Induce DNA Strand Scission Research Articles

Bioactive peptides from shrimp shell processing discards: Antioxidant and biological activities

Protein hydrolysates were prepared from shrimp discards, using various enzymes, which were subsequently analyzed for their antioxidant potential and biological activities. Raw shrimp shell discards hydrolysates (RSH) and isolated shrimp shell protein hydrolysates (SPH) exhibited radical scavenging activities (ABTS, DPPH and hydroxyl), reducing power and ferrous ion chelating ability. In addition, RSH and SPH inhibited beta-carotene bleaching in an oil-in-water emulsion, cupric ion induced LDL cholesterol peroxidation as well as peroxyl and hydroxyl radical induced DNA strand scission effectively. In-vitro determination of ACE inhibitory activity by RSH and SPH showed that these hydrolysates could be used in supplements, foods or in the purified form as potential pharmaceuticals for blood pressure control. Moreover, shrimp shell discard protein hydrolysate fractions with highest ACE inhibitory activity were further subjected to gel filtration, mass spectrometry and peptide sequencing. Three potential ACE inhibitory bioactive peptides were identified form shrimp shell discard for the first time.

Internal abstraction of dynemicin A: An MD approach

Dynemicin A has the ability to undergo the Bergman cyclization, forming a para-benzyne moiety with the ability to induce DNA strand scission. This property of dynemicin A makes it a promising anti-tumor agent. Past research has shown conclusively that dynemicin A binds to and abstracts a hydrogen atom (H5′) from the DNA backbone, but the molecular mechanism of the binding event is not fully understood. We have used AMBER Molecular Dynamics simulations to investigate the dynamics associated with the reaction mechanisms. Previously, two binding mechanisms have been proposed, of which the second is more supported: (1) dynemicin A intercalates between two base pairs and directly abstracts a hydrogen atom from DNA and (2) dynemicin A inserts into the minor groove and directly abstracts a hydrogen atom from DNA. We propose a third mechanism, where dynemicin A intercalates, then undergoes a proximate, intramolecular hydrogen atom abstraction (internal abstraction). While not studied here, the resulting radical would then subsequently abstract a hydrogen atom from DNA.

Antioxidant Activity and Protective Effect Against Plasmid DNA Strand Scission of Leaf, Bark, and Heartwood Extracts from Acacia catechu

The antioxidant activity of methanol extract/fractions of leaf, bark, and heartwood of Acacia catechu was evaluated by various antioxidant assays, including free radical, superoxide and hydroxyl radical, reducing power, metal ion chelation, as well as hydroxyl radical induced DNA strand scission. The leaf, bark, and heartwood powder was extracted in methanol and the lyophilized methanol extract was fractionated with different solvents in the order of increasing polarity. The results indicate that ethyl acetate fraction of heartwood has the highest antioxidant capacities, presenting lower EC(50) values particularly in free radical scavenging activity, including DPPH radicals (4.76 ± 0.14 μg/mL), superoxide anions (26.21 ± 0.79 μg/mL), and hydroxyl radicals (33.69 ± 1.42 μg/mL), in direct assay systems. Reducing power was also highest in ethyl acetate fraction of heartwood (EC(50) of 79.05 ± 1.02 μg/mL). As for the chelating power on ferrous ions, leaf extract was more effective than bark and heartwood extracts. Furthermore, the ethyl acetate and acetone fractions of heartwood significantly protected pBR322 supercoiled plasmid DNA against strand scission induced by hydroxyl radicals in a Fenton's reaction mixture. The present investigation suggests that the three organs of A. catechu differ significantly in their antioxidant potential as seen in the DPPH radical scavenging assay, reducing power assay, metal ion chelating assay, superoxide radical scavenging assay and hydroxyl radical scavenging assay. Further, our results showed that crude methanol extract and ethyl acetate fraction of heartwood of A. catechu might have a good potential as a source for natural health products due to its antioxidant and DNA protective activities.

Synthesis and Characterization of Substituted Ethyl 2-(1-aminocyclobutyl)- 5-(benzoyloxy)-6-hydroxypyrimidine-4-carboxylate Derivatives as Antioxidant Agents

A series of novel ethyl 2-(1-aminocyclobutyl)-5-(benzoyloxy)-6-hydroxy-pyrimidine-4-carboxylate derivatives 8(a-i) were synthesized by nucleophilic substitution reaction of ethyl 2-(1-aminocyclobutyl)-5-(benzoyloxy)-6-hydroxypyrimidine- 4-carboxylate (7) with different substituted aliphatic/aromatic sulfonyl chlorides (R-SO2-Cl). All the synthesized compounds were characterized by IR, 1H NMR and LC/MS analysis. The synthesized compounds were evaluated for their antioxidant activity by diphenylpicrylhydrazyl (DPPH) assay and also by hydroxy radical induced DNA strand scission assay. Among the synthesized compounds 8c, 8f and 8i showed promising antioxidant activity. Keywords: Benzyl 1-cyanocyclobutylcarbamate, Pyrimidine, Hydroxy radical, DPPH Assay, Antioxidant

