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.     

Photosensitization of 2'-deoxyadenosine-5'-monophosphate by pterin

UV-A radiation (320-400 nm) induces damages to the DNA molecule and its components through photosensitized reactions. Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. We have investigated the photosensitization of 2'-deoxyadenosine-5'-monophosphate (dAMP) by pterin (PT) in aqueous solution under UV-A radiation. The effect of pH was evaluated, the participation of oxygen was investigated and the products analyzed. Kinetic studies revealed that the reactivity of dAMP towards singlet oxygen (1O2) is very low and that this reactive oxygen species does not participate in the mechanism of photosensitization, although it is produced by PT upon UV-A excitation. In contrast, analysis of irradiated solutions by means of electrospray ionization mass spectrometry strongly suggested that 8-oxo-7,8-dihydro-2'-deoxyadenosine-5'-monophosphate (8-oxo-dAMP) was produced, indicating that the photosensitized oxidation takes place via a type I mechanism (electron transfer).     

FORMATION OF 7,8-DIHYDRO-8-OXOGUANINE IN THE 1,2-DIOXETANE-INDUCED OXIDATION OF CALF THYMUS DNA: EVIDENCE FOR PHOTOSENSITIZED DNA DAMAGE BY THERMALLY GENERATED TRIPLET KETONES IN THE DARK

Abstract— Isolated calf thymus DNA was treated with the 1,2-dioxetanes 3-acetoxymethyl-3,4,4-tri-methyl-1,2-dioxetane, 2,3-dimethylbenzofuran dioxetane, 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxeta-ne (HTMD), 3,3,4,4-tetramethyl-1,2-dioxetane and 3,4,4-trimethyl-1,2-dioxetane (TrMD), which on thermal decomposition generate triplet-excited carbonyl products. To monitor quantitatively the formation of the mutagenic oxidation product 7,8-dihydro-8-oxoguanine (8-oxoGua), a sensitive and selective HPLC electrochemical assay was used after acidic hydrolysis (HF/pyridine) of the dioxetane-treated DNA. High yields of 8-oxoGua (up to ca 4% of the available guanine) were obtained for HTMD and TrMD. Both were investigated in detail with respect to effects of concentration, time and temperature. The oxidative reactivity of 1,2-dioxetanes was compared with several type I (benzophenone and riboflavin) and type II (methylene blue and rose bengal) photooxidants and disodium 1,4-etheno-2,3-ben-zodioxin-1,4-dipropionate as a chemical source of singlet oxygen. The persistence of 8-oxoGua towards oxidation by HTMD was examined in the reaction with 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) and with oxidized DNA. It was shown that, indeed, 8-oxoGua is consumed in the oxidized DNA on prolonged exposure to an excess of HTMD. The reaction of 8-oxodGuo with HTMD afforded the two 4R* and 4S* diastereomers of 9-(2-deoxy-ß-D-erythropentofuranosyl)-4,8-dihydro-4-hydroxy-8-oxoguanine as main oxidation products. Trapping experiments with teft-butanol confirmed that hydroxyl radicals are not involved, whereas the use of the triplet quenchers sodium 9,10-dibromo-anthra-cene-2-sulfonate and 2,3-diazabicyclo[2.2.1]hept-2-ene established that triplet-excited states are mainly responsible for the observed DNA oxidation through type I action (electron transfer chemistry). The role of singlet oxygen was tested by means of deuterium isotope effects in D₂O versus H₂O, but no definitive conclusion could be reached in regard to the involvement of ¹0₂ in these oxidations. The present results reveal that 1,2-dioxetanes are efficient DNA oxidants and excellent tools to study photooxidation reactions of DNA in the dark.     

PHOTOBINDING OF PSORALENS TO BACTERIAL MACROMOLECULES IN SITU AND INDUCTION OF GENETIC EFFECTS IN A BACTERIAL TEST SYSTEM. EFFECTS OF SINGLET OXYGEN DIAGNOSTIC AIDS D2O AND DABCO

