Efficient photoinduced DNA damage by coralyne

The photoinduced DNA damage by the berberine derivative coralyne is presented. The irradiation of coralyne in the presence of plasmid DNA namely, pBR322, leads to remarkably fast DNA damage by single-strand cleavage, as determined by agarose-gel electrophoresis. Even upon exposure to sunlight, almost all of the supercoiled plasmid is converted to the open circular form in less than a minute [c(pBR322) = 3.5 x 10(-9) M; c(coralyne) = 4.3 x 10(-5) M]. The efficiency of the DNA strand cleavage is not decreased in the presence of radical-trapping reagents such as tert-butanol or DMSO. Moreover, the extent of the DNA damage is the same under aerobic conditions and at reduced oxygen concentration. Thus, the formation of reactive intermediates such as hydroxyl radicals or singlet oxygen is excluded. These results show that the exposure of coralyne and derivatives thereof to light, even with moderate light intensity, needs to be avoided during experiments in which their biological activity is assessed by plasmid unwinding assays.     

Supramolecular Cationic Tetraruthenated Porphyrin Induces Single-Strand Breaks and 8-Oxo-7,8-dihydro-2'-deoxyguanosine Formation in DNA in the Presence of Light

Abstract— The aim of this investigation is the evaluation of DNA interaction of with tetraruthenated porphyrin (TRP) and of DNA damage in the presence of light. Direct-fluorescence and electronic absorption measurements after incubation of DNA with TRP indicate strong binding between pBR322 DNA or calf thymus DNA with the modified porphyrin. Exposure of pBR322 DNA to TRP (up to 3 μ M ) and light leads to single-strand break formation as determined by the conversion of the supercoiled form (form I) of the plasmid into the nicked circular form (form II). Oxidative DNA base damage was evaluated by the detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after irradiation of calf thymus DNA in the presence of the TRP. The data demonstrated a dose and time dependence with each type of DNA damage. These data indicate (1) a specificity of the binding mode and (2) type I and II photoinduced mechanisms leading to strand scission activity and 8-oxodGuo formation. Accordingly, singlet molecular oxygen formation, after TRP excitation, was confirmed by near-infrared emission. From these investigations a potential application of TRP in photodynamic therapy is proposed.     

Ferric-ion-photosensitized damage to DNA by hydroxyl and non-hydroxyl radical mechanisms.

Iron(III) and UVA (320-400 nm) light strongly diminished the transforming activity of Haemophilus influenzae DNA in the presence of oxygen. Iron(III) alone in the absence of light had no measurable effect on the transforming activity. The chelating agent ethylenediaminetetraacetic acid (EDTA) conferred virtually complete protection, but hydroxyl radical scavengers (mannitol, methanol, ethanol, isopropanol and dimethyl sulfoxide) inhibited only a small fraction of the inactivation. Treatment of plasmid DNA (pBR322) with iron(III) results in the conversion of the covalently closed circular form of the plasmid to open circles and ultimately to the linear form. Concomitant with the alteration in the conformation of the plasmid, the ability to transform Escherichia coli was reduced. In model systems, iron(III) photoreacted with the DNA backbone causing nicking and double-strand breakage. The results are consistent with a mechanism involving a preliminary complexation of iron(III) by DNA followed by the generation of reactive free radicals other than .OH. We suggest that bound iron, or other UV-absorbing transition metal complexes, may be chromophores capable of causing DNA damage in the long-wave near-UV region.     

PHOTOCLEAVAGE OF DNA IN THE PRESENCE OF SYNTHETIC WATER-SOLUBLE PORPHYRINS

In the presence of oxygen and visible light, various synthetic water-soluble porphyrins cleave pBR 322 plasmid supercoiled DNA (form I) producing relaxed (form II) and linear (form III) DNA corresponding to single-strand and double-strand breaks respectively. Large variations are observed in the efficiency of the porphyrins containing a diamagnetic metal or no metal at all. Singlet oxygen (¹O₂) seems to be involved in the mechanism of cleavage consistent with the inhibitory effect of the azide anion, N^–₃. The higher efficiency of cationic porphyrins (as compared to anionic ones) is due to their greater affinity for DNA as shown by experiments carried out at either high ionic strength or in the presence of the surfactant, sodium dodecyl sulfate.     

