Photo-induced DNA Cleavage Research Articles

Photoinduced DNA Cleavage and Cellular Damage in Human Dermal Fibroblasts by 2,3-Diaminophenazine¶

Aromatic amines, such as o-phenylenediamine (OPD), have been used extensively in commercial hair dyes and in the synthesis of agricultural pesticides. Air oxidation of OPD results in the formation of 2,3-diaminophenazine (DAP). Although the mutagenic toxicity of DAP has been shown in both prokaryotic and eukaryotic systems, its phototoxicity remains largely unexplored. This study focuses on the pH-dependent photophysical properties of DAP and demonstrates its ability to photoinduce DNA damage to pUC19 plasmid in vitro. The photocytotoxicity of DAP toward human skin fibroblasts was also measured. DAP exhibits weak intercalative binding to double-stranded DNA with a binding constant Kb = 3.5 × 103 M−1. Furthermore, upon irradiation with visible light, DAP is able to nick plasmid DNA in the presence of oxygen. The concentration of DAP that resulted in 50% cell death was 172 ± 9 μM in the dark and 13 ± 1 μM after irradiation of the DAP-treated cell cultures with visible light (400–700 nm, 30 min, 5 J/cm2). The 13-fold increase in toxicity upon exposure to visible light shows the need for further study of the photocytotoxicity of contaminants such as DAP.

Photoinduced DNA Cleavage and Cellular Damage in Human Dermal Fibroblasts by 2,3‐Diaminophenazine¶

ABSTRACTAromatic amines, such as o‐phenylenediamine (OPD), have been used extensively in commercial hair dyes and in the synthesis of agricultural pesticides. Air oxidation of OPD results in the formation of 2,3‐diaminophenazine (DAP). Although the mutagenic toxicity of DAP has been shown in both prokaryotic and eukaryotic systems, its phototoxicity remains largely unexplored. This study focuses on the pH‐dependent photophysical properties of DAP and demonstrates its ability to photoinduce DNA damage to pUC19 plasmid in vitro. The photocytotoxicity of DAP toward human skin fibroblasts was also measured. DAP exhibits weak intercalative binding to double‐stranded DNA with a binding constant Kb= 3.5 × 103M−1. Furthermore, upon irradiation with visible light, DAP is able to nick plasmid DNA in the presence of oxygen. The concentration of DAP that resulted in 50% cell death was 172 ± 9 μM in the dark and 13 ± 1 μM after irradiation of the DAP‐treated cell cultures with visible light (400–700 nm, 30 min, 5 J/cm2). The 13‐fold increase in toxicity upon exposure to visible light shows the need for further study of the photocytotoxicity of contaminants such as DAP.

Metal-assisted red light-induced DNA cleavage by ternary l-methionine copper(ii) complexes of planar heterocyclic bases

Ternary copper(II) complexes [Cu(l-met)B(Solv)](ClO4) (1-4), where B is a N,N-donor heterocyclic base like 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3) and dipyrido[3,2-a:2'],3'-c]phenazene (dppz, 4), are prepared and their DNA binding and photo-induced DNA cleavage activity studied (L-Hmet =L-methionine). Complex 2, structurally characterized by X-ray crystallography, shows a square pyramidal (4 + 1) coordination geometry in which the N,O-donor L-methionine and N,N-donor heterocyclic base bind at the basal plane and a solvent molecule is coordinated at the axial site. The complexes display a d-d band at approximately 600 nm in DMF and exhibit a cyclic voltammetric response due to the Cu(II)/Cu(I) couple near -0.1 V in DMF-Tris-HCl buffer. The complexes display significant binding propensity to the calf thymus DNA in the order: 4(dppz) > 3(dpq) > 2(phen>> 1(bpy). Control cleavage experiments using pUC19 supercoiled DNA and distamycin suggest major groove binding for the dppz and minor groove binding for the other complexes. Complexes 2-4 show efficient DNA cleavage activity on UV (365 nm) or red light (632.8 nm) irradiation via a mechanistic pathway involving formation of singlet oxygen as the reactive species. The DNA cleavage activity of the dpq complex is found to be significantly more than its dppz and phen analogues.

