Oxyresveratrol protects against DNA damage induced by photosensitized riboflavin

Riboflavin can be photosensitized to produce reactive oxygen species. In the present study, a DNA damage assay was developed based on the photo reaction of riboflavin. In this test system, oxyresveratrol showed higher DNA protective effect than the well-known antioxidants Trolox and ascorbic acid. The results suggest potential applications for oxyresveratrol as an anti-aging agent and a riboflavin stabilizer.     

Chemopreventive action of xanthone derivatives on photosensitized DNA damage

Photosensitized DNA damage participates in solar-UV carcinogenesis, photogenotoxicity and phototoxicity. A chemoprevention of photosensitized DNA damage is one of the most important methods for the above phototoxic effects. In this study, the chemopreventive action of xanthone (XAN) derivatives (bellidifolin [BEL], gentiacaulein [GEN], norswertianin [NOR] and swerchirin [SWE]) on DNA damage photosensitized by riboflavin was demonstrated using [32P]-5'-end-labeled DNA fragments obtained from genes relevant to human cancer. GEN and NOR effectively inhibited the formation of piperidine-labile products at consecutive G residues by photoexcited riboflavin, whereas BEL and SWE did not show significant inhibition of DNA damage. The four XAN derivatives decrease the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), an oxidative product of G, by photoexcited riboflavin. The preventive action for the 8-oxodGuo formation of these XAN derivatives increased in the following order: GEN>NOR>BEL>SWE. A fluorescence spectroscopic study and ab initio molecular orbital calculations suggested that the prevention of DNA photodamage is because of the quenching of the triplet excited state of riboflavin by XAN derivatives through electron transfer. This chemoprevention is based on neither antioxidation nor a physical sunscreen effect; rather, it is based on the quenching of a photosensitizer. In conclusion, XAN derivatives, especially GEN, may act as novel chemopreventive agents by the quenching mechanism of an excited photosensitizer.     

Consecutive adenine sequences are potential targets in photosensitized DNA damage

Based on direct spectroscopic measurements of hole transfer in DNA and quantification of the yield of DNA oxidative damage, consecutive adenine sequences were found to be a good launching site for photosensitizers to inject a hole in DNA, where the following rapid hole transfer between adenines causes a long-lived charge-separated state leading to DNA oxidative damage. According to the results, the essential requisites for an efficient and/or harmful photosensitizer are determined as follows: to be able to oxidize adenine to trigger hole transfer between adenines, and react rapidly with molecular oxygen following its reduction, avoiding charge recombination and making the reaction irreversible. These results will greatly help us to classify photosensitizers harmful to human health, and to design an improved photosensitizer for biochemical applications.     

Hole transfer in DNA and photosensitized DNA damage: importance of adenine oxidation

Photosensitized DNA damage reactions were investigated for two well-known DNA-damaging photosensitizers (Sens), naphthalimide (NI) and napthaldiimide (NDI), which have similar photophysical properties but differ in their redox properties. NI and NDI derivatives (NIN, NDIN), which have cationic side chains and electrostatically binding to DNA due to favorable electrostatic interactions between the negatively charged phosphate groups of DNA and cationic groups, and NIP and NDIP, which possess phosphate groups and do not bind to DNA, were synthesized. NIN and NDIN can oxidize A and G via their singlet excited state, and NDIP oxidizes A and G via its triplet excited state, whereas NIP oxidizes only G. A combination of laser flash photolysis kinetic studies and quantitative HPLC analyses of photosensitized DNA damage was performed for several DNA sequences in the presence of Sens. NIN, NDIN, and NDIP, which oxidizes A, caused significant DNA damage upon photoirradiation, and DNA damage yield increased with the length of the consecutive A stretch. In contrast, NIP, which oxidizes only G, caused only moderate damage to DNA and showed no preference for the consecutive A sequences. These results clearly demonstrate the importance of A-oxidation, especially in consecutive A sequences, which triggers the rapid hole transfer between A's.     

