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.     

Effect of polyelectrolytes on aggregation of a cyanine dye

The effect of poly(acrylic acid) and poly(methacrylic acid) on the molecular state of a cationic cyanine dye (3,3′-diethyl-2,2′-thiacarbocyanine iodide) was studied. Gels based on the above polymers were shown to efficiently absorb an oppositely charged dye. The absorption of dye ions by a gel induced their aggregation. It was shown that, in the acrylic acid-based gels, H-aggregates, dimers, and single ions of the dye coexist. In gels containing methacrylic acid units, dye ions mainly formed H-aggregates. A comparison of aggregation of dye ions in the gels with that in aqueous solutions of corresponding polyelectrolytes was performed. When dye ions were immobilized in a gel network, the proportion of the ions forming the H-aggregates was considerably larger than that in solutions. The effect of the gel network charge density on the aggregation of dye ions was investigated. It was shown that an increase in the fraction of charged units in network chains facilitates aggregation.     

Photohydrolysis of methotrexate produces pteridine,which induces poly-G-specific DNA damage through photoinduced electron transfer

Methotrexate (MTX), an antineoplastic agent, demonstrates phototoxicity. The mechanism of damage to biomacromolecules induced by photoirradiated MTX was examined using 32P-labeled DNA fragments obtained from a human gene. Photoirradiated MTX caused DNA cleavage specifically at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA only when the DNA fragments were treated with piperidine, which suggests that DNA cleavage was caused by base modification with little or no strand breakage. With denatured single-stranded DNA the damage occurred at most guanine residues. The amount of formation of 8-hydroxy-2'-deoxyguanosine (8-oxodGuo), an oxidative product of 2'-deoxyguanosine, in double-stranded DNA exceeded that in single-stranded DNA. These results suggest that photoirradiated MTX participates in 8-oxodGuo formation at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA through electron transfer, and then 8-oxodGuo undergoes further oxidation into piperidine-labile products. Fluorescence measurement, high-pressure liquid chromatography and mass spectrometry have demonstrated that photoexcited MTX is hydrolyzed into 2,4-diamino-6-(hydroxymethyl)pteridine (DHP). DNA damage induced by DHP was observed in a similar manner as was the damage induced by MTX. The extent of DNA damage and the formation of 8-oxodGuo by DHP were much larger than those induced by MTX. The kinetic analysis, based on the time course of DNA oxidation by photoirradiated MTX, suggests that DNA damage is caused by photoexcited DHP rather than by photoexcited MTX. In conclusion, photoexcited MTX undergoes hydrolysis through intramolecular electron transfer, resulting in the formation of DHP, which exhibits a phototoxic effect caused by oxidation of biomacromolecules through photoinduced electron transfer.     

Comparison of DNA damage photoinduced by ketoprofen, fenofibric acid and benzophenone via electron and energy transfer.

Ketoprofen (KP) and fenofibrate, respectively, anti-inflammatory and hypolipidemiant agents, promote anormal photosensitivity in patients and may induce photoallergic cross-reactions correlated to their benzophenone-like structure. Here, their ability to photosensitize the degradation of biological targets was particularly investigated in DNA. The photosensitization of DNA damage by KP and fenofibric acid (FB), the main metabolite of fenofibrate, and their parent compound, benzophenone (BZ), was examined on a 32P-end-labeled synthetic oligonucleotide in phosphate-buffered solution using gel sequencing experiments. Upon irradiation at lambda > 320 nm, piperidine-sensitive lesions were induced in single-stranded oligonucleotides by KP, FB and BZ at all G sites to the same extent. This pattern of damage, enhanced in D2O is characteristic of a Type-II mechanism. Spin trapping experiments using 2,2,6,6-tetramethyl-4-piperidone have confirmed the production of singlet oxygen during drug photolysis. On double-stranded oligonucleotides, highly specific DNA break occurred selectively at 5'-G of a 5'-GG-3' sequence, after alkali treatment. Prolonged irradiation led to the degradation of all G residues, with efficiency decreasing in the order 5'-GG > 5'-GA > 5'-GC > 5'-GT, in good agreement with the calculated lowest ionization potentials of stacked nucleobase models supporting the assumption of a Type-I mechanism involving electron transfer, also observed to a lesser extent with adenine. Cytosine sites were also affected but the action of mannitol which selectively inhibited cytosine lesions suggests, in this case, the involvement of hydroxyl radical, also detected by electronic paramagnetic resonance using 5,5-dimethyl-1-pyrrolidine-1-oxide as spin trap. On a double-stranded 32P-end-labeled 25-mer oligonucleotide containing TT and TTT sequences, the three compounds were found to photosensitize by triplet-triplet energy transfer the formation of cyclobutane thymine dimers detected using T4 endonuclease V.     