Red-light photosensitized cleavage of DNA by (l-lysine)(phenanthroline base)copper(ii) complexes

Ternary copper(II) complexes [Cu(l-lys)B(ClO4)](ClO4)(1-4), where B is a heterocyclic base, viz. 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3) and dipyrido[3,2-a:2',3'-c]phenazene (dppz, 4), are prepared and their DNA binding and photo-induced DNA cleavage activity studied (l-lys =l-lysine). Complex 2, structurally characterized by X-ray crystallography, shows a square-pyramidal (4 + 1) coordination geometry in which the N,O-donor l-lysine and N,N-donor heterocyclic base bind at the basal plane and the perchlorate ligand is bonded at the elongated axial site. The crystal structure shows the presence of a pendant cationic amine moiety -(CH2)4NH3+ of l-lysine. The one-electron paramagnetic complexes display a d-d band in the range of 598-762 nm in DMF and exhibit cyclic voltammetric response due to Cu(II)/Cu(I) couple in the range of 0.07 to -0.20 V vs. SCE in DMF-Tris-HCl buffer. The complexes having phenanthroline bases display good binding propensity to the calf thymus DNA giving an order: 4 (dppz) > 3 (dpq) > 2 (phen)>> 1 (bpy). Control cleavage experiments using pUC19 supercoiled DNA and distamycin suggest major groove binding for the dppz and minor groove binding for the other complexes. Complexes 2-4 show efficient DNA cleavage activity on UV (365 nm) or visible light (694 nm ruby laser) irradiation via a mechanistic pathway involving formation of singlet oxygen as the reactive species. The amino acid l-lysine bound to the metal shows photosensitizing effect at red light, while the heterocyclic bases are primarily DNA groove binders. The dpq and dppz ligands display red light-induced photosensitizing effects in copper-bound form.

Ternary iron(ii) complex with an emissive imidazopyridine arm from Schiff base cyclizations and its oxidative DNA cleavage activity

The ternary iron(II) complex [Fe(L')(L")](PF6)3(1) as a synthetic model for the bleomycins, where L' and L" are formed from metal-mediated cyclizations of N,N'-(2-hydroxypropane-1,3-diyl)bis(pyridine-2-aldimine)(L), is synthesized and structurally characterized by X-ray crystallography. In the six-coordinate iron(ii) complex, ligands L' and L" show tetradentate and bidentate chelating modes of bonding. Ligand L' is formed from an intramolecular attack of the alcoholic OH group of L to one imine moiety leading to the formation of a stereochemically constrained five-membered ring. Ligand L" which is formed from an intermolecular reaction involving one imine moiety of L and pyridine-2-carbaldehyde has an emissive cationic imidazopyridine pendant arm. The complex binds to double-stranded DNA in the minor groove giving a Kapp value of 4.1 x 10(5) M(-1) and displays oxidative cleavage of supercoiled DNA in the presence of H2O2 following a hydroxyl radical pathway. The complex also shows photo-induced DNA cleavage activity on UV light exposure involving formation of singlet oxygen as the reactive species.

Photosensitisation and photoinduced DNA cleavage by four naturally occurring anthraquinones

Photosensitising properties of four naturally occurring anthraquinones, viz., erythroglaucin (ERG), teloschistin (TEL), 1-hydroxy-2-methylanthraquinone (HYQ) and 1-methoxy-2-hydroxyanthraquinone (MEQ) are studied. Photogeneration of singlet oxygen was monitored by both optical RNO (para-nitrosodimethylaniline) bleaching and electron paramagnetic resonance spectroscopy (EPR)-2,2,6,6-tetramethyl piperidinol (TEMPL) methods. In comparison with rose bengal (RB), singlet oxygen generating efficiencies of ERG, TEL, HYQ and MEQ are determined to be 0.10, 0.14, 0.02 and 0.13, respectively. The formation of O2− was monitored by optical spectroscopy using SOD inhibitable cytochrome c reduction assay. Photolysis of ERG and MEQ in DMSO, in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) generated a multiline EPR spectrum characteristic of mixture of O2−, OH and OCH3 adducts. Both electron transfer (Type I) and energy transfer (Type II) paths are involved in the photosensitisation of ERG, TEL, HYQ and MEQ. Quantum efficiencies of the quinones exhibit correlation with their half-wave potentials. The photoinduced DNA cleavage by quinones was examined with covalently closed circular plasmid DNA, in vitro. Observed DNA photo strand scission correlates with the reactive oxygen species (ROS) generating efficiencies of the quinones.

Porphyrin?DNA cross-linking agent hybrids: chemical synthesis and biological studies

Three new porphyrin–DNA cross-linking conjugates 8, 9, and 10 have been synthesized. Their photoinduced DNA cleavage activity have been studied. The IC50 values to THP-1 cells in the presence of porphyrin derivatives 8, 9, and 10 with photoirradiation were 5.6, 88.4, and 61.8 nM, respectively.

