Zinc(II) Protects Against Metal-Mediated Free Radical Induced Damage: Studies on Single and Double-Strand DNA Breakage

M13 DNA was used as a source for single and double-stranded DNA. Free radical-induced damage to single and double stranded DNA was caused by asorbateliron and ascorbate/copper oxidative systems. The degree of breakage was estimated by running samples on an agarose gel and staining with ethidium bromide, followed by photographic analysis. DflA breakage was dependent on time and concentration of iron or copper ions. Zincions protected against damage caused by iron/asorbate both to single-stranded and double-stranded DNA. In contrast, in the copper/ascorbate system zinc ions protected only against the double-stranded DNA (replicative form of M13) breakage, and not against copper-mediated single-stranded DNA breakages. It seemed to amplify the efficiency of breakage. The protection provided to the replicative form in the copper/ascorbate system is much less effective than the protection to DNA in the iron/ascorbate system. These results support the notion that redox-inactive metal ions, that compete for iron or copper binding sites, could provide protection against transition metal-mediated and free radical-induced damage.     

Zinc(II) Protects Against Metal-Mediated Free Radical Induced Damage: Studies on Single and Double-Strand DNA Breakage

M13 DNA was used as a source for single and double-stranded DNA. Free radical-induced damage to single and double stranded DNA was caused by asorbateliron and ascorbate/copper oxidative systems. The degree of breakage was estimated by running samples on an agarose gel and staining with ethidium bromide, followed by photographic analysis. DflA breakage was dependent on time and concentration of iron or copper ions. Zincions protected against damage caused by iron/asorbate both to single-stranded and double-stranded DNA. In contrast, in the copper/ascorbate system zinc ions protected only against the double-stranded DNA (replicative form of M13) breakage, and not against copper-mediated single-stranded DNA breakages. It seemed to amplify the efficiency of breakage. The protection provided to the replicative form in the copper/ascorbate system is much less effective than the protection to DNA in the iron/ascorbate system. These results support the notion that redox-inactive metal ions, that compete for iron or copper binding sites, could provide protection against transition metal-mediated and free radical-induced damage.     

脱氧核糖核酸对谷胱甘肽过氧化物酶活性影响的初步研究

谷胱甘肽过氧化物酶是机体内重要抗氧酶系之一。它的活力和含量,反映机体清除自由基的能力。自由基对细胞结构的损伤很大,随着年龄的增长,抗氧化酶活力逐渐下降,从而引起自由基及脂质过氧化产物日益增多,最终导致机体衰老和老年性疾病的发生^[1]。本试验试图探讨DNA对谷胱甘肽过氧化物酶活性影响从而探索DNA对抗自由基的作用。     

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Copper(II) complexes of diclofenac: Spectroscopic studies and DNA strand breakage

Copper(II) complexes of diclofenac with interesting anti-inflammatory profiles have been prepared and studied by infrared and electronic spectroscopy. In the solid state and in polar and coordinating solvents, all the complexes are solvated binuclear carboxylato-bridged complexes, [Cu(L)²(S)]², where L is monodeprotonated diclofenac and S is the axially bonded solvent. The effect of the copper(II) complexes on the in vitro DNA strand breakeage was studied by agarose gel electrophoresis. Relaxation or double stranded scissions of pDNA were observed leading to the formation of linear pDNA. Treatment of pDNA with high concentrations of these compounds caused a disappearance of pDNA. For the parent drug, sodium diclofenac, no effect on the pDNA was observed. This study presents some indications that the binuclear copper(II) complexes, [Cu(L)²(S)]², could have some relevance in the treatment of tumor cell lines.     

Role of Amino Thiols in Luminol Chemiluminescence Coupled with Copper(II)-catalysed Oxidation of Cysteine and Glutathione

The Cu(II)-catalysed oxidation of the amino thiols cysteine and glutathione with oxygen was examined in the presence of luminol. Light emission was markedly suppressed when the concentration of amino thiols was greater than that of Cu(II). Cysteine caused a delay of chemiluminescence (CL) and no CL emission was observed at high concentrations of GSH. The suppression in CL could be explained in terms of the complexation of the Cu(II) catalyst by formation of a Cu(II)–dicysteine complex in case of cysteine, and of a Cu(II)–GSSG complex in case of GSH.     