Induced and photoinduced DNA damage by quinolones: ciprofloxacin, ofloxacin and nalidixic acid determined by comet assay

Quinolones are degraded by light with loss of their antimicrobial activity, generating active species or radicals. Evidence exists that some fluoroquinolones (lomefloxacin, fleroxacin and enoxacin) induce damage to the cellular membrane and DNA cleavage by photosensitization. In this study, the genotoxic potential of the quinolones ofloxacin, nalidixic acid and ciprofloxacin (three antimicrobials frequently used in therapy) was evaluated upon irradiation with UV light by using the comet assay on cells of the Jurkat line. The results demonstrate that there are significant differences between the control groups (positive control with 50 microM H2O2, negative controls without drug and with and without irradiation) and the groups of irradiated quinolones (ofloxacin 2.76 x 10(-5) M, nalidixic acid 2.15 x 10(-4) M and ciprofloxacin 2.01 x 10(-5) M), indicating that, at the dose of irradiation employed (necessary to produce 50% photodegradation), the photodecomposition of the quinolones enhanced DNA damage. The unirradiated drugs also exhibited genotoxicity significantly different from that of the negative control.     

Characterization of photoinduced isomerization and intersystem crossing of the cyanine dye Cy3

Several important photophysical properties of the cyanine dye Cy3 have been determined by laser flash photolysis. The triplet-state absorption and photoisomerization of Cy3 are distinguished by using the heavy-atom effects and oxygen-induced triplet --> triplet energy transfer. Furthermore, the triplet-state extinction coefficient and quantum yield of Cy3 are also measured via triplet-triplet energy-transfer method and comparative actinometry, respectively. It is found that the triplet --> triplet (T1-->Tn) absorptions of trans-Cy3 largely overlap the ground-state absorption of cis-Cy3. Unlike what occurred in Cy5, we have not observed the triplet-state T1-->Tn absorption of cis-Cy3 and the phosphorescence from triplet state of cis-Cy3 following a singlet excitation (S0-S1) of trans-Cy3, indicating the absence of a lowest cis-triplet state as an isomerization intermediate upon excitation in Cy3. The detailed spectra of Cy3 reported in this paper could help us interpret the complicated photophysics of cyanine dyes.     

Group VA effects on surfactant aggregation number and dye solubilization efficiency

The effects of changing the charge-bearing atom in the polar head of a surfactant ion on micelle size and dye solubilization efficiency were investigated. Replacement of nitrogen in decyltrimethylammonium bromide by phosphorus or arsenic increases the micellar aggregation number by at least 20% in 0.5 m NaBr solutions. The ability of the surfactants to solubilize the dye Orange-OT is only slightly affected by the substitution of phosphorus for nitrogen, but it is lowered appreciably by the substitution of arsenic.     

Spacer-controlled aggregation and surface morphology of a selenacarbocyanine dye on gemini monolayers

