Double-strand DNA cleavage induced by oxindole-Schiff base copper(II) complexes with potential antitumor activity

Some oxindole-Schiff base copper(II) complexes have already shown potential antitumor activity towards different cells, inducing apoptosis in a process modulated by the ligand, and having nuclei and mitochondria as main targets. Here, three novel copper(II) complexes with analogous ligands were isolated and characterized by spectroscopic techniques, having their reactivity compared to the so far most active complex in this class. Cytotoxicity experiments carried out toward human neuroblastoma SH-SY5Y cells confirmed its pro-apoptosis property. DNA cleavage studies were then performed in the presence of these complexes, in order to verify the influence of ligand structural features in its nuclease activity. All of them were able to cause double-strand DNA scissions, giving rise to nicked circular Form II and linear Form III species, in the presence of hydrogen peroxide. Additionally, DNA Form II was also detected in the absence of peroxide when the most active complex, [Cu(isaepy)₂]²⁺ 1, was used. In an effort to better elucidate their interactions with DNA, solutions of the different complexes titrated with DNA had their absorption spectra monitored. An absorbance hyperchromism observed at 260 nm pointed to the intercalation of these complexes into the DNA structure. Further, investigations of 2-deoxy-d-ribose (DR) oxidation catalyzed by each of those complexes, using 2-thiobarbituric acid reactive species (TBARS) method, and detection of reactive oxygen species (ROS) formation by spin-trapping EPR, suggested that their mechanism of action in performing efficiently DNA cleavage occurs preferentially, but not only by oxidative pathways.     

Synthesis and DNA cleavage activities of mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes which linked with uracil

Mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes, which could attach to peptide nucleic acid (PNA), were synthesized as DNA cleavage agents. The structures of these new mononuclear complexes were identified by MS and (1)H NMR spectroscopy. The catalytic activities on DNA cleavage of these mononuclear complexes with different central metals were subsequently studied, which showed that copper complex was better catalyst in the DNA cleavage process than zinc and cobalt complexes. The effects of reaction time, concentration of complexes were also investigated. The results indicated that the copper(II) complexes could catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) under physiological conditions to produce selectively nicked DNA (Form II, no Form III produced) with high yields. The mechanism of the cleavage process was also studied.     

Single-stranded DNA modification by an oligonucleotide-phthalocyanine Fe(II) conjugate: the kinetic features and the process mechanism

Catalytic oxidative modification of a single-stranded DNA with hydrogen peroxide and molecular oxygen in the presence of a conjugate containing an oligonucleotide complementary to the DNA fragment and tetra-4-carboxyphthalocyanine Fe(II) was studied. The conjugate examined was found to be active in the reaction of oxidative DNA cleavage in the presence of hydrogen peroxide, like the earlier studied oligonucleotide conjugates containing metallocomplexes tetra-4-carboxyphthalocyanine Co(II) and 2,4-di-[2-(2-hydroxyethyl)]deuteroporphyrin IX Fe(III) generating active oxygen forms. The new conjugate was more active in the case of oxidation with molecular oxygen. Kinetic features and optimal regimes of DNA oxidation with hydrogen peroxide were found.     

Prooxidant and antioxidant activities of bilirubin and its metabolic precursor biliverdin: a structure-activity study