MECHANISMS OF CITRAL PHOTOTOXICITY

Citral, a monoterpene aldehyde synthesized by several plant genera, has been reported to exhibit antimicrobial activity. For the first time, we report that critral exhibits UV-A (315-400 nm) light enhanced oxygen-dependent toxicity against a series of Escherichia coli strains differing in DNA repair and catalase proficiency. Those E. coli strains carrying a gene leading to catalase deficiency (katF) are particularly sensitized to inactivation by citral and UV-A treatment when compared to catalase proficient strains (katF+). Consistent with these in vivo observations, citral when treated with UV-A in vitro produces H2O2. When tested against Fusarium oxysporum and F. solani, fungal root pathogens of Citrus, enhanced toxicity by citral in the presence of UV-A was demonstrated, while dark toxicity was negligible. When the plasmid pBR322 was treated with citral in the presence of UV-A, a change in conformation from the covalently closed circular to the open circular and, ultimately, the linear form was observed. The change in plasmid conformation corresponded to a reduction in transforming activity. Holding plasmid DNA which had been treated with UV-A light in the presence of citral at 4 degrees C for 22 h in the dark resulted in continued degradation of the DNA and loss of transforming activity. Holding plasmid DNA treated with UV-A or citral alone under identical conditions had no detectable effect on either plasmid conformation or transforming activity.     

SINGLE-STRAND BREAKS IN SUPERCOILED DNA INDUCED BY VACUUM-UV RADIATION IN AQUEOUS SOLUTION

We studied the induction of single-strand breaks in the DNA of plasmid pBR 322 by vacuum-UV radiation above 145 nm in aqueous solutions in relation to the production of OH-radicals in water. The similarity and dissimilarity were examined of the wavelength dependence between the two effects. The maximum of single strand breaks at 150 nm could be explained by the action of OH-radicals derived from direct water photolysis: the maximum at 180 nm remains unexplained. There was no indication that the direct absorption of photon by the DNA molecule plays an important role in the production of single-strand breaks.     

In vitro DNA damage characterisation studies on plasmid pBR322 after exposure to γ radiation by <Superscript>60</Superscript>Co

When a biological system is either accidentally or intentionally exposed to radiation, the energy absorbed triggers a number of successive events including damage to living tissues. Major radiation damage is due to the aqueous free radicals generated by the radiolysis of water. These free radicals act as molecular marauders and in turn damage DNA, mitochondrial membrane, lipid, cellular protein, resulting in cellular dysfunction and mortality. In view of the above mentioned facts an experiment was conducted to study the genotoxic effects of γ radiation and its dose effectiveness. The present experiment was conducted on samples of plasmid pBR322 DNA as the in vitro experimental model devoid of any DNA repair and replication machinery. The samples were exposed to different doses of gamma radiations from 1 to 200 Gy. Exposure of plasmid pBR322 DNA to γ radiation resulted in production of single strand breaks as a result of which, the supercoiled (SC) form was converted to relaxed form (RL). Exposure of radiation, even at very low dose of 1 Gy, exhibited a significant damage to DNA resulting in about 70% SC form and 30% RL form of DNA. At a dose of 10 Gy the SC form was reduced to about 37% and further 5% at a dose of 50 Gy with about 88.5 and 6.5% RL and linear (L) forms of DNA respectively. Thus, the disappearance of supercoiled form of plasmid pBR322 DNA was found to be directly related to radiation dose and exhibited a radiation dose dependent pattern.     

Synthesis of innovative biochemical active mixed ligand metal(II) complexes with thiazole containing Schiff base: In vitro antimicrobial profile

An unique Schiff base ligand, formed by the condensation reaction of 2‐aminobenzothiazole with curcumin and its Cu(II), Ni(II), Co(II) and Zn(II) complexes incorporating 2,2′‐bipyridine as coligand were synthesised. They were characterized via analytical and spectroscopic methods. The complexes adopt square planar geometry. Their antimicrobial activity and photocatalytic efficiency on Congo red dye molecule were explored. It is found that all the complexes are antimicrobial active and show higher activity than the ligand. The nuclease activity of the above metal complexes was also assessed by absorption titration, fluorescence, viscosity and gel electrophoresis assay. The complexes bind CT DNA through intercalation mode. The data reveal that the above synthesised metal(II) complexes are found to be effective metallonucleases. The gel electrophoresis results exhibit that the metal complexes cleave pBR322 plasmid DNA in presence of hydrogen peroxide effectively compared to the ligand. The synthesised metallonucleases should lead to a new era for the logical sketch of dominant agents for probing and targeting nucleic acids. This exploration reveals that Cu(II) complex has a valued biological and photochemical profile.     