The adsorption and aggregation of a selenacarbocyanine dye (3,3-disulfopropyl-9-methyl-selenacarbocyanine, SeCy) onto Langmuir monolayers of a series of gemini amphiphiles with different methylene spacers were investigated. When the monolayers of the gemini amphiphiles were spread on the dye-containing subphase, the dye could be easily adsorbed and aggregated onto the monolayers through the electrostatic and pi-pi interaction. The dye formed complexes with gemini amphiphiles and stacked as J-aggregates in all the transferred multilayers, regardless of the structure of gemini amphiphiles. However, the surface morphologies of the complex monolayers showed a significant dependence on the spacer length of the gemini amphiphiles and the temperature of the subphase. Nanorods were observed for the complex films with spacer lengths ranged from 4 to 10 methylenes. With the temperature of the subphases increased from 20 degrees C to 30 degrees C, aligned longer nanofibers were formed instead. Although both the gemini amphiphiles and the dye were achiral, strong circular dichroism (CD) signals were observed for the transferred complex films. However, the CD signals could be just opposite in different places of the transferred films, suggesting that a resolved enantiomeric micro/nanostructure coexisted in the films. On the other hand, for the complex film of the dye with the gemini amphiphile of two methylenes spacer, neither CD signal nor ordered surface morphologies were detected in any place of the film although the dye itself stilled formed J-aggregate in the film. It was suggested that regular nanoarchitectures and resolved chiral domains could be observed only when the spacer of gemini amphiphiles is comparative to the distance between the two SO3- groups in the dye.     

Effect of UVC-induced damage to DNA on the intercalation of thiazole orange: a convenient reporter for DNA damage

We report a novel method of identifying damage to DNA leading to the loss of intercalation sites. Thiazole orange (TO), an intercalating cyanine dye, fluoresces strongly when intercalated in DNA, but not free in solution. Upon UVC-induced damage to DNA, the change in TO fluorescence is greater than the change in any of the other spectral or biochemical indicators (absorbance, circular dichroism and agarose gel electrophoresis), thus providing a fast screening method to identify damage to DNA. The method is geared toward high levels of damage, such as those that may result during radiation treatment of food products.     

Effect of nanointegration on photoinduced hydrogen-generating nanogel systems

The nanointegration mechanism for photoinduced hydrogen nanogenerators using nanogels is described. By spatially integrating poly(N-isopropylacrylamide-co-Ru(bpy)(3)) nanogels as a photosensitizer and Pt nanoparticles as a catalyst, a mechanism using electrostatic interactions and the shrinking behavior of the thermosensitive polymer network is revealed. In addition, to evaluate the sensitivity to exterior energy, light, and heat, the integrated nanospace is controlled by using thermosensitive nanogels, which drastically shrink above the volume phase transition temperature. Such nanospatial control of multiple kinds of functional molecules in a photochemical reaction is important for the realization of artificial photosynthetic systems.     

Neutral histidine and photoinduced electron transfer in DNA photolyases

The two major UV-induced DNA lesions, the cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts, can be repaired by the light-activated enzymes CPD and (6-4) photolyases, respectively. It is a long-standing question how the two classes of photolyases with alike molecular structure are capable of reversing the two chemically different DNA photoproducts. In both photolyases the repair reaction is initiated by photoinduced electron transfer from the hydroquinone-anion part of the flavin adenine dinucleotide (FADH(-)) cofactor to the photoproduct. Here, the state-of-the-art XMCQDPT2-CASSCF approach was employed to compute the excitation spectra of the respective active site models. It is found that protonation of His365 in the presence of the hydroquinone-anion electron donor causes spontaneous, as opposed to photoinduced, coupled proton and electron transfer to the (6-4) photoproduct. The resulting neutralized biradical, containing the neutral semiquinone and the N3'-protonated (6-4) photoproduct neutral radical, corresponds to the lowest energy electronic ground-state minimum. The high electron affinity of the N3'-protonated (6-4) photoproduct underlines this finding. Thus, it is anticipated that the (6-4) photoproduct repair is assisted by His365 in its neutral form, which is in contrast to the repair mechanisms proposed in the literature. The repair via hydroxyl group transfer assisted by neutral His365 is considered. The repair involves the 5'base radical anion of the (6-4) photoproduct which in terms of electronic structure is similar to the CPD radical anion. A unified model of the CPD and (6-4) photoproduct repair is proposed.     