Bilirubin, which is derived from its metabolic precursor biliverdin, is the end product of heme catabolism. It has been proposed as a physiological antioxidant present in human extracellular fluids. We have earlier shown that bilirubin in the presence of the transition metal ion Cu(II) causes strand cleavage in DNA through generation of reactive oxygen species, particularly the hydroxyl radical. Thus bilirubin possesses both antioxidant and prooxidant properties. In order to understand the chemical basis of various biological properties of bilirubin, we have studied the structure-activity relationship between bilirubin and its precursor biliverdin. The latter has also been reported to possess both antioxidant and toxic properties. In the present studies bilirubin was found to be more effective in the DNA cleavage reaction and a more efficient reducer of Cu(II). The rate of formation of hydrogen peroxide and hydroxyl radicals by the compounds also showed a similar pattern. The relative antioxidant activity was also examined by studying the effect of these compounds on DNA cleavage by a hydroxyl radical generating system and their quenching effect on hydroxyl radicals. The results indicate that bilirubin is more active both as an antioxidant as well as an oxidative DNA cleaving agent. A model for binding of copper to bilirubin has been proposed where two copper ions are bound to two molecules of bilirubin through their terminal pyrrole nitrogens. In order to account for the enhanced copper reducing capacity of bilirubin we have further proposed that an additional copper binding site is provided for in the case of bilirubin due to the absence of a double bond between pyrrole rings II and III. Further it would appear that the structural features of the bilirubin molecule which are important for its prooxidant action are also the ones that render it a more effective antioxidant.     

Enhanced nucleophilicity and depressed electrophilicity of peroxide by zinc(II), aluminum(III) and lanthanum(III) ions

The binuclear zinc(II) complex, [Zn2(HPTP)(CH3COO)]2+ was found highly active to cleave DNA (double-strand super-coiled DNA, pBR322 and phix174) in the presence of hydrogen peroxide. However, no TBARS (2-thiobarbituric acid reactive substance) formation was detected in a solution containing 2-deoxyribose (or 2'-deoxyguanosine, etc); where (HPTP) represents N,N,N'-N'-tetrakis(2-pyridylmethyl)-1,3-diamino-2-propanol. These facts imply that DNA cleavage reaction by the binuclear Zn(II)/H2O2 system should be due to a hydrolytic mechanism, which may be attributed to the enhanced nucleophilicity but depressed electrophilicity of the peroxide ion coordinated to the zinc(II) ion. DFT (density-functional theory) calculations on the peroxide adduct of monomeric zinc(II) have supported the above consideration. Similar DFT calculations on the peroxide adducts of the Al(III) and La(III) compounds have revealed that electrophilicity of the peroxide ion in these compounds is strongly reduced. This gives an important information to elucidate the fact that La3+ can enhance the growth of plants under certain conditions.     

ESR evidence for superoxide, hydroxyl radicals and singlet oxygen produced from hydrogen peroxide and nickel(II) complex of glycylglycyl-L-histidine

ESR studies using spin traps, 5,5-dimethylpyrroline-N-oxide and alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone, revealed that hydroxyl radical adducts are produced by the decomposition of hydrogen peroxide in the presence of nickel(II) oligopeptides. Order of catalytic activities of nickel(II) oligopeptides used in the production of hydroxyl radical adducts was tetraglycine greater than pentaglycine greater than triglycine greater than GlyGly, GlyHis. Ni(II) GlyGlyHis plus hydrogen peroxide produced superoxide in addition to hydroxyl radical adduct. Trapping experiments with 2,2,6,6-tetramethyl-4-piperidone suggested that singlet oxygen was generated by the reaction of hydrogen peroxide with Ni(II) GlyGlyHis, but not in the case of tetraglycine, pentaglycine, triglycine, GlyGly or GlyHis.     

Copper(II)/H(2)O(2)-mediated DNA cleavage: involvement of a copper(III) species in H-atom abstraction of deoxyribose units.

A new bis-amido-copper(II) complex 2 has been prepared. In the presence of reducing agents (ascorbate or DTT) under air atmosphere or hydrogen peroxide, complex 2 exhibited interesting nuclease activities in the 1-10 microM concentration range. For explaining the activity observed with hydrogen peroxide, we propose the occurrence of a bis-amido-copper(III) intermediate and an oxidation mechanism involving a H-atom abstraction of deoxyribose moieties of DNA.     

The effects of nitroxide radicals on oxidative DNA damage

The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2, 6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1-0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H(2)O(2). The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.     