Guanine-specific DNA damage photosensitized by the dihydroxo(tetraphenylporphyrinato)antimony(V) complex

The dihydroxo(tetraphenylporphyrinato)antimony(V) complex (SbTPP) demonstrates bactericidal activity under visible-light irradiation. This phototoxic effect could be caused by photodamage to biomolecules, but the mechanism has not been well understood. In this study, to clarify the mechanism of phototoxicity by SbTPP, DNA damage photosensitized by SbTPP was examined using [(32)P]-5'-end-labeled DNA fragments. SbTPP induced markedly severe photodamage to single-stranded rather than to double-stranded DNA. Photo-irradiated SbTPP frequently caused DNA cleavage at the guanine residue of single-stranded DNA after Escherichia coli formamidopyrimidine-DNA glycosylase or piperidine treatment. HPLC measurement confirmed the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an oxidation product of 2'-deoxyguanosine, and showed that the content of 8-oxodG in single-stranded DNA is larger than that in double-stranded DNA. The effects of scavengers of reactive oxygen species on DNA damage suggested the involvement of singlet oxygen. These results have shown that the mechanism via singlet oxygen formation mainly contributes to the phototoxicity of SbTPP. On the other hand, SbTPP induced DNA damage specifically at the underlined G of 5'-GG, 5'-GGG, and 5'-GGGG in double-stranded DNA. The sequence-specificity of DNA damage is quite similar to that induced by the type I photosensitizers, suggesting that photo-induced electron transfer slightly participates in the phototoxicity of SbTPP. In conclusion, SbTPP induces DNA photodamage via singlet oxygen formation and photo-induced electron transfer. A similar mechanism can damage other biomacromolecules, such as protein and the phospholipid membrane. The damage to biomacromolecules via these mechanisms may participate in the phototoxicity of SbTPP.     

Mechanisms of photosensitized DNA cleavage

Photosensitization may promote DNA damages such as nucleic acid oxidation or single strand breaks via three main pathways: hydroxyl radicals attack, electron transfer process or oxidation by singlet oxygen. While direct production of OH^. by photosensitization is rarely observed, the mechanism of DNA attack by OH^. is now well established on the basis of informations provided by water radiolysis experiments. Some dyes may also induce single strand breaks via an electron transfer occurring from a nucleobase to the sensitizer in the excited state. This process generates base radical cations identical to those arising from DNA photoionisation. These radicals may undergo deprotonation or dehydration to form the same neutral radicals as those produced by OH^. but with a slightly different pattern. In contrast, while many sensitizers produce singlet oxygen, the mechanism of DNA damages induced by this way is still unclear. In this case the guanine moiety in nucleosides or in DNA is selectively altered leading to the formation of 8 oxoG or 8 oxodG and FapyGua. The mechanism of single strand breaks formation by singlet oxygen is discussed in this overview.     

Mechanism of DNA damage photosensitized by trisbipyrazyl ruthenium complex. Unusual role of Cu/Zn superoxide dismutase

Trisbipyrazyl ruthenium(II) (Ru[bpz]3(2+)) was examined as DNA photosensitizer. Damage resulting from the photolysis of synthetic oligonucleotides has been monitored by polyacrylamide gel electrophoresis. Photoadduct formation is found on both single- and double-stranded oligonucleotides. On oligonucleotide duplex, oxidative damage occurs selectively at the 5'G of the 5'GG3' site and to a lesser extent at the 5'G of a GA sequence. These findings suggest the involvement of electron transfer and show that this mechanism is the main DNA damaging process involved in Ru(bpz)3(2+) photosensitization. In addition, photoadducts and oxidative damage are both highly affected by an increase of salt concentration in the reaction medium, stressing the importance of direct interactions between nucleic acid bases and the excited ruthenium complex for efficient electron transfer. On single-stranded oligonucleotides, all the guanines are oxidized to the same extent. In this case, oxidative damage, which is not affected by an increase of salt in the solution, has been attributed, in part, to singlet oxygen. More importantly, Cu/Zn superoxide dismutase (SOD) strongly enhances the yield of all damage, correlated to an increase of both electron transfer and singlet oxygen production. This original activity of SOD is the first example of bioactivation of a polyazaaromatic ruthenium complex.     

Control of singlet oxygen generation photosensitized by meso-anthrylporphyrin through interaction with DNA

To control the activity of photosensitized singlet oxygen ((1)O(2)) generation, the electron donor-connecting porphyrin, 5-(9'-anthryl)-10,15,20-tris(p-pyridyl)porphyrin (AnTPyP), was designed and synthesized. AnTPyP became water-soluble by the protonation of the pyridyl moieties in the presence of 5 mM trifluoroacetic acid (pH 2.3). The photoexcited state of the porphyrin ring in an AnTPyP molecule was effectively deactivated by intramolecular electron transfer from the anthracene moiety within 0.04 ns in an aqueous solution. The deactivation was suppressed by the interaction with a DNA strand, resulting in the elongation of the lifetime of the porphyrin excited state and the enhancement of the fluorescence intensity. Furthermore, it was confirmed that the interaction enabled the photoexcited AnTPyP to generate (1)O(2). Selective (1)O(2) generation by forming a complex with DNA should be the initial step to realize the target selective photodynamic therapy.     

