 V vs Ag/AgCl [or 0.95 and 0.28 V vs normal hydrogen electrode (NHE)], respectively. More importantly, for the first time, the Abeta-Cu(I) complex has been generated electrochemically and was found to catalyze the reduction of oxygen to produce hydrogen peroxide. The voltammetric behaviors of the three Abeta segments suggest that diffusion of oxygen to the metal center can be affected by the length and hydrophobicity of the Abeta peptide. The determination and assignment of the redox potentials clarify some misconceptions in the redox reactions involving Abeta and provide new insight into the possible roles of redox metal ions in the Alzheimer's disease (AD) pathogenesis. In cellular environments, the reduction potential of the Abeta-Cu(II) complex is sufficiently high to react with antioxidants (e.g., ascorbic acid) and cellular redox buffers (e.g., glutathione), and the Abeta-Cu(I) complex produced could subsequently reduce oxygen to form hydrogen peroxide via a catalytic cycle. Using voltammetry, the Abeta-Cu(II) complex formed in solution was found to be readily reduced by ascorbic acid. Hydrogen peroxide produced, in addition to its role in damaging DNA, protein, and lipid molecules, can also be involved in the further consumption of antioxidants, causing their depletion in neurons and eventually damaging the neuronal defense system. Another possibility is that Abeta-Cu(II) could react with species involved in the cascade of electron transfer events of mitochondria and might potentially sidetrack the electron transfer processes in the respiratory chain, leading to mitochondrial dysfunction.