A new trick (hydroxyl radical generation) for an old vitamin (B12)

Photolysis of hydroxocobalamin in the presence of plasmid DNA (pBR322) results in DNA cleavage. Temporal control of hydroxyl radical production and DNA strand scission by hydroxocobalamin was demonstrated using a 2-deoxyribose assay and a plasmid relaxation assay, respectively. The light-driven hydroxocobalamin-mediated catalytic formation of hydroxyl radicals was demonstrated using radical scavenging studies of DNA cleavage and via recycling of a hydroxocobalamin-resin conjugate several times without loss of efficacy.     

THE PHOTOTOXICITY OF 8-M-ETHOXYTHIONEPSORALEN AND 6-M-ETHYLTHIONECOUMARIN

The phototoxicity of 8-methoxythionepsoralen (8-MOTP) and 6-methylthione coumarin (6-MTC) when activated by UV-A has been investigated using a variety of Escherichia coli strains, Haemophilus influenzae transforming DNA and Escherichia coli pBR322 plasmid DNA. The results demonstrate that 8-MOTP is a strictly oxygen independent photosensitizer that is about 500-fold less efficient in forming lesions leading to equivalent lethality than is the parent compound from which it is derived (8-MOP). As is true for 8-MOP, 8-MOTP is capable of inducing histidine independent mutations in E. coli and inactivating transforming DNA consistent with DNA being a target for lesions induced by this molecule in the presence of UV-A. 6-MTC is a strongly oxygen dependent photosensitizer activated by UV-A when tested with either E. coli cells or transforming DNA in contrast to the parent compound (6-methylcoumarin; 6-MC) which is not phototoxic when treated with UV-A. These results imply that the membrane may be an important target leading to lethality. 6-MTC in the presence of UV-A can inactivate pBR322 plasmid and Haemophilus influenzae transforming DNA activity in vitro suggesting that DNA is a potential target for this molecule when activated by UV-A.     

Formation of pyrazine derivatives from D-glucosamine and their deoxyribonucleic acid (DNA) strand breakage activity

Two pyrazine derivatives [fructosazine (3) and deoxyfructosazine (6)] were simultaneously formed in a solution of D-glucosamine hydrochloride under various conditions. They showed deoxyribonucleic acid (DNA) strand breakage activity in plasmid pBR322 comparable to that of D-glucosamine. The DNA strand breakage by fructosazine (3) was stimulated by Cu2+.     

DNA interaction studies of ruthenium(II) polypyridyl complex : [Ru(dmb)2(ITAP)](ClO4)2 (ITAP = isatino [1,2-b]-1,4,8,9-tetraazatriphenylene)

A new ligand ITAP and its complex [Ru(dmb)₂(ITAP)](ClO₄)₂ (ITAP = isatino [1,2-b]-1,4,8, 9-tetraazatriphenylene, dmb = 4,4′-dimethyl-2,2′-bipyridine) have been synthesized and characterized by elemental analysis, Fast atom bombardment mass spectra, Electrospray mass spectra, and ¹H NMR. Thermal denaturation and absorption titration experiments show the complex binds to calf thymus DNA (CT-DNA) with moderate affinities. Viscosity measurements and thermal denaturation indicate that the DNA-binding mode could be intercalative interaction. The Ru(II) complex in the presence of plasmid pBR322 DNA has been found to promote the cleavage of plasmid pBR322 DNA from the supercoiled Form I to the open circular Form II upon irradiation. Mechanisms for DNA cleavage by the complex were also investigated.     