Ultrafast photoinduced relaxation dynamics of the indoline dye D149 in organic solvents

The relaxation dynamics of the indoline dye D149, a well-known sensitizer for photoelectrochemical solar cells, have been extensively characterized in various organic solvents by combining results from ultrafast pump-supercontinuum probe (PSCP) spectroscopy, transient UV-pump VIS-probe spectroscopy, time-correlated single-photon counting (TCSPC) measurements as well as steady-state absorption and fluorescence. In the steady-state spectra, the position of the absorption maximum shows only a weak solvent dependence, whereas the fluorescence Stokes shift Δν?(F) correlates with solvent polarity. Photoexcitation at around 480 nm provides access to the S(1) state of D149 which exhibits solvation dynamics on characteristic timescales, as monitored by a red-shift of the stimulated emission and spectral development of the excited-state absorption in the transient PSCP spectra. In all cases, the spectral dynamics can be modeled by a global kinetic analysis using a time-dependent S(1) spectrum. The lifetime τ(1) of the S(1) state roughly correlates with polarity [acetonitrile (280 ps) < acetone (540 ps) < THF (720 ps) < chloroform (800 ps)], yet in alcohols it is much shorter [methanol (99 ps) < ethanol (178 ps) < acetonitrile (280 ps)], suggesting an appreciable influence of hydrogen bonding on the dynamics. A minor component with a characteristic time constant in the range 19-30 ps, readily observed in the PSCP spectra of D149 in acetonitrile and THF, is likely due to removal of vibrational excess energy from the S(1) state by collisions with solvent molecules. Additional weak fluorescence in the range 390-500 nm is observed upon excitation in the S(0)→S(2) band, which contains short-lived S(2)→S(0) emission of D149. Transient absorption signals after excitation at 377.5 nm yield an additional time constant in the subpicosecond range, representing the lifetime of the S(2) state. S(2) excitation also produces photoproducts.     

Complexes of polyelectrolyte hydrogels with organic dyes: Effect of charge density on the complex stability and intragel dye aggregation

The swelling behavior of polyelectrolyte gels based on poly(diallyldimethylammonium chloride) (copolymers of diallyldimethylammonium chloride and acrylamide with the variable composition) and poly(methacrylic acid, sodium salt) in the presence of organic water soluble dyes (alizarin, naphthol blue black, rhodamine) was studied. The collapse of the polyelectrolyte gels in the presence of oppositely charged dyes together with the effective absorption of dyes was observed. The shrinking degree and the dye absorption by the gel depend on the charges of the polymer network and the dye, and also on the dye concentration. Stability of the gel–dye complexes in a salt solution of NaCl and Al₂(SO₄)₃ was studied. It was shown that the complex stability in the salt solution depends on the charge density of the polymer chains forming the gel. The increase of charge density of polymer generally leads to the enhancement of the complex stability. For the systems with the fraction of charged poly(diallyldimethylammonium chloride) monomer units above 0.5 the release of alizarin to the external solution of Al₂(SO₄)₃ reservoir is practically completely suppressed. The obtained results show that oppositely charged dyes are generally from stable complexes with polyelectrolyte gels. © 1999 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 37: 1209–1217, 1999     

Selective surface-enhanced fluorescence and dye aggregation with layer-by-layer film substrates

One of the avenues for the development of the analytical applications of surface enhanced spectroscopy is the engineering of enhancing substrates that would be selective and target specific. In the present report, the proof of this concept is demonstrated using the layer-by-layer (LbL) technique to fabricate portable selective substrates containing metal nanoparticles which can provide surface enhanced fluorescence (SEF) or surface enhanced Raman scattering (SERS). The selectivity to ionic species is attained by adding metal-free top layers of polymer electrolytes to an LbL SERS enhancing substrate. In addition, it was observed that the surface charge of the top layer determines the dye aggregation, leading to the formation of adsorbed J or H aggregates.     