Copper-1,10-Phenanthroline-Induced Apoptosis in Liver Carcinoma Bel-7402 Cells Associates with Copper Overload,Reactive Oxygen Species Production,Glutathione Depletion and Oxidative DNA Damage

The mechanism of cytotoxicity on liver carcinoma Bel-7402 cells induced by copper-1,10-phenanthroline, Cu(OP)₂, has been studied. Cell viability and apoptotic rate were examined in cells treated with Cu(OP)₂ or Cu²⁺ alone. It was found that the apoptosis induced by Cu(OP)₂ could not be induced by Cu²⁺ or OP alone in our experimental conditions. Total copper content in cells was measured by atomic absorption spectrophotometry, and the abnormal elevation of intracellular copper transported by lipophilic OP ligand may play the role of initial factor in the apoptosis, which caused subsequent redox state changes in cells. Intracellular levels of reactive oxygen species (ROS) were detected by fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Reduced (GSH) and total glutathione (GSSG + GSH) were determined by High-performance liquid chromatography (HPLC) after derivatization, and the ratios of GSH/GSSG were subsequently calculated. The overproduction of ROS and the decreased GSH/GSSG ratio were observed in cells which represented the occurrence of oxidative stress in the apoptosis. Oxidative DNA damage was also found in cells treated with Cu(OP)₂ in the early stage of the apoptosis, and it suggests that the activation of DNA repair system may be involved in the pathway of the apoptosis induced by Cu(OP)₂.     

辐射及活性氧对DNA的损伤以及芥子碱的保护作用

在X射线照射下,小牛胸腺DNA的碱基损伤及链断裂随着剂量升高而增加,其损伤主要集中于链断裂;活性氧可以引起DNA损伤,H2O2仅造成少量伤害,当在含有H2O2的体系中加入微量的Cu2＋、Fe2＋时损伤急剧增加,这是由反应产生的·OH所致,Cu2＋的致损伤效果明显高于Fe2＋。·OH清除剂芥子碱具有很强的抗辐射及抗氧化作用,且对DNA无伤害。这说明·OH在DNA的氧化损伤中起重要作用。     

Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used ( 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.     

DNA binding and oxidative DNA damage induced by a quercetin copper(II) complex: potential mechanism of its antitumor properties

The interaction of a quercetin copper(II) complex with DNA was investigated using UV–vis spectra, fluorescence measurement, viscosity measurement, agarose gel electrophoresis, and thiobarbituric acid reactive substances assay. The results indicate that the quercetin copper(II) complex can promote the cleavage of plasmid DNA, producing single and double DNA strand breaks, and intercalate into the stacked base pairs of DNA. Moreover, the complex can induce oxidative DNA damage involving generation of reactive oxygen species such as H₂O₂ and Cu(I)OOH. In addition, the cytotoxicity experiments carried out with A549 cells confirmed its apoptosis-inducing activity. And we also demonstrate that the levels of survivin protein expression in A549 cells decreased, and that relative activity of caspase-3 increased significantly after treatment with the complex. So our results suggest that the antitumor mechanism of the quercetin copper(II) complex involves not only its oxidative DNA damage with generation of reactive oxygen species but also its specific interaction with DNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

DNA cleavage promoted by trigonal-bipyramidal zinc(II) and copper(II) complexes formed by asymmetric tripodal tetradendate 2-[bis(2-aminoethyl)amino]ethanol

Asymmetric trigonal-bipyramidal Zn(II) complex 1 formed by 2-[bis(2-aminoethyl)amino]ethanol (L) was found to be able to promote the cleavage of supercoiled plasmid DNA pBR322 to the nicked and linear DNA via a hydrolytic manner but only in neutral Tris-HCl buffer, no cleavage was observed in HEPES or NaH₂PO₄/Na₂HPO₄ buffer. However, the copper complex 2 of L, possessing the similar coordination geometry, can only promote DNA cleavage via an oxidative mechanism in the presence of ascorbic acid. ESI-MS study implies that complex 1 exist mainly as [Zn(L)]²⁺/[Zn(L-H)]⁺ in neutral Tris-HCl buffer. Moreover, there is no discriminable species for complex 1 in HEPES or NaH₂PO₄/Na₂HPO₄ buffer. A phosphate activation mechanism via phosphate coordinating to Zn(II) center of [Zn(L)]²⁺/[Zn(L-H)]⁺ to form the stable trigonal-bipyramidal structure is proposed for the hydrolytic cleavage promote by complex 1. For complex 2, the abundance of [Cu(L)Cl]⁺ is higher than that of [Cu(L)]²⁺/[Cu(L-H)]⁺ in Tris-HCl buffer. The lower phosphate binding/activating ability of Cu(II) in complex 2 may be the origin for its incapability to promote the hydrolytic DNA cleavage. However, the readily accessible redox potential of Cu(II) makes complex 2 promote the oxidative DNA cleavage. Although the DNA cleavage promoted by complex 1 has no specificity, trigonal-bipyramidal Zn(II) complexes formed by asymmetric tripodal polyamine with ethoxyl pendent should be a novel potential model for practical artificial nuclease.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