Antioxidant Activity of a Series of FluorinatedPyrano-nucleoside Analogues of N4-benzoyl Cytosine andN6-benzoyl Adenine

Fluorinated nucleoside analogues are known as antitumor, antiviral andchemotherapeutic agents, although the antioxidant activity of this kind of molecules is notyet investigated. In this study we have tested the antioxidant activity of a series of modifiedpyrano-nucleoside analogues using three in vitro assays. Firstly, the antioxidant capacity ofthe products was assessed using the DPPH assay and secondly, in order to examine theability of the products to protect DNA from the activity of reactive oxygen species (ROS),a peroxyl radical (ROO•) and a hydroxyl radical (OH•) induced DNA strand scission assaywere used. None of the molecules showed the ability to scavenge DPPH radical andprevent OH• induced DNA strand breakage. Although, most of the tested nucleosideanalogues, had the ability to prevent ROO• induced DNA damage.

Structural basis for DNA-cleaving activity of resveratrol in the presence of Cu(II)

Resveratrol ( 1, 3,5,4′-trihydroxy- trans-stilbene), a polyphenol found in grapes and other food products, is known as an antioxidant and cancer chemopreventive agent. However, 1 was shown to induce genotoxicity through a high frequency of micronucleus and sister chromatid exchange in vitro and DNA-cleaving activity in the presence of Cu(II). The present study was designed to explore the structure–activity relationship of 1 in DNA strand scission and to characterize the substrate specificity for Cu(II) and DNA binding. When pBR322DNA was incubated with 1 or its analogues differing in the number and positions of hydroxyl groups in the presence of Cu(II), the ability of 4-hydroxystilbene analogues to induce DNA strand scission is much stronger than that of 3-hydroxy analogues. The high binding affinity with both Cu(II) and DNA was also observed by 4-hydroxystilbene analogues. The reduction of Cu(II) which is essential for activation of molecular oxygen proceeded by addition of 1 to the solution of the Cu(II)–DNA complex, while such reduction was not observed with the addition of isoresveratrol, in which the 4-hydroxy group of 1 is changed to the 3-position. The results show that the 4-hydroxystilbene structure of 1 is a major determinant of generation of reactive oxygen species that was responsible for DNA strand scission.

Antioxidant activity and protective effect on DNA strand scission of Rooibos tea (Aspalathus linearis)

Rooibos tea (Aspalathus linearis) was extracted by refluxing with water and 75% ethanol as a solvent. Antioxidant activity and protective effect on DNA strand scission were investigated by using different antioxidant assay systems and DNA strand nicking assay, respectively. 75% Ethanol extract has higher content of total soluble phenolics and flavonoid than water extract. Antioxidant activities such as hydrogen donating capacity and scavenging activity of hydrogen peroxide were higher in 75% ethanol extract than in water extract except the rate constant with hydroxyl radical. Peroxyl radical induced DNA strand scission was prevented by both 75% ethanol and water extract and hydroxyl radical induced DNA strand scission was not. This result indicates that total soluble phenolics, specially flavonoid, of Rooibos tea are responsible for several kinds of antioxidant activities and preventive activity on peroxyl radical induced DNA strand scission.

Strand cleavage of supercoiled DNA by water-soluble peroxyl radicals. The overlooked importance of peroxyl radical charge.

It is well established that the peroxyl radicals formed during the thermal decomposition of 2,2'-azobis(amidinopropane), ABAP, in oxygenated water can cleave double-stranded DNA, from which fact it has been concluded that peroxyl radicals, as a general class, can induce DNA strand scission. However, the ABAP-derived radicals are positively charged, and DNA is a negatively charged polyanion. Moreover, the relatively small and, therefore, free to diffuse peroxyl radicals likely to be formed in vivo will generally be negatively charged or neutral. Plasmid supercoiled DNA [pBR 322, 4361 base pairs (bp)] was reacted with known, equal fluxes of two positively charged peroxyl radicals, a negatively charged peroxyl radical, and a neutral peroxyl radical. The two positively charged peroxyl radicals degraded >/=80% of the supercoiled pBR 322 at a flux of 4 radicals/bp, but the negatively charged and neutral peroxyl radicals had no significant effect even at a flux as high as 24 radicals/bp. The same lack of effect on the DNA was also observed with high fluxes of superoxide/hydroperoxyl radicals. Similar results were obtained with another supercoiled DNA, pUC 19, except that pUC 19 is somewhat more sensitive to strand scission by positively charged peroxyl radicals than pBR 322. We conclude that most of the peroxyl radicals likely to be formed in vivo have little or no ability to induce DNA strand scission and that the potential role of electrostatics in radical/DNA reactions should always be considered.