The photobinding of radiolabeled psoralen and 8-methoxypsoralen (8-MOP) to biological macromolecules under conditions that affect the lifetime of singlet oxygen (¹O₂) is reported. These conditions are: increase of ¹O₂ lifetime in D₂O and ¹O₂ quenching with DABCO. The photobinding to calf thymus DNA was studied in vitro and the covalent photobinding to DNA and other biological macromolecules (RNA, proteins) was also studied in intact bacteria. The results of the DNA photobinding experiments have been related to the induction of genetic damage in a bacterial test system. In addition, laser flash photolysis has been used to measure the effect of D₂O and DABCO on the psoralen and 8-MOP triplet lifetimes. In general D₂O increases the triplet lifetimes and DABCO quenches the triplet states with the probable formation of radicals. The results suggest that the covalent photobinding of 8-MOP to various biological macromolecules in situ is a basis for cell damage occurring at various cellular targets. Analysis of the results of the mutagenicity test suggests that in the presence of D₂O the mechanism of induction of genetic lesions is not changed and therefore largely seems to be independent of singlet oxygen.     

CERTAIN SINGLET OXYGEN QUENCHERS AFFECT THE PHOTOREACTION BETWEEN 8-MOP and DNA

The effect of certain chemicals known as singlet oxygen quenchers on the photoreaction between 8-MOP and DNA has been studied in vitro; sodium azide, l,4-diazabicyclo-(2,2,2)-octane, p-carotene and dimethylsolfoxide (used as a solvent) appeared to be capable of reducing significantly the 8-MOP ability to induce both monoadducts and cross-links in DNA. Therefore, these chemicals seem to be not useful in studying the singlet oxygen implication in the induction of biological effects of 8-MOP sensitization.     

PHTHALOCYANINE AND NAPHTHALOCYANINE PHOTOSENSITIZED OXIDATION OF 2'-DEOXYGUANOSINE: DISTINCT TYPE I AND TYPE II PRODUCTS

Abstract
The photodynamic properties of the di-and tetrasulfonated zinc and aluminium phthalocyanines and a tetrasulfonated aluminium napththalocyanine were studied using 2'-deoxyguanosine as a DNA model compound. The major photooxidation products of this nucleoside were identified and classified according to their formation through a radical mechanism (type I) or a singlet oxygen mediated mechanism (type II). The major type I product was obtained and identified as 2,2-diamino [(2-deoxy-β- d - erythro pentofuranosyl)-4-amino]-5( 2H )-oxazolone. Two major type II products were characterized as the 4R* and 4S* diastereomers of 9-(2-deoxy-β- d - erythro pentofuranosyl)-7,8-dihydro-4-hydroxy-8-oxoguanine. In addition a third product, also resulting from a type II photooxidation, was identified as 8-oxo-7,8-dihydro-2'-deoxyguanosine. Quantification of these products provided a means to estimate the contribution of type I and type II pathways during the phthalocyanine and naphthalocyanine mediated photooxidation of 2'-deoxyguanosine, confirming the major role of singlet oxygen in these processes.     

8-oxoguanine and 8-oxodeoxyguanosine Biomarkers of Oxidative DNA Damage: A Review on HPLC–ECD Determination

Reactive oxygen species (ROS) are continuously produced in living cells due to metabolic and biochemical reactions and due to exposure to physical, chemical and biological agents. Excessive ROS cause oxidative stress and lead to oxidative DNA damage. Within ROS-mediated DNA lesions, 8-oxoguanine (8-oxoG) and its nucleotide 8-oxo-2′-deoxyguanosine (8-oxodG)—the guanine and deoxyguanosine oxidation products, respectively, are regarded as the most significant biomarkers for oxidative DNA damage. The quantification of 8-oxoG and 8-oxodG in urine, blood, tissue and saliva is essential, being employed to determine the overall effects of oxidative stress and to assess the risk, diagnose, and evaluate the treatment of autoimmune, inflammatory, neurodegenerative and cardiovascular diseases, diabetes, cancer and other age-related diseases. High-performance liquid chromatography with electrochemical detection (HPLC–ECD) is largely employed for 8-oxoG and 8-oxodG determination in biological samples due to its high selectivity and sensitivity, down to the femtomolar range. This review seeks to provide an exhaustive analysis of the most recent reports on the HPLC–ECD determination of 8-oxoG and 8-oxodG in cellular DNA and body fluids, which is relevant for health research.     