Oxidative DNA damage of mixed copper(II) complexes with sulfonamides and 1,10-phenanthroline. Crystal structure of [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)2(1,10-phenanthroline)

Mixed coordination compounds of Cu(II) with sulfonamides and 1,10-phenanthroline as ligands have been prepared and characterised. Single crystal structural determination of the complex [Cu(N-quinolin-8-yl-p-toluenesulfonamidate)(2)(phen)] shows Cu(II) ions are located in a highly distorted octahedral environment, probably as a consequence of the Jahn-Teller effect. The FT-IR and electronic paramagnetic resonance (EPR) spectra are also discussed. The mixed complexes prepared undergo an extensive DNA cleavage in the presence of ascorbate and hydrogen peroxide. Two of the complexes have higher nucleolytic efficiency than the bis(o-phenanthroline)copper(II) complex.     

The conjugates of uracil-cyclen Zn(II) complexes: synthesis, characterization, and their interaction with plasmid DNA

As an important nucleobase in RNA, uracil was introduced into the side chain of cyclen (1,4,7,10-tetraazacyclododecane) by using phenylene dimethylene group as bridge. The target compounds 5 were obtained in high yields. Subsequent experiments demonstrated that the uracil-cyclen conjugates can bind Zn(2+) cation rapidly in water, and the catalytic activities of their Zn(II) complexes 6 in DNA cleavage were also studied. The results showed that Zn(II) complexes can catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) to produce nicked DNA (Form II and Form III) with high selectivity. In water solution, complex 6b may form a unique and stable supramolecular structure, which benefits the DNA cleavage process.     

Synthesis and DNA‐Cleavage Activities of Dinuclear Copper(II) Complexes of Urea‐Bridged Macrocyclic Polyamines

Two dinuclear macrocyclic polyamine copper(II) (Cu^II) complexes, which have two cyclen units linked by urea, were synthesized as DNA‐cleavage agents. The structures of these new dinuclear complexes were identified by HR‐ESI‐MS and IR analyses. The catalytic activities of DNA cleavage of these dinuclear Cu^II complexes were subsequently studied. The results show that 6a was the better catalyst in the DNA‐cleavage process than 6b . The effects of reaction time and concentration of complexes were also investigated. The results indicate, that the Cu^II complexes could catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA; Form I) under physiological conditions to produce selectively nicked DNA (Form II; no Form III was produced) with high yields (nearly 100%) in short time in the absence of reductant or oxidant.     

DNA cleavage by pentadentate iron(II) complexes containing fluoro-substituted phenyl groups.

Four pentadentate iron(II) complexes containing non- or fluoro-substituted phenyl group (2b-2e) were synthesized and cleaving activity of them to pUC19 DNA was evaluated in the presence of hydrogen peroxide. DNA cleavage activity increased with the number of substituted fluorine atoms on the phenyl group of 2b.     

DNA and RNA damage by Cu(II)-amikacin complex.

The oxidation-promoting reactivity of copper(II) complex of aminoglycosidic antibiotic amikacin [Cu(II)-Ami] in the presence of hydrogen peroxide, was studied at pH 7.4, using 2'-deoxyguanosine (dG), pBR322 plasmid DNA and yeast tRNAPhe as target molecules. The mixtures of complex with H2O2 were found to be efficient oxidants, converting dG to its 8-oxo derivative, generating strand breaks in plasmid DNA and multiple cleavages in tRNAPhe. The complex underwent autooxidation as well, with amikacin hydroperoxides as likely major products. This reactivity pattern was found to be due to a combination of metal-bound and free hydroxyl radicals.     

Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin

Upon irradiation with 365-nm light, copper(II)-camptothecin significantly produced single- and double-strand breaks of DNA and also induced a marked inactivation of bacteriophage. The nucleotide sequence analysis exhibited considerably random DNA cleavage. The DNA strand scission by the camptothecin-Cu(II)-UV light system, as well as the phage inactivation, was strongly suppressed by bathocuproine and catalase, indicating participation of cuprous species and hydrogen peroxide in the reaction. The present results suggest that (1) Cu(II) ion may play an important role as a cofactor in antitumor action of camptothecin and (2) the combination of copper-camptothecin plus long-wave ultraviolet light is useful against certain cancer treatment as a new photochemotherapy.     

