Photochemical and Photobiological Studies of a Furonaphthopyranone as a Benzo-spaced Psoralen Analog in Cell-free and Cellular DNA

Abstract— Photobiological activities of the benzo-spaced psoralen analog furonaphthopyranone 3 have been investigated in cell-free and cellular DNA. The molecular geometry parameters of 3 suggest that it should not form interstrand crosslinks with DNA. With cell-free DNA no evidence for crosslinking but also not for monoadduct formation was obtained; rather, the unnatural furocoumarin 3 induces oxidative DNA modifications under near-UVA irradiation. The enzymatic assay of the photosensitized damage in cell-free PM2 DNA revealed the significant formation of lesions sensitive to formamidopyrimidine DNA glyco-sylase (Fpg protein). In the photooxidation of calf thymus DNA by the furonaphthopyranone 3, 0.29±0.02% 8-oxo-7,8-dihydroguanine (8-oxoGua) was observed. With 2'-deoxyguanosine (dGuo), the guanidine-releasing photooxidation products oxazolone and oxoimidazolidine were formed predominately, while 8-oxodGuo and 4-HO-8-oxodGuo were obtained in minor amounts. The lack of a significant D₂O effect in the photooxidation of DNA and dGuo reveals that singlet oxygen (type II process) plays a minor role; control experiments with tert -butanol and mannitol confirm the absence of hydroxyl radicals as oxidizing species. The furonaphthopyranone 3 (E_red= -1.93±0.03V) should act in its singlet-excited state as electron acceptor for the photooxidation of dGuo (δG_ET ca – kcal/mol), which corroborates photoinduced electron transfer (type I) as a major DNA-oxidizing mechanism. A comet assay in Chinese hamster ovary (CHO) AS52 cells demonstrated that the psoralen analog 3 damages cellular DNA upon near-UVA irradiation; however, no photosensitized mutagenicity was observed in CHO AS52 cell cultures     

The roles of hydroxyl radicals, photo-generated holes and oxygen in the photocatalytic degradation of humic acid

This article is aimed at studying on the roles of the hydroxyl radicals, photo-generated holes, and oxygen in the photocatalytic degradation of humic acid (HA) in acid and alkaline conditions. The results indicate that hydroxyl radicals?? scavenger alone can inhibit the photocatalytic degradation process completely in alkaline condition, which implies that photo-generated holes cannot directly degrade the organic matter in alkaline condition. Moreover, the reaction sites between hydroxyl radicals and HA is on the TiO₂ surface in acid condition. But in alkaline condition, hydroxyl radicals diffuse and react with HA in the solution. The generation of hydroxyl radicals almost stops and the photocatalytic degradation is inhibited seriously without oxygen, which illustrates that oxygen plays an important role in the photocatalytic degradation of HA.     

Photochemical reactions of aromatic ketones with nucleic acids and their components. 3. Chain breakage and thymine dimerization in benzophenone photosensitized DNA

Abstract— Excitation of benzophenone in the presence of calf thymus and E. coli DNA leads to photosensitized damages to the macromolecule. Two main reactions are observed: thymine dimerization and chain break formation. Benzophenone photosensitized chain breaks are also observed in polyadenylic acid. The melting temperature of DNA decreases with the duration of irradiation. Under our experimental conditions, the ratio of the yields of dimers and single-chain breaks produced in DNA is about 1. Photosensitized damage to deoxyribose residues leading to chain breakage is shown to be similar to that produced by X or γ ray irradiation. The oxygen effect upon chain break production is studied and discussed in relation with its effect upon intermediate species. Thymine dimers are formed following energy transfer from benzophenone in its triplet state. In previous flash-photolysis studies we showed that benzophenone in its triplet state reacts with water molecules to give ketyl and OH radicals. Ketyl radicals are not involved in reactions with DNA. It is proposed that OH radicals produced in the above reaction are responsible for the production of single-chain breaks by attack on the deoxyribose residues.     

Reactive molecular dynamics study on the first steps of DNA damage by free hydroxyl radicals

We employ a large scale molecular simulation based on bond-order ReaxFF to simulate the chemical reaction and study the damage to a large fragment of DNA molecule in the solution by ionizing radiation. We illustrate that the randomly distributed clusters of diatomic OH radicals that are primary products of megavoltage ionizing radiation in water-based systems are the main source of hydrogen abstraction as well as formation of carbonyl and hydroxyl groups in the sugar moiety that create holes in the sugar rings. These holes grow up slowly between DNA bases and DNA backbone, and the damage collectively propagates to a DNA single and double strand break.     