PHTHALOCYANINE AND NAPHTHALOCYANINE PHOTOSENSITIZED OXIDATION OF 2'-DEOXYGUANOSINE

Abstract— The photodynamic properties of the di-and tetrasulfonated zinc and aluminium phthalocyanines and a tetrasulfonated aluminium napththalocyanine were studied using 2'-deoxyguanosine as a DNA model compound. The major photooxidation products of this nucleoside were identified and classified according to their formation through a radical mechanism (type I) or a singlet oxygen mediated mechanism (type II). The major type I product was obtained and identified as 2,2-diamino [(2-deoxy-β- d - erythro pentofuranosyl)-4-amino]-5( 2H )-oxazolone. Two major type II products were characterized as the 4R* and 4S* diastereomers of 9-(2-deoxy-β- d - erythro pentofuranosyl)-7,8-dihydro-4-hydroxy-8-oxoguanine. In addition a third product, also resulting from a type II photooxidation, was identified as 8-oxo-7,8-dihydro-2'-deoxyguanosine. Quantification of these products provided a means to estimate the contribution of type I and type II pathways during the phthalocyanine and naphthalocyanine mediated photooxidation of 2'-deoxyguanosine, confirming the major role of singlet oxygen in these processes.     

LASER FLASH PHOTOKINETIC STUDIES OF ROSE BENGAL SENSITIZED PHOTODYNAMIC INTERACTIONS OF NUCLEOTIDES AND DNA

Abstract— Laser flash photolysis studies of the production of the triplet state of the xanthene dye, rose bengal (RB), have been carried out. The reactions of this state with oxygen to form singlet oxygen and the superoxide anion radical have been observed and yields measured. Quenching of RB(T₁) by oxygen leads to approximately 75% singlet oxygen and 20% superoxide. The reactivity of these species-RB(T₁), O₂(¹Δ_g) and O₂^-—with four nucleotides and DNA have been determined. Only guanine residues showed any noticeable reaction at neutral pH. At higher pH guanine rate constants increased. The consequences to biological photodynamic processes are discussed.     

ALTERATION OF GUANINE RESIDUES DURING PROFLAVINE MEDIATED PHOTOSENSITIZATION OF DNA

Abstract— Mild photodynamic treatments of proflavine-calf thymus DNA complexes induce a unique and quantitatively important alteration of the guanine residues which can be related to the lethal lesions due to the combined action of proflavine and light on phages. The 'altered guanine' is destroyed by HClO₄ but is recovered after partial DNA depurination under the form of two photoproducts. The first product, G_ox, elutes as guanine on a Sephadex column but has a modified UV absorbance spectrum. It gives rise by further irradiation to another product, X, which elutes at pH 9.7 as a pyrimidine compound and presented a maximal UV absorbance at 246 nm. Product X is also selectively released by piperidine fixation onto the photo-damaged DNA. The guanine degradation process is markedly decreased in the presence of the singlet oxygen quencher, NaN₃. The photodynamic lesion inhibits the enzymatic degradation of the DNA but generates locally denatured regions that are sensitive to S1 endonuclease.     

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.     

Singlet oxygen oxidation of isolated and cellular DNA: product formation and mechanistic insights

This survey focuses on recent aspects of the singlet oxygen oxidation of the guanine moiety of nucleosides, oligonucleotides, isolated and cellular DNA that has been shown to be the exclusive DNA target for this biologically relevant photogenerated oxidant. A large body of mechanistic data is now available from studies performed on nucleosides in both aprotic solvents and aqueous solutions. A common process to both reaction conditions is the formation of 8-oxo-7,8-dihydroguanine by reduction of 8-hydroperoxyguanine that arises from the rearrangement of initially formed endoperoxide across the 4,8-bond of the purine moiety. However, in organic solvent the hydroperoxide is converted as a major degradation pathway into a dioxirane that subsequently decomposes into a complex pattern of oxidation products. A different reaction that involved the formation of a highly reactive quinonoid intermediate consecutively to the loss of a water molecule from the 8-hydroperoxide has been shown to occur in aqueous solution. Subsequent addition of a water molecule at C5 leads to the generation of a spiroiminodihy-dantoin compound via a rearrangement that involves an acyl shift. However, in both isolated and cellular DNA the latter decomposition pathway is at the best a minor process, because only 8-oxo-7,8-dihydroguanine has been found to be generated. It is interesting to point out that singlet oxygen has been shown to contribute predominantly to the formation of 8-oxo-7,8-dihydroguanine in the DNA of bacterial and human cells upon exposure to UVA radiation. It may be added that the formation of secondary singlet-oxygen oxidation products of 8-oxo-7,8-dihydroguanine, including spiroiminodihydantoin and oxaluric acid that were characterized in nucleosides and oligonucleotide, respectively, have not yet been found in cellular DNA.     