The synthesis and antitumor activity of metal complexes of amine–carboxyborane adducts

The amine carboxyborane metal complexes, tetrakis - μ - (trimethylamine - boranecarboxylato)acetonitrile dicopper(II) and his-μ-(morpholine-boranecarboxylato)zinc(II) dihydrate demonstrated cytotoxic activity against human Tmolt 3 , HeLa-S 3 and MB-9812 cell growth. Tetrakis-μ-(trimethylamine-boranecarboxylato)-acetonitrile dicopper(II) and bis - μ - (morpholine - boranecarboxylato)zinc (II) dihydrate inhibited L 1210 DNA, RNA and protein syntheses, with greatest inhibitory effects on DNA synthesis. The reduction in DNA synthesis correlates well with inhibition of de novo purine synthesis and the key enzymes involved in this pathway, i.e. IMP dehydrogenase and PRPP amido transferase. These compounds were also observed to induce DNA strand scission but did not appear to intercalate between base pairs of DNA, alkylate bases or cause cross-linking of the strands of DNA. Tetrakis-μ-(trimethylamine - boranecarboxylato)acetonitrile dicopper(II) also demonstrated the ability to inhibit L 1210 DNA topoisomerase II activity.

Role of the central metal ion and ligand charge in the DNA binding and modification by metallosalen complexes.

Several metal complexes of three different functionalized salen derivatives have been synthesized. The salens differ in terms of the electrostatic character and the location of the charges. The interactions of such complexes with DNA were first investigated in detail by UV-vis absorption titrimetry. It appears that the DNA binding by most of these compounds is primarily due to a combination of electrostatic and other modes of interactions. The melting temperatures of DNA in the presence of various metal complexes were higher than that of the pure DNA. The presence of additional charge on the central metal ion core in the complex, however, alters the nature of binding. Bis-cationic salen complexes containing central Ni(II) or Mn(III) were found to induce DNA strand scission, especially in the presence of co-oxidant as revealed by plasmid DNA cleavage assay and also on the basis of the autoradiogram obtained from their respective high-resolution sequencing gels. Modest base selectivity was observed in the DNA cleavage reactions. Comparisons of the linearized and supercoiled forms of DNA in the metal complex-mediated cleavage reactions reveal that the supercoiled forms are more susceptible to DNA scission. Under suitable conditions, the DNA cleavage reactions can be induced either by preformed metal complexes or by in situ complexation of the ligand in the presence of the appropriate metal ion. Also revealed was the fact that the analogous complexes containing Cu(II) or Cr(III) did not effect any DNA strand scission under comparable conditions. Salens with pendant negative charges on either side of the precursor salicylaldehyde or ethylenediamine fragments did not bind with DNA. Similarly, metallosalen complexes with net anionic character also failed to induce any DNA modification activities.

Bifunctional antitumor agents: a DNA interactive enediyne from the pyrrolo[9,10-b]phenanthrene template

Regioselective substitution of the intercalative pyrrolo[9,10-b]phenanthrene template was used to prepare an enediyne conjugate. The enediyne induced DNA strand scission at relevent concentration but proteolysis was not significant.

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases.

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O(2-)-, to O2 and H2O2, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may be injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage phi X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H2O2. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.

Syntheses, structures and reactivities of designed analogues of cobalt(III)-bleomycinsz: Insight into the mechanism of sequence-specific DNA cleavage upon illumination

Three Co (III) complexes of a designed ligand PMAH that mimics the metal-binding domain of the antitumor antibioticbleomycin (BLM) have been isolated and structurally characterized. The coordination structures of the various forms of Co(III)-BLMs have been established on the basis of spectral similarities between these synthetic analogues and the corresponding Co(III)-BLMs. All three analogues, like Co(III)-BLMs, induce DNA strand scission upon UV illumination. Both DNA cleavage and spin trapping experiments demonstrate that UV irradiation of the analogues generates a C/N-based radical on the ligand framework which rapidly reacts with water to produce -OH radical near the DNA helix and causes strand scission. A similar mechanism could account for the photoactivity of the Co(III)-BLMs. Covalent attachment of DNA-binding groups to these analogues enhances the DNA-affinities and photocleavage efficiencies to a great extent. The hybrid analogues promote sequence-specific DNA photodamage at micromolar concentrations. The metallated cores of the hybrid analogues are the primary determinant of the observed sequence-specificity. Details of the mode of binding of the hybrid analogues to DNA have been explored by NMR techniques.