Interaction between dyes and polyelectrolytes. XIII. Effect of dye aggregation on the excitation energy transfer between bound dyes

Effect of polyanions on the aggregation of methylene blue (MB) was investigated spectrophotometrically. The following polyanions were used: potassium poly(vinyl sulfate), potassium poly(styrenesulfonate), sodium poly(methacrylate), and sodium poly(acrylate). The state of aggregation was largely dependent on the kind of polyanion and polyanion-MB ratio. MB-photo-sensitized isomerization of cis-p-(phenylazo)phenyltrimethylammonium iodide(cis-PTA) to the transisomer was used advantageously to investigate the effect of dye aggregation on the triplet excitation energy transfer between cationic dyes bound to polyanions. Although the efficiency of the excitation energy transfer between MB and cis-PTA was enhanced by the addition of polyanions, the formation of highly aggregated MB reduced the efficiency of the excitation energy transfer. Correlation with the dye aggregation induced by polyanions and the efficiency of excitation energy transfer between dyes was discussed.     

Effect of layer charge density on orientation and aggregation of a cationic laser dye incorporated in the interlayer space of montmorillonites

Effect of layer charge density of clay on the orientation and aggregation state of a laser dye, oxazine 4, in dye/clay complexes was investigated using a series of layer-charge-controlled montmorillonites as host materials. By the combination of polarized UV-vis spectroscopy and powder X-ray diffraction methods, it was revealed that the higher layer charge caused the formation of higher-order H-aggregates with the molecular axis nearly perpendicular to the silicate layer, and that the basal spacing was mostly governed by the degree of dye aggregation.     

Nickel, copper, and zinc centered ruthenium-substituted porphyrins: effect of transition metals on photoinduced DNA cleavage and photoinduced melanoma cell toxicity

Coordination of two [Ru(bipy)(2)Cl](+) moieties (where bipy = 2,2'-bipyridine) to the pyridyl nitrogens in the 5,10-positions of meso-5,10,15-(4-pyridyl)-20-(pentafluorophenyl)porphyrin gives the diruthenium porphyrin complex I. Insertion of nickel(II), copper(II), and zinc(II) into the porphyrin center gives the complexes II-IV, respectively. Electronic transitions associated with the ruthenium porphyrin include an intense Soret band and four less intense Q-bands in the visible region of the spectrum. An intense π-π* transition in the UV region associated with the bipyridyl groups and a metal-to-ligand charge transfer (MLCT) band appearing as a shoulder to the Soret band are also observed. A shift of the Soret band and collapse of the Q-bands into one band is observed upon insertion of the metal ions into the porphyrin center. Electrochemical properties associated with the complexes include a redox couple in the cathodic region attributed to the porphyrin and a redox couple in the anodic region due to the Ru(III/II) couple. DNA titrations of the complexes indicate that they interact strongly with DNA potentially through an intercalation mechanism. Irradiation of aqueous solutions of the complexes and supercoiled DNA at a 5:1 base pair to complex ratio with visible light above 400 nm shows nicking of DNA for the nickel(II) and copper(II) complexes and photocleavage of DNA for the zinc(II) complex. Cell studies with dermal skin (normal) fibroblast and melanoma cells indicate that the free base porphyrin(I) is toxic to both normal and melanoma cells, while the nickel(II) and copper(II) complexes, II and III, are non-toxic to both cell lines when irradiated with a tungsten lamp. The zinc(II) complex, IV, is non-toxic to normal cells but toxic to melanoma cells when irradiated under the same conditions.     

Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base.

Fluorescently labeled oligonucleotide probes have been widely used in biotechnology, and fluorescence quenching by the interaction between the dyes and a nucleobase has been pointed out. This quenching causes big problem in analytical methods, but is useful in some other cases. Therefore, it is necessary to estimate the fluorescence quenching intensity under various conditions. We focused on the redox properties of some commercially available fluorescent dyes, and investigated dye-nucleotide interactions between a free dye and a nucleotide in aqueous solution by electrochemical and spectroscopic techniques. Our results suggested that the quenching was accompanied by photoinduced electron transfer between a thermodynamically quenchable excited dye and a specific base. Several kinds of fluorescent dyes labeled to the 5'-end of oligonucleotide C10T6 were prepared, and their quenching ratios compared upon hybridization with the complementary oligonucleotide A6G10. The quenching was completely reversible and their efficiencies depended on the attached fluorophore types. The fluorescence of 5-FAM, BODIPY FL or TAMRA-modified probe was strongly quenched by hybridization.