DNA cleavage mediated by copper superoxide dismutase via two pathways

The known action of Cu, Zn superoxide dismutase (Cu(2)Zn(2)SOD) that converts O(2)(-) to O(2) and H(2)O(2) plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of Cu(2)Zn(2)SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much Cu(2)Zn(2)SOD may be injurious to the cells. The present study examined the DNA cleavage activity mediated by a Cu(n)SOD that contains 1-4 copper ions, in order to obtain an insight into the aberrant copper-mediated oxidative chemistry in the enzyme. A high SOD activity was observed upon metallation of the apo-form of Cu(2)Zn(2)SOD with Cu(II), indicating that nearly all of the Cu(II) in the Cu(n)SOD is as active as the Cu(II) in the copper site of fully active Cu(2)Zn(2)SOD. Using a supercoiled DNA as substrate, significant DNA cleavage was observed with the Cu(n)SOD in the presence of hydrogen peroxide or mercaptoethanol, whereas DNA cleavage with free Cu(II) ions can occur only <5% under the same conditions. Comparison with other proteins shows that the DNA cleavage activity is specific to some proteins including the Cu(n)SOD. The steady state study suggests that a cooperative action between the SOD protein and the Cu(II)may appear in the DNA cleavage activity, which is independent of the number of Cu(II) in the Cu(n)SOD. The kinetic study shows that a two-stage reaction was involved in DNA cleavage. The effects of various factors including EDTA, radical scavengers, bicarbonate anion, and carbon dioxide gas molecules on the Cu(n)SOD-mediated DNA cleavage activity were also investigated. It is proposed that DNA cleavage occurs via both hydroxyl radical oxidation and hydroxide ion hydrolysis pathways. This work implies that any form of the copper-containing SOD enzymes (including Cu(2)Zn(2)SOD and its mutants) might have the DNA cleavage activity.     

Unusual reactivity in a commercial chromium supplement compared to baseline DNA cleavage with synthetic chromium complexes

Commercially available chromium supplements were tested for their DNA cleavage ability compared with synthetic chromium(III) complexes, including chromium(III) tris-picolinate [Cr(pic)3], basic chromium acetate [Cr3O(OAc)6]+, model complexes, and recently patented Cr-complexes for use in supplements or therapy. Four different supplements (P1-P4) were tested for their DNA cleaving activity in the presence and the absence of H2O2, dithiothreitol (DTT) or ascorbate. One supplement, P1, showed nicking of DNA in the absence of oxidant or reductant at 120 microM metal concentration. Different lot numbers of P1 were also tested for DNA cleavage activity with similar results. Commercial supplements containing Cr(pic)3 nicked DNA at 120 microM metal concentrations in the presence of 5 mM ascorbate or with excess hydrogen peroxide, analogous to reactions with synthetic Cr(pic)3 reported elsewhere. Another chromium (non-Cr(pic)3) supplement, P2, behaves in a comparable manner to simple Cr(III) salts in the DNA nicking assay. Chromium(III) malonate [Cr(mal)2] and chromium(III) acetate [Cr(OAc)] can nick DNA in the presence of ascorbate or hydrogen peroxide, respectively, only at higher metal concentrations. The Cr(III) complexes of histidine, succinate or N-acetyl-L-glutamate do not nick DNA to a significant degree.     