Copper (II) acetate improves the quality of pear (Pyrus) DNA during extraction

Isolation of high-quality DNA from Pyrus is particularly difficult because of high endogenous levels of polysaccharides, phenolics, and other organic constituents that interfere with DNA isolation and purification. Presence of phenolic compounds caused browning of tissue and supernatant during the extraction process, despite supplementation with PVP, as suggested by standard methods. By modifying the CTAB extraction procedure of Aldrich and Cullis (1993) by including copper (II) acetate treatment, we obtained high-quality DNA. Copper (II) acetate enabled fixation and removal of tannins. The DNA yield from this procedure is high (up to 1.25 μg of DNA/mg of leaf tissue). This DNA is completely digestible with restriction endonucleases and suitable for generation of molecular markers, such as random-amplified polymorphic DNA and amplified fragment length polymorphism analyses, indicating freedom from common contaminating polyphenolic compounds.     

Double-strand hydrolysis of DNA by a magnesium(II) complex with diethylenetriamine

The development of artificial nucleases that hydrolyze DNA or RNA is of great interest in molecular biology, biotechnology, and medicine. We now report that a magnesium(II) complex of diethylenetriamine (Mg-dien) can effectively promote the double-stranded cleavage of plasmid DNA and the dideoxynucleotide dApdA under physiological conditions of pH and temperature. Experiments performed in the presence of hydrogen peroxide, radical scavengers, or under rigorously anaerobic conditions indicate that DNA cleavage mediated by Mg-dien occurs via a hydrolytic path. Mg-dien efficiently hydrolyzes supercoiled pBR322 DNA and the pseudo-first-order rate constant at 37 degrees C and pH 8.0 is estimated to be 1.60 h(-1). The dinucleotide dApdA hydrolysis, with Mg-dien at 170 microM, shows a rate enhancement factor of ca. 5 x 10(8). 1H and 31P(1H) NMR studies show that Mg-dien effectively hydrolyzes 5'-dAMP to give deoxyadenosine and inorganic phosphate. While Mg2+ has been found at the catalytic sites of many natural nucleases, Mg-dien appears to be the first synthetic Mg2+-containing system capable of hydrolyzing dideoxynucleotides and DNA and thus may provide a simple model system to assist mechanistic studies of naturally occurring nucleases.     

Protein Restriction Without Strong Caloric Restriction Decreases Mitochondrial Oxygen Radical Production and Oxidative DNA Damage in Rat Liver

Previous studies have shown that caloric restriction decreases mitochondrial oxygen radical production and oxidative DNA damage in rat organs, which can be linked to the slowing of aging rate induced by this regime. These two characteristics are also typical of long-lived animals. However, it has never been investigated if those decreases are linked to the decrease in the intake of calories themselves or to decreases in specific dietary components. In this study the possible role of the dietary protein was investigated. Using semipurified diets, the ingestion of proteins of Wistar rats was decreased by 40% below that of controls while the other dietary components were ingested at the same level as in animals fed ad libitum. After seven weeks in this regime the liver of the protein restricted animals showed 30–40% decreases in mitochondrial production of reactive oxygen species (ROS) and in oxidative damage to nuclear and mitochondrial DNA. The decreases in ROS generation occurred specifically at complex~I. They also occurred without changes in mitochondrial oxygen consumption. Instead, there was a decrease in the percent free radical leak (the percentage of total electron flow leading to ROS generation in the respiratory chain). These results are strikingly similar to those previously obtained after 40% caloric restriction in the liver of Wistar rats. Thus, the results suggest that part of the decrease in aging rate induced by caloric restriction can be due to the decreased intake of proteins acting through decreases in mitochondrial ROS production and oxidative DNA damage. Interestingly, these tissue oxidative stress-linked parameters can be lowered by restricting only the intake of dietary protein, probably a more feasible option than caloric restriction for adult humans.     