Cu(II) Ions and the Stilbene-Chroman Hybrid with a Catechol Moiety Synergistically Induced DNA Damage, and Cell Cycle Arrest and Apoptosis of HepG2 Cells: An Interesting Acid/Base-Promoted Prooxidant Reaction

Development of potential cancer treatment strategies by using an exogenous reactive oxygen species (ROS)-generating agent (prooxidant) or redox intervention, has attracted much interest. One effective ROS generation method is to construct a prooxidant system by polyphenolic compounds and Cu(II) ions. This work demonstrates that Cu(II) and the stilbene-chroman hybrid with a catechol moiety could synergistically induce pBR322 plasmid DNA damage, as well as cell cycle arrest and apoptosis of HepG2 cells. Additionally, an interesting acid/base-promoted prooxidant reaction was found. The detailed chemical mechanisms for the reaction of the hybrid with Cu(II) in acid, neutral and base solutions are proposed based on UV/Vis spectral changes and identification of the related oxidative intermediates and products.     

A copper(II) complex of the Schiff base from l-valine and 2-hydroxy-1-naphthalidene plus 1,10-phenanthroline: synthesis, crystal structure, and DNA interaction

A new Cu(II) complex, [Cu(naph-val)phen] (naph-val = Schiff base derived from 2-hydroxy-1-naphthaldehyde and l-valine, phen = 1,10-phenanthroline), has been synthesized and characterized by physicochemical methods. The crystal structure of the complex showed that there are four independent molecular structures in the crystallographic asymmetric unit, and each of them shows a distorted square-pyramidal CuN₃O₂ coordination geometry. In the crystal, the π − π stacking and intermolecular hydrogen bonds form a 2D network. The interactions between the Cu(II) complex and calf thymus DNA (CT-DNA) have been studied by spectroscopic methods, as well as viscosity and thermal denaturation measurements. The results indicate that the Cu(II) complex binds to CT-DNA in an intercalative mode. The cleavage reaction on pBR322 plasmid DNA has been investigated by agarose gel electrophoresis in the absence and presence of mercaptopropionic acid. The Cu(II) complex exhibits an efficient DNA cleavage activity.     

α-TERTHIENYL PHOTOSENSITIZES DAMAGE TO pBR322 DNA

alpha-Terthienyl photosensitizes single strand breaks in pBR322 DNA. Almost identical results were observed under oxygen and under argon. In the presence of oxygen, this DNA nicking was enhanced by histidine and was not affected by superoxide dismutase, catalase, or the antioxidant BHT. Although chemical damage to DNA treated with alpha-terthienyl plus near-UV was clearly demonstrated in vitro, transformation in E. coli with this damaged pBR322 DNA still took place. Likewise, Haemophilus influenzae DNA transforming activity was not significantly decreased by photosensitization with alpha-terthienyl.     

Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker

This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.     

Novel 13,14‐dimethyl‐5,8‐dioxa‐2,11,13,14‐tetraaza‐1,12‐diphosphabicyclo [10.1.1] tetradecane 1,12‐dioxide ligand and its Ni(II), Co(II), and Cu(II) complexes: Synthesis,characterization, antimicrobial,DNA cleavage,and computational studies

A novel diazadiphosphetidine ligand derived from the reaction of 2,4‐dichloro‐1,3‐dimethyl‐1,3,2,4‐diazadiphosphetidine‐2,4‐dioxide and 2,2′‐(ethane‐1,2‐diylbis[oxy])bis(ethan‐1‐amine) and its Ni(II), Cu(II), and Co(II) complexes have been synthesized, characterized by spectroscopic, elemental analyses, magnetic susceptibility, and conductivity methods, and screened for antimicrobial, DNA binding, and cleavage properties. Spectroscopic analysis and elemental analyses indicate the formula [M(H₂L)Cl₂] for the Cu(II), Co(II), Ni(II), and Zn(II) complexes and octahedral geometry for all the complexes. The non‐electrolytic nature of the complexes in dimethyl sulfoxide (DMSO) was confirmed by their molar conductance values, which are in the range 12.32–6.73 Ω^?1 cm² mol^?1. Computational studies have been carried out at the density functional theory (DFT)‐B3LYP/6‐31G(d) level of theory on the structural and spectroscopic properties of diazadiphosphetidine H₂L and its binuclear Cu(II), Co(II), Ni(II), and Zn(II) complexes. Six tautomers and geometrical isomers of the diazadiphosphetidine ligand were confirmed using semiempirical AM1 and DFT method from DMOL³ calculations. The copper complex had the best antibacterial activity against Staphylococcus aureus (ATCC 29213). DNA cleavage activities of the compounds, evaluated on pBR322 DNA by agarose gel electrophoresis in the presence and absence of an oxidant (H₂O₂) and a free‐radical scavenger (DMSO), indicated no activity for the ligand and moderate activity for the complexes, with the copper complex cleaving pBR322 DNA more efficiently in the presence of H₂O₂.     