Effects of reaction rate of radical anion of a photosensitizer with molecular oxygen on the photosensitized DNA damage

Based on the synthesis of DNA modified with photosensitizers, direct spectroscopic measurements of the hole transfer in DNA, and quantification of the yield of the DNA oxidative damage, the reaction rate of the radical anion of the photosensitizer was demonstrated to be critically important in determining the efficiency of photosensitized DNA damage.     

DNA damage by fasicularin

Fasicularin is a structurally novel thiocyanate-containing alkaloid isolated from the ascidian Nephteis fasicularis. Early biological experiments suggested that this compound's cytotoxic properties may stem from its ability to damage cellular DNA. Sequence gel analysis reveals that treatment of a 5'-32P-labeled DNA duplex with fasicularin in pH 7.0 buffer causes strand cleavage selectively at guanine residues. Further experiments indicate that production of these base-labile lesions in DNA involves alkylation of guanine residues by a fasicularin-derived aziridinium ion. This work reveals fasicularin as the first natural product found to generate a DNA-alkylating aziridinium ion via a mechanism analogous to the clinically used anticancer drugs mechlorethamine, melphalan, and chlorambucil.     

Sequence diversity, metal specificity, and catalytic proficiency of metal-dependent phosphorylating DNA enzymes

Although DNA has not been found responsible for biological catalysis, many artificial DNA enzymes have been created by "in vitro selection." Here we describe a new selection approach to assess the influence of four common divalent metal ions (Ca(2+), Cu(2+), Mg(2+), and Mn(2+)) on sequence diversity, metal specificity, and catalytic proficiency of self-phosphorylating deoxyribozymes. Numerous autocatalytic DNA sequences were isolated, a majority of which were selected using Cu(2+) or Mn(2+) as the divalent metal cofactor. We found that Cu(2+)- and Mn(2+)-derived deoxyribozymes were strictly metal specific, while those selected by Ca(2+) and Mg(2+) were less specific. Further optimization by in vitro evolution resulted in a Mn(2+)-dependent deoxyribozyme with a k(cat) of 2.8 min(-1). Our findings suggest that DNA has sufficient structural diversity to facilitate efficient catalysis using a broad scope of metal cofactor utilizing mechanisms.     

Sequence specificity of hydrogen-bonded molecular duplexes

Hydrogen-bonded molecular duplexes, 1.3 and 1.4, each of which contains a mismatched binding site (acceptor-to-acceptor in 1.3, and donor-to-donor in 1.4), were designed and synthesized based on duplex 1.2. One- and two-dimensional NMR studies demonstrated that, despite their single mismatched binding sites, the backbones of duplexes 1.3 and 1.4 still stayed in register through the formation of the remaining five H-bonds. The backbones of 1.3 and 1.4 adjusted to the presence of the mismatched binding sites by slightly twisting around these sites, which alleviate any head-on repulsive interactions between two H-bond donors (amide O) or between two acceptors (amide H). After 1 equiv of single strand 2, which forms a perfectly matched duplex 1.2 with single strand 1, was added into the solution of either 1.3 or 1.4, only 1.2 and single strand 3 or 4, were detected. Isothermal titration calorimetry (ITC, in chloroform containing 5% DMSO) indicated that duplexes 1.3 and 1.4 were significantly (>40 times) less stable than the corresponding perfectly hydrogen-bonded duplex 1.2. These NMR and ITC results indicate that the pairing of two complementary single strands is not affected by another very similar single strand that contains only one wrong H-bond donor or acceptor, which demonstrates that the self-assembly of this class of H-bonded duplexes is a highly sequence-specific process. The role of these H-bonded duplexes as predictable and programmable molecular recognition units for directing intermolecular interactions has thus been established.     

Polymerization photosensitized by carbonyl compounds

The polymerization of methyl methacrylate and styrene photosensitized by acetone, aldehydes, ethyl pyruvate, 2,3-butanedione, and 2,3-pentanedione has been investigated and the effect of several additives (carbon tetrachloride, cumene, diethyl amine, triethyl amine, 2-pentanol, and tetrahydrofuran) on initiation efficiency has been evaluated. The initiation efficiency of a given system depends on several factors, the most important of which are the relative rates of quenching by the monomers and the additives and type of product obtained.     