In situ DNA oxidative damage by electrochemically generated hydroxyl free radicals on a boron-doped diamond electrode

In situ DNA oxidative damage by electrochemically generated hydroxyl free radicals has been directly demonstrated on a boron-doped diamond electrode. The DNA-electrochemical biosensor incorporates immobilized double-stranded DNA (dsDNA) as molecular recognition element on the electrode surface, and measures in situ specific binding processes with dsDNA, as it is a complementary tool for the study of bimolecular interaction mechanisms of compounds binding to DNA and enabling the screening and evaluation of the effect caused to DNA by radicals and health hazardous compounds. Oxidants, particularly reactive oxygen species (ROS), play an important role in dsDNA oxidative damage which is strongly related to mutagenesis, carcinogenesis, autoimmune inflammatory, and neurodegenerative diseases. The hydroxyl radical is considered the main contributing ROS to endogenous oxidation of cellular dsDNA causing double-stranded and single-stranded breaks, free bases, and 8-oxoguanine occurrence. The dsDNA-electrochemical biosensor was used to study the interaction between dsDNA immobilized on a boron-doped diamond electrode surface and in situ electrochemically generate hydroxyl radicals. Non-denaturing agarose gel-electrophoresis of the dsDNA films on the electrode surface after interaction with the electrochemically generated hydroxyl radicals clearly showed the occurrence of in situ dsDNA oxidative damage. The importance of the dsDNA-electrochemical biosensor in the evaluation of the dsDNA-hydroxyl radical interactions is clearly demonstrated.     

In vivo effects of porphyrins on bacterial DNA.

The DNA damage in intact Staphylococcus aureus and E. coli cells induced by photosensitized deuteroporphyrin or hemin is described. Treatment of S. aureus cultures with hemin or photosensitized deuteroporphyrin (Dp) caused time-dependent changes in the plasmidial DNA profiles. The major observation was the disappearance of the plasmid supercoiled fraction. The chromosomal DNA was also affected by hemin and by photosensitized Dp, since its degradation products were detected after exposing the bacterial cells to the porphyrin drugs. Photosensitization of E. coli cells, pretreated with Dp and polymyxin B nonapeptide (PMBNP), also resulted in plasmidial damage. No such damage occurred when E. coli cultures were treated with hemin and PMBNP. The above results can be tightly correlated with the antimicrobial action of porphyrins. Their damage to the bacterial DNA seems to reflect one of the in vivo effects of these porphyrins.     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

Oxidative DNA Damage in the Photolysis of Af-Hydroxy-2-Pyridone, a Specific Hydroxyl-Radical Source

Abstract The photooxidative DNA damage by iV-hydroxy-2-pyri-done (1) is caused by hydroxyl radicals, as confirmed by electron paramagnetic resonance studies with the spin trap 5,5-dimethylpyrroline JV-oxide. Irradiation of the pyridone 1 at 300 nm induced strand breaks in super-coiled pBR322 DNA, while in calf-thymus DNA and 2'-deoxyguanosine (dG), respectively, 8-oxoguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine were formed. Time-dependent control experiments disclosed that photoprod-ucts of pyridone 1, e.g. 2-pyridone (3), are not responsible for the modification of DNA. Also the photosensitization by the pyridine-2-one chromophore was excluded, because JV-methylpyridine-2-one (2), which cannot generate hydroxyl radicals, was ineffective in the photooxidation of DNA and dG. Thus, the photolysis of pyridone 1 serves as a specific source of hydroxyl radicals for DNA damage, both strand breaks and base modifications.     

PHOTOSENSITIZATION OF SUPERCOILED DNA DAMAGE BY 5,6-DIHYDROXYINDOLE-2-CARBOXYLIC ACID, A PRECURSOR OF EUMELANIN

The photosensitizing or photoprotecting action of 5,6-dihydroxyindole-2-carboxylic acid (DICA), an intermediate in the biosynthesis of eumelanins, was investigated. Under irradiation at 313 nm, aqueous buffered solutions of DICA (22.5 μW) photosensitized the cleavage of phage φX174 DNA. The number of single strand breaks (SSB) depended on the dose of irradiation and was more important in the absence than in the presence of oxygen. In the presence of oxygen, the quantum yield of SSB was around 6′10 ⁷ (Φ_SSB) The influence of specific scavengers, such as mannitol, sodium azide or superoxide dismutase, indicated that hydroxyl radicals, superoxide anions and perhaps singlet oxygen were involved in these processes. The increase in SSB in D₂O was also indicative of the participation of singlet oxygen. Comparative experiments performed with indole-2-carboxylic acid (IC), a dehydrox-ylated analog of DICA, showed that this compound, although lacking a phenol group, also photosensitized DNA cleavage via a mechanism involving hydroxyl radicals. Various sources of these radicals were envisioned. Furthermore, under our conditions, DICA was not found to photoinduce the formation of DNA dimers: No increase in SSB was observed in DNA irradiated in the presence of DICA, after treatment by phage T4 endonuclease V (an enzyme that selectively cuts DNA at dimer sites), whereas, in contrast, a significant increase in SSB was detected after treatment of DNA irradiated alone. So it appears that DICA may both photosensitize DNA cleavage and reduce UV-induced DNA dimer formation.     