Porphyrin–DNA cross-linking agent hybrids: chemical synthesis and biological studies

Three new porphyrin–DNA cross-linking conjugates 8, 9, and 10 have been synthesized. Their photoinduced DNA cleavage activity have been studied. The IC 50 values to THP-1 cells in the presence of porphyrin derivatives 8, 9, and 10 with photoirradiation were 5.6, 88.4, and 61.8 nM, respectively.

Aminyl and iminyl radicals from arylhydrazones in the photo-induced DNA cleavage

Photolytic cleavage of the nitrogen–nitrogen single bond in benzaldehyde phenylhydrazones produced aminyl (R 2N ) and iminyl (R 2CN ) radicals. This photochemical property was utilized in the development of hydrazones as photo-induced DNA-cleaving agents. Irradiation with 350 nm UV light of arylhydrazones bearing substituents of various types in a phosphate buffer solution containing the supercoiled circular φX174 RFI DNA at pH 6.0 resulted in single-strand cleavage of DNA. Attachment of the electron-donating OMe group to arylhydrazones increased their DNA-cleaving activity. Results from systematic studies indicate that both the aminyl and the iminyl radicals possessed DNA-cleaving ability.

Different modes of DNA cleavage activity of dihydroxo-bridged dicopper(II) complexes having phenanthroline bases

Dihydroxo-bridged dicopper(II) complexes [{Cu(phen)} 2(μ-OH) 2](ClO 4) 2 ( 1), [{Cu(dpq)} 2(μ-OH) 2](ClO 4) 2 ( 2) and [{Cu(dppz)(DMF)} 2(μ-OH) 2](PF 6) 2 ( 3), where phen, dpq and dppz are 1,10-phenanthroline, dipyridoquinoxaline and dipyridophenazine, respectively, are prepared and their DNA binding and cleavage properties studied. Complex 3 has been structurally characterized by X-ray crystallography. The complexes have a {Cu 2(μ-OH) 2} 2+ core with an essentially planar arrangement of two CuN 2O 2 basal planes. The complexes are avid binder to calf thymus DNA ( K app value of 4.8 × 10 6 and 5.9 × 10 6 M −1 for 2 and 3, respectively, from ethidium displacement assay) and exhibits significant cleavage of supercoiled (SC) pUC19 DNA in dark in presence of mercaptopropionic acid. Besides, the dpq and dppz complexes display photo-induced DNA cleavage on UV (312 nm) and red light (632.8 nm) irradiations in absence of any additives. Mechanistic investigations reveal minor groove binding for the phen and dpq complexes, and major groove preference for the dppz species. The oxidative DNA cleavage reactions in presence of mercaptopropionic acid as a reducing agent involve hydroxyl radicals. The photo-cleavage reactions at UV light involve singlet oxygen as the reactive species, while similar reactions on red light irradiation (632.8 nm) proceed through the formation of hydroxyl radical. The complexes show significant DNA hydrolase activity in absence of any additives under dark reaction conditions.

Synthesis, crystal structure and photo-induced DNA cleavage activity of ternary copper(II)-thiosemicarbazone complexes having heterocyclic bases

New ternary copper(II) complexes of formulations [Cu(Ph-tsc)B] (B=1,10-phenanthroline, phen ( 1); dipyridoquinoxaline, dpq ( 2); dipyridophenazine, dppz ( 3); Ph-H 2tsc, salicylaldehyde- N(4)-phenylthiosemicarbazone) and [Cu(Me-tsc)(phen)] ( 4, Me-H 2tsc, salicylaldehyde- N(4)-methylthiosemicarbazone) are prepared, and their DNA binding and cleavage properties studied. Complex 1 has been characterized by single crystal X-ray crystallography. The molecular structure shows a distorted square pyramidal (4 + 1) geometry of the complex with the dianionic NSO-donor N(4)-phenyl-substituted thiosemicarbazone binding at the basal plane and the NN-donor planar heterocyclic base (phen) displaying axial–equatorial coordination. The one-electron paramagnetic complexes exhibit axial EPR spectra and show a d–d band near 580 nm for the phen and near 720 nm for the dpq, dppz complexes in their electronic spectra in DMF. The complexes show quasireversible cyclic voltammetric response near 0.08 V vs. SCE in DMF–0.1 M TBAP assignable to the Cu(II)/Cu(I) couple. The Ph-tsc complexes display good binding propensity to calf thymus (CT) DNA. They also show oxidative cleavage of supercoiled (SC) pUC19 DNA in dark under aerobic condition in the presence of mercaptopropionic acid. The complexes exhibit light-induced DNA cleavage activity at 312 and 532 nm. Mechanistic investigations reveal DNA minor groove binding for the phen and dpq complexes, and major groove binding for the dppz species. The complexes are cleavage inactive under argon atmosphere. In the ternary structure, the thiosemicarbazones, dpq and dppz act as photosensitizers, while the planar heterocyclic bases are binder to DNA. The mechanistic pathways involved and the role of metal in the DNA cleavage reactions are discussed.