DNA damage related to increased hydrogen peroxide generation by hypolipidemic drug-induced liver peroxisomes.

Several hypolipidemic drugs and certain industrial plasticizers induce proliferation of peroxisomes, enhance the activity of peroxisome-associated beta-oxidation of fatty acids, and produce hepatocellular carcinomas in the livers of rodents. Because these chemicals themselves are not mutagens and do not covalently modify DNA, unlike the majority of chemical carcinogens, we proposed that the persistent proliferation of peroxisomes, and the induction of associated peroxisomal oxidases, caused a sustained increase in intracellular H2O2 or other reduced oxygen species, which would then introduce mutagenic DNA damage. In the present study, we investigated the ability of peroxisomes purified from the livers of normal and hypolipidemic drug-treated rats to induce DNA strand scission in vitro. Gradient-purified peroxisomes from livers of hypolipidemic drug-treated rats produced a 30- to 70-fold increase in H2O2 generation when compared to controls. The levels of H2O2 generated in incubations containing control or hypolipidemic drug-induced peroxisomes correlated well with the induction of single strand breaks in supercoiled simian virus 40 DNA molecules that were included in these reconstituted peroxisome incubations. Addition of excess catalase to peroxisome incubations failed to prevent strand breaks, suggesting that other reduced oxygen species may be rapidly generated from H2O2. These experimental results are consistent with a mechanism of hepatocarcinogenesis in which hepatocellular genetic damage is introduced by the by-products of peroxisomal fatty acid beta-oxidation, an oxidative pathway that is dramatically increased in hypolipidemic drug-treated livers.

71] Chemical carcinogenesis: Benzopyrene system

Publisher Summary It has been realized that the metabolic activation of chemical carcinogens may lead to free-radical intermediates, which may be related to the action of chemical carcinogens. There is considerable interest in evaluating the importance of such radical intermediates on the toxic, mutagenic, tumor-initiating, and tumor-promoting capacities of the relevant carcinogens. The metabolic activation of benzo[a]pyrene (BP), an important environmental carcinogen, has been extensively investigated. Several major BP metabolites, 6-hydroxy-BP, BP-1,6-dione, BP-3,6-dione, and BP-6,12-dione give rise to free-radical intermediates and reactive reduced oxygen species as the result of autoxidative processes and redox cycles. These metabolites induce DNA strand scission in vitro and are cytotoxic to mammalian cells in culture. The metabolic conversion of BP to reactive electrophiles as well as intermediates that generate reactive oxygen species may account for its ability to serve as a complete carcinogen––that is, having both initiating and promoting activities. This chapter reviews the preparation, purification, and chemical properties of 6-hydroxy-BP and the three isomeric BP diones.

Benzo[a]pyrenedione/benzo[a]pyrenediol oxidation-reduction couples and the generation of reactive reduced molecular oxygen

The ability of the isomeric quinone metabolites of benzo[a]pyrene, benzo[a]pyrene-6,12-dione, benzo[a]pyrene-1,6-dione, and benzo[a]pyrene-3,6-dione to undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo[a]pyrenediols and intermediate semiquinone radicals has been characterized. Under anaerobic conditions, all three benzo[a]pyrenediones are easily reduced to benzo[a]pyrenediols, even by mild biological agents such as NAD(P)H, cysteamine, and glutathione. The benzo[a]pyrenediols, in turn, are very rapidly autoxidized to the benzo[a]pyrenediones when exposed to air. Substantial amounts of hydrogen peroxide are produced during these autoxidations, and other reactive reduced oxygen species, such as the superoxide and hydroxyl radicals, are probably formed transiently as well. The benzo[a]pyrenediol-benzo[a]pyrenedione interconversions proceed by one-electron steps; the corresponsing semiquinone radicals can be monitored by electron spin resonance spectroscopy as inter mediates during these reactions carried out at high pH. Benzo[a]pyrenediones induce DNA strand scission when incubated with bacteriophage T7 DNA. This damage is modified by conditions which indicate that reduced oxygen species propagate the free-radical reactions responsible for the strand scission. Benzo[a]pyrenediones are electron-acceptor substrates for NADH dehydrogenase from Clostridium kluyveri. Catalytic amounds of these benzo[a]pyrene metabolites, together with this respiratory enzyme function as cyclic oxidation-reduction couples which link NADH and molecular oxygen in the continuous production of hydrogen peroxide. These data, together with preliminary results with cells in culture, indicate that benzo[a]pyrenediones are potentially harmful metabolites of benzo[a]pyrene, acting by processes which lead to their regeneration rather than depletion; nucleic acid and call damage is probably produced by the reactive reduced oxygen species resulting from such regenerative oxidation-reduction cycles.