P-3A and (−)-desacetamido P-3A: demonstration and Study of their effective functional cleavage of duplex DNA

A study of the Fe(II) complexes of P-3A (1) and (−)-desacetamido P-3A (2) abilities to cleave duplex DNA was conducted through examination of single-strand and double-strand cleavage of supercoiled φX174 RFI DNA (Form I) in the presence of O₂ to produce relaxed (Form II) and linear (Form III) DNA, respectively. Like Fe(II)-bleomycin A₂ and deglycobleomycin A₂, Fe(II)-1 and 2 effectively produced both single- and double-strand cleavage of supercoiled φX174 DNA. Unlike Fe(II)-bleomycin A₂ or deglycobleomycin A₂, Fe(II)-1 and 2 were found to cleave duplex w794 DNA with no discemible sequence selectively suggesting that the polynucleotide recognition of the C-terminus tetrapeptide S subunit of the bleomycins including the bithiazole may dominate the bleomycin A₂ DNA cleavage selectivity.     

A novel analytical method to evaluate directly catalase activity of microorganisms and mammalian cells by ESR oximetry

Abstract

Electron spin resonance (ESR) oximetry technique was applied for analysis of catalase activity in the present study. Catalase activity was evaluated by measuring oxygen from the reaction between hydrogen peroxide (H₂O₂) and catalase-positive cells. It was demonstrated that the ESR spectra of spin-label probes, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO) and 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (4-maleimido-TEMPO) in the presence of H₂O₂ were broadened with the concentrations of catalase. It was possible to make a calibration curve for catalase activity by peak widths of the spectra of each spin-label probe, which are broadened dependently on catalase concentrations. The broadened ESR spectra were also observed when the catalase-positive micro-organisms or the mammalian cells originally from circulating monocytes/macrophages were mixed with TEMPOL and H₂O₂. Meanwhile, catalase-negative micro-organisms caused no broadening change of ESR spectra. The present study indicates that it is possible to evaluate directly the catalase activity of various micro-organisms and mammalian cells by using an ESR oximetry technique.     

Effects of iron,copper, and chromate ions on the oxidative degradation of cellulose model compounds

Iron(II) and iron(III) ions promote the degradation of the cellulose model 1,5-anhydrocellobiitol by oxygen or hydrogen peroxide; copper and chromate ions have marked and different effects on the iron catalysis. With starch, iron promotes the hydrogen peroxide-induced reaction and copper and chromate ions further enhance the reaction rate. The tensile strength of paper board is reduced by the action of hydrogen peroxide and iron(II) salts, and mixtures of copper, chromate, and arsenate salts (CCA, a timber preservative) also promote degradation in the presence or absence of iron ions. The oxidation of 1,5-anhydrocellobiitol by oxygen in the presence of iron ions is strongly inhibited by CCA and by cetyltrimethylammonium chloride, and is accelerated by phenols and related compounds.     

Singlet oxygen generation from phosphatidylcholine hydroperoxide in the presence of copper

This study pursued whether singlet oxygen ((1)O2) is generated from phosphatidylcholine hydroperoxide (PCOOH), the oxidized modification product of a major constituent of biomembranes and serum lipoproteins. The (1)O2 formation was detected, by utilizing the oxidation of 2,2,6,6-tetramethyl-4-piperidone (TMPD) by (1)O2 to yield 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (TEMPONE), which generates electron spin resonance (ESR) signals. The TEMPONE signal was detected in human plasma with addition of PCOOH by ESR determination after introducing copper(II). The TEMPONE formation was proportional to the amounts of PCOOH added according to moles of active oxygen. The TEMPONE signal intensity was weakened significantly in the presence of beta-carotene and histidine in a concentration-dependent manner, but was not at all decreased by mannitol, Mn-superoxide dismutase and catalase. In addition, HPLC-chemiluminescence analysis demonstrated that incubation with the PCOOH/Cu(II) combination oxidized cholesterol, a relatively oxidation-resistant component, to the cholesterol hydroperoxide. These results reveal that (1)O2 is generated from PCOOH in contact with copper(II). In conclusion, this in-vitro study provides directly the (1)O2 formation in living organisms following the advancement of peroxidation of constitutive lipids.