Photochemical and Photobiotogical Studies with Acridine and Phenanthridine Hydroperoxides in Cell-free DNA

Abstract— The acridine and phenanthridine hydroperoxides 3 and 7 were synthesized as photochemical hydroxyl radical sources for oxidative DNA damage studies. The generation of hydroxyl radicals upon UVA irradiation (Λ. = 350 nm) was verified by trapping experiments with 5,5-di-methyl-1-pyrroline N -oxide and benzene. The enzymatic assays of the damage in cell-free DNA from bacteriophage PM2 caused by the acridine and phenanthridine hydroperoxides 3 and 7 under near-UVA irradiation revealed a wide range of DNA modifications. Particularly, extensive single-strand break formation and DNA base modifications sensitive to formamidopyrimidine DNA glycosylase (Fpg protein) were observed. In the photooxida-tion of calf thymus DNA, up to 0.69±0.03% 8-oxo-7,8-dihydroguanine was formed by the hydroperoxides 3 and 7 on irradiation, whose yield was reduced up to 40% in the presence of the hydroxyl radical scavengers mannitol and fert-butanol. The acridine and phenanthridine hydroperoxides 3 and 7 also induce DNA damage through the type I photooxidation process, for which photoinduced electron transfer from 2'-deoxyguanosine to the singlet states of 3 and 7 was estimated by the Rehm-Weller equation to possess a negative Gibb's free energy of cα -5 kcal/ mol. Control experiments with the sensitizers acridine 1 and the acridine alcohol 4 in calf thymus and PM2 DNA confirmed the photosensitizing propensity of the UVA-ab-sorbing chromophores. The present study emphasizes that for the development of selective and efficient photochemical hydroxyl radical sources, chromophores with low photosensitizing ability must be chosen to avoid type I and type II photooxidation processes.     

CATALASE INACTIVATION FOLLOWING PHOTOSENSITIZATION WITH TETRASULFONATEDMETALLOPHTHALOCYANINES

Abstract— Catalase (CAT) in solution or incorporated in erythrocytes and K562 leukemic cells is inactivated during photosensitization with tetrasulfonated metallophthalocyantnes (MePcS₄). The effect of added scavengers and D₂0 showed that both singlet oxygen and free radical species are involved in this process. Evidence was found that direct interactions of ground or excited-stated photosensitizers with CAT are not responsible for CAT inactivation. Specific techniques to probe early damage to the CAT structure involved optical and EPR spectroscopy, HPLC and polyacrylamide gel electrophoresis analyses. Different primary events of photosensitized protein damage included oxidation of cysteine residues as well as other amino acids, as demonstrated by the formation of carbon-centered free radicals and the loss of absorbance at λ= 275 nm. In parallel, we detected degradation of the CAT heme groups, accompanied by release of Fe(II) ions in solution. These combined phenomena initiate cross-linkages between CAT subunits and subsequent degradation of the protein with formation of irreversible aggregates in solution. Phthalocyanine-mediated photoinactivation of cell-bound CAT results in loss of protection against accumulating H₂0₂, providing an additional pathway of phototoxicity.     

Hoechst-naphthalimide dyad with dual emissions as specific and ratiometric sensor for nucleus DNA damage

A ratiometric fluorescent sensor (Hoe-NI) was developed for high specific nucleus labeling and monitoring of nuclear DNA damage in living cells.     

Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation,

This review article focuses on a critical survey of the main available information on the UVB and UVA oxidative reactions to cellular DNA as the result of direct interactions of UV photons, photosensitized pathways and biochemical responses including inflammation and bystander effects. UVA radiation appears to be much more efficient than UVB in inducing oxidatively generated damage to the bases and 2‐deoxyribose moieties of DNA in isolated cells and skin. The UVA‐induced generation of 8‐oxo‐7,8‐dihydroguanine is mostly rationalized in terms of selective guanine oxidation by singlet oxygen generated through type II photosensitization mechanism. In addition, hydroxyl radical whose formation may be accounted for by metal‐catalyzed Haber–Weiss reactions subsequent to the initial generation of superoxide anion radical contributes in a minor way to the DNA degradation. This leads to the formation of both oxidized purine and pyrimidine bases together with DNA single‐strand breaks at the exclusion, however, of direct double‐strand breaks. No evidence has been provided so far for the implication of delayed oxidative degradation pathways of cellular DNA. In that respect putative characteristic UVA‐induced DNA damage could include single and more complex lesions arising from one‐electron oxidation of the guanine base together with aldehyde adducts to amino‐substituted nucleobases.     

Molecular Mechanism of Photosensitization XI. Membrane Damage and DNA Cleavage Photoinduced by Enoxacin

The photosensitizing activity of enoxacin, 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-1,8-naphthyridine-3-carboxilic acid (ENX), toward membranes and DNA has been studied, taking into account human erythrocyte photohemolysis, unilamellar liposome alterations and plasmid pBR322 DNA photocleavage. Hydroxyl radicals and an aromatic carbene generated from ENX photode-fluorination seem to be the active intermediates involved in the photosensitization process. The steady-state photolysis products do not participate in the process. The mechanism of photosensitization responsible for the membrane damage depends on the oxygen concentration and follows a different path with respect to that operative for DNA cleavage. Between oxygenated radicals, the hydroxyl seems the species mainly responsible for membrane damage, whereas DNA cleavage is mainly produced by the carbene intermediate. A molecular mechanism of the photosensitization induced by ENX is proposed.     

Characterization of free radical-induced damage to DNA by the combined use of enzymatic hydrolysis and gas chromatography-mass spectrometry

The use of gas chromatography-mass spectrometry (GC-MS) for characterization of free radical-induced base damage to DNA is presented. Damage introduced to DNA by reactive oxygen species such as hydroxyl radicals appears to play an important role in mutagenesis, carcinogenesis and aging. Elucidation of the chemical nature of such DNA lesions is necessary for the assessment of their biological consequences and enzymatic repair. DNA exposed to radiation-generated hydroxyl radicals in aqueous solution was hydrolyzed to 2'-deoxyribonucleosides with a mixture of DNase I, venom and spleen exonucleases and alkaline phosphatase. The hydrolysate was subsequently trimethylsilylated and analyzed by GC-MS. A large number of DNA lesions were separated and identified. Mass spectra obtained were interpreted on the basis of the typical fragmentation pathways of trimethylsilylated nucleosides. The use of GC-MS with selected-ion monitoring facilitated the detection of these lesions at the very low quantities and radiation doses (below 10 Gray) that might be relevant to those in biological systems.     

Oxidation of 2-deoxyribose by benzotriazinyl radicals of antitumor 3-amino-1,2,4-benzotriazine 1,4-dioxides

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is the lead bioreductive drug in clinical trials as an anticancer agent to kill refractory hypoxic cells of solid tumors. It has long been known that, upon metabolic one-electron reduction, tirapazamine induces lethal DNA double strand breaks in hypoxic cells. These strand breaks arise from radical damage to the ribose moiety of DNA, and in this pulse radiolysis and product analysis study we examine mechanistic aspects of the dual function of tirapazamine and analogues in producing radicals of sufficient power to oxidize 2-deoxyribose to form radicals, as well as the ability of the compounds to oxidize the resulting deoxyribose radicals to generate the strand breaks. Both the rate of oxidation of 2-deoxyribose and the radical yield increase with the one-electron reduction potentials of the putative benzotriazinyl radicals formed from the benzotriazine 1,4-dioxides. Subsequent oxidation of the 2-deoxyribose radicals by the benzotriazine 1,4-dioxides and 1-oxides proceeds through adduct formation followed by breakdown to form the radical anions of both species. The yield of the radical anions increases with increasing one-electron reduction potentials of the compounds. We have previously presented evidence that oxidizing benzotriazinyl radicals are formed following one-electron reduction of the benzotriazine 1,4-dioxides. The reactions reported in this work represent the kinetic basis of a short chain reaction leading to increased oxidation of 2-deoxyribose, a process which is dependent on the one-electron reduction potential of the benzotriazinyl radicals that are above a threshold value of ca. 1.24 V.     