Photoinduced DNA Cleavage and Cellular Damage in Human Dermal Fibroblasts by 2,3-Diaminophenazine

Aromatic amines, such as o-phenylenediamine (OPD), have been used extensively in commercial hair dyes and in the synthesis of agricultural pesticides. Air oxidation of OPD results in the formation of 2,3-diaminophenazine (DAP). Although the mutagenic toxicity of DAP has been shown in both prokaryotic and eukaryotic systems, its phototoxicity remains largely unexplored. This study focuses on the pH-dependent photophysical properties of DAP and demonstrates its ability to photoinduce DNA damage to pUC19 plasmid in vitro. The photocytotoxicity of DAP toward human skin fibroblasts was also measured. DAP exhibits weak intercalative binding to double-stranded DNA with a binding constant K(b) = 3.5 x 10(3) M(-1). Furthermore, upon irradiation with visible light, DAP is able to nick plasmid DNA in the presence of oxygen. The concentration of DAP that resulted in 50% cell death was 172 +/- 9 microM in the dark and 13 +/- 1 microM after irradiation of the DAP-treated cell cultures with visible light (400-700 nm, 30 min, 5 J/cm(2)). The 13-fold increase in toxicity upon exposure to visible light shows the need for further study of the photocytotoxicity of contaminants such as DAP.

Photo-Induced DNA cleavage reaction characteristics of propargylic sulfones possessing anthraquinone chromophore

DNA cleavage potency of propargylic sulfones possessing anthraquinone chromophore 1 under UV-irradiation was evaluated in comparison with the dark reaction. 1 showed inefficient DNA cleavage activity, while having considerably strong DNA binding ability. This result is accounted for by spatial conditions that the activated alkylating allenic site of intercalated 1 could not effectively approach to DNA bases, most probably guanine moiety, and thereby led to insufficient DNA strand cleavage. In contrast, the DNA cleavage activity of 1 was notably enhanced upon UV-irradiation (λ ex=365 nm) followed by incubation. Under UV-irradiation, further DNA cleavage were occurred primary at 5′-G of GG steps within DNA. A DNA cleavage mechanism for 1, by which photo-induced one-electron oxidation of 5′-G of GG steps may occur along with ordinary alkylation, has been proposed.

Active oxygen species generated from photoexcited fullerene (C60) as potential medicines: O2-* versus 1O2.

To characterize fullerenes (C(60) and C(70)) as photosensitizers in biological systems, the generation of active oxygen species, through energy transfer (singlet oxygen (1)O(2)) and electron transfer (reduced active oxygen radicals such as superoxide anion radical O(2)(-)* and hydroxyl radical *OH), was studied by a combination of methods, including biochemical (DNA-cleavage assay in the presence of various scavengers of active oxygen species), physicochemical (EPR radical trapping and near-infrared spectrometry), and chemical methods (nitro blue tetrazolium (NBT) method). Whereas (1)O(2) was generated effectively by photoexcited C(60) in nonpolar solvents such as benzene and benzonitrile, we found that O(2)(-)* and *OH were produced instead of (1)O(2) in polar solvents such as water, especially in the presence of a physiological concentration of reductants including NADH. The above results, together with those of a DNA cleavage assay in the presence of various scavengers of specific active oxygen species, indicate that the active oxygen species primarily responsible for photoinduced DNA cleavage by C(60) under physiological conditions are reduced species such as O(2)(-)* and *OH.

Photo-induced DNA cleavage reaction characteristics of propargylic sulfones possessing an anthraquinone chromophore.