Structures and DNA-binding and cleavage properties of ternary copper(II) complexes of glycine with phenanthroline, bipyridine, and bipyridylamine

The crystal structures of the series of three complexes, [Cu(Gly)(bpy)Cl].2H2O (1) (Gly=glycine; bpy=2,2'-bipyridine), [Cu(Gly)(phen)Cl]2.7H2O (2) (phen=1,10-phenanthroline), and [Cu(Gly)(bpa)(H2O)Cl] (3) (bpa=2,2'-bipyridylamine) were determined, and the coordination modes of Cu(II) ternary complexes were compared. The central Cu(II) atoms of complexes 1 and 3 have a similar distorted octahedral coordination geometry, while the Cu(II) atom of complex 2 has a distorted square pyramidal coordination. In all complexes, the aromatic heterocyclic compounds bpy, phen, and bpa, behave as a bidentate N,N' ligand, and Gly behaves as a bidentate N,O ligand. DNA-binding properties of the complexes to calf thymus (CT) DNA were studied by using the fluorescence method. Each of the complexes showed binding propensity to CT DNA with the relative order 2>3> or =1. DNA cleavage studies indicate that each of the complexes, especially 2, can cleave plasmid supercoiled pBR322 DNA in the presence of H2O2 and ascorbic acid with cleavage efficiency in the order 2>3 approximately 1. The degradation of the conformation of CT DNA by the complexes was also reflected in the decrease in the intensities of the characteristic CD bands with the relative order 2>3 approximately 1.     

DNA-binding and photocleavage studies of metallofluorescein–porphyrin complexes of zinc(II) and copper(II)

The DNA-binding behaviors of the fluorescein?Cporphyrinatozinc(II) complex Zn(Fl-PPTPP) (Fl-PPTPP?=?5-(4-fluoresceinpropyloxy)phenyl-10,15,20-triphenylporphyrin) and fluorescein?Cporphyrinatocopper(II) complex Cu(Fl-PPTPP) with calf thymus DNA (CT-DNA) were investigated by UV?CVis absorption titrations, fluorescence spectra, viscosity measurements, thermal denaturation and circular dichroism. The results suggest that both complexes interact with CT-DNA by intercalation. In addition, their photocleavage reactions with pBR322 supercoiled plasmid DNA were investigated. Both complexes exhibit significant DNA cleavage activity, and singlet oxygen may play an important role in these reactions.     

Spectroscopy: the study of DNA cleavage by newly synthesized polydentate macrocyclic ligand and its copper(II) complexes

A novel hexadentate nitrogen donor [N6] macrocyclic ligand, i.e. 2,6,12,16,21,22-hexaaza-3,5,13,15-tetramethyl-4,14-diethyl-tricyclo-[15.3.1.1(7-11)]docosane-1(21),2,5,7(22),8,10,12,15,17,19-decaene (L), has been synthesized. Copper(II) complexes with this ligand have been prepared and subjected to elemental analyses, molar conductance measurements, magnetic susceptibility measurements, mass, 1H NMR (ligand), IR, electronic, and EPR spectral studies. On the basis of molar conductance the complexes may be formulated as [Cu(L)X2] [X = Cl(-), Br(-), NO3(-) and CH3COO(-)] due to their nonelectrolytic nature in N,N'-dimethylformamide (DMF). All the complexes are of the high spin type and are six coordinated. On the basis of IR, electronic and EPR spectral studies tetragonal geometry has been assigned to the Cu(II) complexes. The interaction of these complexes with calf thymus DNA has been explored by using absorption, emission, viscosity measurements, electrochemical studies and DNA cleavage. All the experimental results suggest that the complexes bind to DNA and also promote the cleavage plasmid pBR 322, in the presence of H2O2 and ascorbic acid.