Synthesis of C3' modified nucleosides for selective generation of the C3'-deoxy-3'-thymidinyl radical: a proposed intermediate in LEE induced DNA damage

DNA damage pathways induced by low-energy electrons (LEEs) are believed to involve the formation of 2-deoxyribose radicals. These radicals, formed at the C3' and C5' positions of nucleotides, are the result of cleavage of the C-O phosphodiester bond through transfer of LEEs to the phosphate group of DNA oligomers from the nucleobases. A considerable amount of information has been obtained to illuminate the identity of the unmodified oligonucleotide products formed through this process. There exists, however, a paucity of information as to the nature of the modified lesions formed from degradation of these sugar radicals. To determine the identity of the damage products formed via the 2',3'-dideoxy-C3'-thymidinyl radical (C3'(dephos) sugar radical), phenyl selenide and acyl modified sugar and nucleoside derivatives have been synthesized, and their suitability as photochemical precursors of the radical of interest has been evaluated. Upon photochemical activation of C3'-derivatized nucleosides in the presence of the hydrogen atom donor tributyltin hydride, 2',3'-dideoxythymidine is formed indicating the selective generation of the C3'(dephos) sugar radical. These precursors will make the identification and quantification of products of DNA damage derived from radicals generated by LEEs possible.     

Mechanisms of photoinitiated cleavage of DNA by 1,8-naphthalimide derivatives.

Using water-soluble 1,8-naphthalimide derivatives, the mechanisms of photosensitized DNA damage have been elucidated. Specifically, a comparison of rate constants for the photoinduced relaxation of supercoiled to circular DNA, as a function of dissolved halide, oxygen and naphthalimide concentration, has been carried out. The singlet excited states of the naphthalimide derivatives were quenched by chloride, bromide and iodide. In all cases the quenching products were naphthalimide triplet states, produced by induced intersystem crossing within the collision complex. Similarly, the halides were found to quench the triplet excited state of the 1,8-naphthalimide derivatives by an electron transfer mechanism. Bimolecular rate constants were < 10(5) M-1 s-1 for quenching by bromide and chloride. As expected from thermodynamic considerations quenching by iodide was 6.7 x 10(9) and 8.8 x 10(9) M-1 s-1 for the two 1,8-naphthalimide derivatives employed. At sufficiently high ground-state concentration self-quenching of the naphthalimide triplet excited state also occurs. The photosensitized conversion of supercoiled to circular DNA is fastest when self-quenching reactions are favored. The results suggest that, in the case of 1,8-naphthalimide derivatives, radicals derived from quenching of the triplet state by ground-state chromophores are more effective in cleaving DNA than reactive oxygen species or radicals derived from halogen atoms.     

Biosensor with Protective Membrane for the Detection of DNA Damage and Antioxidant Properties of Fruit Juices

With the purpose to prepare a DNA biosensor protected with an outer‐sphere membrane against high molecular weight interferences, a carbon film electrode was layer‐by‐layer modified with dsDNA and chitosan. Using cyclic and square‐wave voltammetry and impedance spectroscopy, the oxidative damage of DNA by the hydroxyl and superoxide anion radicals was detected which consists of opening of the helix structure followed by deep DNA chain degradation. The biosensor has been applied to the detection of the antioxidant effect of apple and orange juices. The investigation of the novel biosensor with a protective membrane represents a significant contribution to the field of DNA biosensors utilization.     

X-ray and UV Radiation Damage of dsDNA/Protein Complexes

Simple SummaryOne of the most common diseases in the world is cancer. The development of an appropriate treatment pathway for cancer patients seems to be crucial to fight this disease. Therefore, solving the problem that affects more and more people in an aging society is crucial. The study presents the results of radiation and photochemical damage to DNA interacting with proteins (specifically/non-specifically). The obtained results of the analysis of photoliths and radiolites by means of the LC-MS technique allowed to identify possible mechanisms of degradation of DNA interacting with proteins. Results suggest the protective action of protein against hydroxyl radicals or solvated electrons and increased damaging effect when sensitized DNA is irradiated by UV light (280 or 320 nm) compared to the DNA alone (without protein interaction).AbstractRadiation and photodynamic therapies are used for cancer treatment by targeting DNA. However, efficiency is limited due to physico-chemical processes and the insensitivity of native nucleobases to damage. Thus, incorporation of radio- and photosensitizers into these therapies should increase both efficacy and the yield of DNA damage. To date, studies of sensitization processes have been performed on simple model systems, e.g., buffered solutions of dsDNA or sensitizers alone. To fully understand the sensitization processes and to be able to develop new efficient sensitizers in the future, well established model systems are necessary. In the cell environment, DNA tightly interacts with proteins and incorporating this interaction is necessary to fully understand the DNA sensitization process. In this work, we used dsDNA/protein complexes labeled with photo- and radiosensitizers and investigated degradation pathways using LC-MS and HPLC after X-ray or UV radiation.