Propargylic sulfones (PS) are known as pH dependent DNA cleaver. DNA cleavage by PS was considered to proceed by alkylation of G base to allenic sulfones formed from PS in basic condition. We designed PS posessing naphthalene and anthraquinone (AQ) unit and investigated DNA cleavage characteristics. Although these compounds showed high intercalating abilities, this high intercalating ability did not lead to DNA cleaving activity. This result indicates that spatial arrangement of activated allene against guanine base is very important in DNA cleavage by PS. In addition, UV-irradiation to PS possessing AQ unit leads to efficient DNA cleavage at 5'-G of GG sequence. This cleavage pattern exhibited typical cleavage of one-electron oxidation of B-form DNA. Therefore, this result suggests that PS possessing AQ unit cleave DNA by both the alkylation mechanism and the photooxidaization mechanism.

Photoinduced cleavage of DNA by bromofluoroacetophenone–pyrrolecarboxamide conjugates

Bromofluoroacetophenone derivatives which produce fluorine substituted phenyl radicals that cleave DNA upon excitation were investigated as a novel photonuclease. Pyrrolecarboxamide-conjugated bromofluoroacetophenones; 4′-bromo-2′-fluoroacetophenone and 2′-bromo-4′-fluoroacetophenone were synthesized and their DNA cleaving activities and sequence selectivities were determined. Bromofluoroacetophenone–pyrrolecarboxamide conjugates were found to be effective DNA cleaving agents upon irradiation in concentration dependent manner based on plasma relaxation assay. The DNA cleaving activities of 2′-bromo-4′-fluoroacetophenone derivatives were larger than those of 4′-bromo-2′-fluoroacetophenone derivatives.

Photo-induced DNA cleavage in self-assembly multilayer films

Electrostatic self-assembly multilayer films have been successfully fabricated from a C60–ethylenediamine adduct (C60–EDA), and DNA. Under visible light irradiation, the DNA is cleaved and the films are destroyed. The photo-induced reaction follows a direct electron-transfer mechanism. A high oxygen concentration in the solution enhances the reaction, while separation of C60–EDA and DNA retards the reaction dramatically.

Photoinduced DNA cleavage by cyclopentadienyl metal complexes conjugated to DNA recognition elements.

[structure: see text]. DNA recognition elements have been attached to CpW(CO)3CH3 and CpW(CO)3Ph, which produce methyl and phenyl radicals that cleave DNA upon photolysis. The inclusion of binding moieties in 3 increases the efficiency but not the selectivity of strand scission over that seen in the simple unfunctionalized complex, while 11 cleaves preferentially at T sites within AT-rich tracts.

Solution properties and photonuclease activity of cationic bis-porphyrins linked with a series of aliphatic diamines.

The development of efficient photo-induced DNA cleavage agents has been of particular interest for biomedical applications such as cancer photodynamic therapy (PDT). Toward this objective, we synthesized a series of cationic bis-porphyrins with various lengths of diamino alkyl linkage, N,N'-bis[4-[10,15,20-tris(1-methylpyridinium-4-yl)porphyrin-5-yl]benzoyl]oligomethylenediamine hexaiodide. They were expected to show more efficient photocleavage of DNA than unichromophore meso-tetrakis(4-N-methylpyridiniumyl)porphine (TMPyP), which is well known to cleave DNA effectively on illumination. The cationic bis-porphyrins were found to self-aggregate in aqueous solution, and the aggregation property was accounted for by the formation of an intermolecular dimer. Because conservative-type circular dichroism spectra of the bis-porphyrins were induced in the Soret region on binding to calf thymus DNA, we assigned their binding mode to outside self-stacking on the DNA surface. Their photonuclease activity using plasmid DNA decreased as the number of their linker hydrocarbons increased, and was well correlated with their tendency for dimerization. The inhibitory effect of azide anion, N3-, and the enhancement effect of D2O suggest that singlet oxygen was probably involved in the photocleavage of DNA.

Photoinduced cleavage of plasmid DNA in the presence of pterin

Summary The photoinduced cleavage of plasmid DNA by UV -A light in the presence of pterin was investigated. Electrophoretic analysis of the irradiated plasmid pUCI8 in the presence of pterin showed that UV light of 350 nm induced the transformation of a significant proportion of the supercoiled plasmid to its relaxed form. A minor proportion of plasmid forms are also converted to the linear plasmid isomer at the longer irradiation times. All these transformations during irradiation can be observed in the absorption spectrum of DNA as function of time. Such spectral modifications correlated with the extent and the kinetics of plasmid relaxation, but not with the appearance of the linear plasmid. None of the exchanges were operative without the irradiation with UV-A light. Control experiments with pterin or plasmid DNA irradiated separately, showed no photochemical changes. Results taken together suggest that the observed changes in the supercoiled plasmid as well as the spectral modifications both derive from the generation of single-strand break in the DNA.