DNA binding behaviors and cleavage properties of a Ru(II) polypyridyl complex

A novel complex, [Ru(phen)₂pzip]²⁺1 (phen = 1,10-phenanthroline; pzip = 2-(pyrazine-2-yl)imidazo-[4,5-f][1,10]phenanthroline]), has been synthesized and characterized by elemental analysis, ES-MS, ¹H NMR. The DNA-binding behaviors of this complex were studied by spectroscopic methods and viscosity measurements. The results indicate that the complex can bind to CT-DNA in an intercalative mode. When irradiated at 365 nm, complex 1 can promote the cleavage of plasmid pBR322DNA. Furthermore, Zn²⁺ can trigger the DNA cleavage of complex 1 without irradiation. The mechanism studies revealed that the DNA cleavage by complex 1 in the presence of Zn²⁺ is likely to proceed via a hydrolytic cleavage process.     

Free radical mediated interaction of ascorbic acid and ascorbate/Cu(II) with viral and plasmid DNAs

Previous studies indicate that ascorbic acid, when combined with copper or iron cleaves several viral DNA. ln this study, we generated the ascorbate radical anion electrochemically in a simple chemical environment without the participation of a metal ion. This solution possesses viral DNA scission activity. Ohe absence of catalytic metal ions [Fe (III) and Cu(II)] in the incubation medium was evidenced by metal chelating agents such as desferrioxamine and EDTA. Ohe radical quenching at high EDTA concentration was attributed to ionic strength of EDTA rather than metal chelation. Ohe effects of antioxidants, radical scavangers, catalase, superoxide dismutase and some proteins on DNA cleavage have been tested. Cleavage may not arise directly from ascorbate free radical but the reaction of the radical form of ascorbate with oxygen may produce the actual reactive species. Aerobic oxidation of ascorbate itself strictly requires transition metal catalysts, however electrochemically produced ascorbyl radical avoided the kinetic barrier that prevented direct oxidation of ascorbic acid with oxygen and eliminated the need for the transition metal ion catalysts.     

RNF8/RNF168信号通路在DNA损伤修复中的作用

细胞内DNA会受部分外界因素(如紫外辐射,电离辐射和化学毒素)和内部因素(如复制错误)的影响而发生损伤,包括DNA双链断裂、DNA错配和DNA交链等。DNA损伤发生后,损伤部位会被一些蛋白识别,进而招募一系列蛋白至损伤部位,形成一个修复系统。DNA双链断裂是最严重的一种DNA损伤,错误修复往往导致疾病的发生。DNA双链断裂(double strand break, DSB)后,细胞启动RNF8/RNF168信号通路进行修复。RNF8和RNF168是这条通路的枢纽蛋白;53BP和BRCA1是关键的效应蛋白,决定着DSB修复的方式;组蛋白泛素化、磷酸化和甲基化等翻译后修饰是这条通路顺利进行的基本条件;染色质重塑、泛素化酶/去泛素化酶平衡和蛋白稳定性是这条通路的主要调节方式。本综述对RNF8/RNF168信号通路进行了梳理总结,希望其能对相关研究者起到参考作用。     

Copper(II) complexes with tridentate pyrazole-based ligands: synthesis, characterization, DNA cleavage activity and cytotoxicity

Tridentate pyrazole-containing ligands of the Schiff base type, SalPz — HL¹, Cl₂SalPz — HL² and I₂SalPz — HL³, were used to prepare a series of new Cu(II) complexes (CuSalPz — 1, CuCl₂SalPz — 2 and CuI₂SalPz — 3). These new complexes have been studied by different analytical techniques (electrospray ionization mass spectrometry (ESI-MS), elemental analysis, FT-IR and EPR). The spectroscopic properties of 1-3 are consistent with the formation of Cu(II) complexes coordinated by monoanionic and tridentate (N,N,O)-chelators, behaving as monomeric species in aqueous solution, as shown by EPR studies. Crystals of 2 and 3, obtained by slow concentration of methanolic solutions of the compounds, were also analyzed by X-ray diffraction analysis. The X-ray structural study has shown that 2 crystallized as a dinuclear compound, [Cu₂(μ-Cl)₂(Cl₂SalPz)₂], while the solid state structure determined for 3 is best described by monomeric units of [CuCl(I₂SalPz)] displaying short Cu···Cl intermolecular contacts. The in vitro evaluation of 1-3 comprised the study of their DNA-cleaving ability using plasmid DNA and the assessment of their cytotoxic activity against several human cancer cell lines (PC-3 prostate, MCF-7 breast and A2780 and A2780cisR-ovary). The studies with plasmid DNA have shown that 2 and 3 induce extensive DNA cleavage in the presence of different additives. The cytotoxic activity of 2 and 3 is comparable to the one presented by cisplatin, with the exception of the A2780 cell line where cisplatin is more active. It has been found that the introduction of halogen substituents in the phenolate rings of the chelators enhanced the cytotoxicity of the respective Cu(II) complexes.