Confocal fluorescence imaging of photosensitized DNA denaturation in cell nuclei

The double-stranded helical structure of DNA is maintained in part by hydrogen bonds between strands and by stacking interactions between adjacent purine and pyrimidine bases in one strand. The transition (denaturation) from a double-stranded (ds) to a single-stranded (ss) form can be induced in isolated DNA or fixed cells by exposure to elevated temperatures, alkali or acids, aprotic or nonpolar solvents or some drugs. We report here that DNA denaturation can occur in situ in cell nuclei as a result of interaction between light and an intercalated dye, acridine orange or ethidium bromide. This DNA photodenaturation was probed using metachromatic properties of acridine orange and imaged by fluorescence confocal microscopy. Furthermore, an empirical kinetic model was developed to separate changes of acridine orange luminescence intensities caused by photobleaching from those that were a result of DNA denaturation. We investigated the influence of oxygen on these phenomena and propose a mechanism by which photodenaturation may occur.     

Cyanine dyes as protectors of K562 cells from photosensitized cell damage

Several cyanine dyes were found to protect K562 leukemia cells against toxicity mediated by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and light. Most cyanine dyes derived from dimethylindole were better photoprotectors than cyanine dyes with other structures. This correlated with the fact that cyanine dyes derived from dimethylindole were predominately monomeric at millimolar concentrations within K562 cells, while other cyanine dyes formed aggregates. For cyanine dyes that are derived from dimethylindole and have absorption band wavelengths greater than 700 nm, fluorescence-energy transfer from TPPS2 to the cyanine dye was the most important mechanism for photoprotection. There was no spectroscopic evidence for complex formation between the cyanine dyes and TPPS2. The dimethylindole derivative, 1,1',3,3,3',3'-hexamethylindodicarbocyanine, was an excellent photoprotector, but a poor quencher of TPPS2 fluorescence and a relatively poor singlet-oxygen quencher. This cyanine dye may act by quenching excited triplet TPPS2. Singlet-oxygen quenching may contribute to the photoprotection provided by cyanine dyes not derived from dimethylindole. Differences in the subcellular distribution of the various cyanine dyes studied may have contributed to the different apparent mechanisms of photoprotection.     

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.     

Determination of the enantiomers of suprofen and [2H3]suprofen in plasma by capillary gas chromatography-mass spectrometry

A method for the stereoselective assay of the (+)- and (-)-enantiomers of suprofen and [2H3]suprofen in human plasma was developed using gas chromatography-mass spectrometry-selected-ion monitoring. (+/-)-[2H7]Suprofen was used as an internal standard. The method involved diethyl ether extraction and chiral derivatization with S-(-)-1-(naphthyl)ethylamine to form diastereomeric amide. The diastereoisomers were separated on a capillary gas chromatograph-mass spectrometer. Quantitation was achieved by selected-ion monitoring of the quasi-molecular ions of the diastereoisomers. The sensitivity, specificity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application to pharmacokinetic studies of suprofen enantiomers.     

The microenvironment of DNA switches the activity of singlet oxygen generation photosensitized by berberine and palmatine

The effect of the interaction between DNA and the photosensitizer on photosensitized singlet oxygen (1O2) generation was investigated using DNA-binding alkaloids, berberine and palmatine. These photosensitizers were bound to DNA by electrostatic force. Near-infrared luminescence measurement demonstrated that the photoexcited alkaloids can generate 1O2 only when the photosensitizers are bound to DNA. A fluorescence decay study showed significant enhancement of the lifetime of their photoexcited state with the DNA binding. A calculation study suggested that the electrostatic interaction with DNA inhibits the quenching of the photoexcited state of these alkaloids via intramolecular electron transfer, leading to the prolongation of the lifetime of their excited state. This effect should enhance their intersystem crossing and the yield of energy transfer to molecular oxygen. The results show that the electrostatic interaction with DNA significantly affects the 1O2 generation activity of a photosensitizer. In addition, this interaction may be applied to the control and the design of photosensitizers for medical applications such as photodynamic therapy.     

Sequence specific DNA cross-linking triggered by visible light

A new biocompatible strategy for photoinduced DNA interstrand cross-linking is presented. Methylene blue induced (1)O(2) formation triggers furan oxidation; the resulting aldehyde then rapidly reacts with complementary A or C with formation of stable adducts. Easily accessible furan modified nucleosides, a commercially available photosensitizer, and visible light irradiation constitute the necessary tools to achieve selective duplex interstrand cross-linking.