钌(II)多吡啶配合物光断裂DNA的实验研究

研究了一系列钌(II)多吡啶配合物对pBR 322 DNA 的光断裂作用, 并与光谱法和粘度法的研究结果进行了对比. 实验结果表明, 钌(II)多吡啶配合物光断裂DNA的能力不仅与配合物与DNA相互作用的结合模式和结合强度有关, 还与配合物自身的电子结构有关; 钌(II)多吡啶配合物对DNA的光断裂存在立体选择性; 其断裂机理是激发态的配合物与溶液中的氧分子发生能量转移生成单线态氧活性氧化物种, 将鸟嘌呤碱基氧化而导致DNA断裂. 本研究对于遗传工程中的化学核酸酶以及以DNA为靶标的药物设计有重要的意义.     

HPLC-an Effective Method of DNA Fragment Determination

The role played by DNA in molecular biology is clearly recognized to be vital to life on this planet. 8-oxo-7,8-dihydro-2deoxyguanosine(=8-OHdG), is probably the most important product of "oxidative stress” in DNA. Its concentration in DNA is, in fact. a quantitative analysis of the degree of DNA damage that an organism has undergone. Due to the importance of 8-OHdG of nucleic acidg in mutagenesis, carcinogenesis and aging, numerous chemical and biological investigations have been made on this subject in the past time. Kuchino and co-workers have found that 8-OHdG residue in DNA is misreading during the process of DNA replication. Recently, some reports have been presented on high 8-OHdG levels in patients suffering from various diseases such as chronic hepatitis, Fanconi s anemia, diabetes mellitus and Helicobacter pylori infections. As a result, 8-OHdG is a useful marker for the study of DNA damage arising from reactive oxygen species and is of great significance for cancer research. The 8-OHdG levels in DNA can help understand the mechanism of carcinogens and lead to more effective treatments for many different types of cancer. For these reasons, an analysis of 8-OHdG with quickness, low cost and high accuracy is required.     

SINGLET OXYGEN INDUCES FRANK STRAND BREAKS AS WELL AS ALKALI- AND PIPERIDINE-LABILE SITES IN SUPERCOILED PLASMID DNA

A covalently closed, circular, supercoiled plasmid was exposed to singlet oxygen by a separated-surface sensitizer. For each exposure, the quantity of single oxygen entering the DNA target solution was estimated by its oxidation of histidine. After singlet oxygen exposure, some DNA samples were treated to disclose occult lesions. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed circular and linear species, and all bands were quantitated fluorometrically. Exposure of supercoiled plasmid DNA to singlet oxygen induced frank DNA strand breaks, alkali-labile sites (pH 12.5, 90 degrees C, 30 min), and piperidine-labile sites (0.4 M, 60 degrees C, 30 min), all in a dose-dependent manner. Yields of alkali-labile and piperidine-labile sites ranged from one to four times the frank strand break yield. Replacement of buffered H2O by buffered D2O as the DNA solvent for singlet oxygen exposures increased DNA lesion yields by a factor of 2.6 (averaged over lesion classes). Our data for the detection of frank strand breaks is at variance with published results from studies in which singlet oxygen was derived from a thermolabile endoperoxide dissolved in the DNA solution.     

Does the DNA Binding Mode of a Molecule Affect its Ability to Interact With Singlet Oxygen?

Singlet oxygen is known to be a potent mutagenic agent and several biologically relevant molecules have been proposed to act as scavengers for this noxious species. However, numerous studies have been conducted in homogenous solution and the reactivity of singlet oxygen scavengers known to bind DNA has never been investigated in double-stranded DNA. In the following paper, we present the results obtained regarding the interaction between 4',6-diamidino-2-phenylindole (DAPI) and singlet oxygen. We show the molecule to be a potent scavenger of singlet oxygen in aqueous solution with an absolute rate constant (chemical and physical quenching of singlet oxygen) of (1.7 ± 0.3) × 10⁷  m ⁻¹ s⁻¹. In addition, we demonstrate that the binding mode of a singlet oxygen scavenger to DNA can strongly influence its reactivity toward singlet oxygen. In the case of DAPI, while the molecule exhibits a chemical reaction with singlet oxygen when the molecule is free in aqueous solution or intercalated in GC sequences of DNA, DAPI becomes chemically unreactive toward singlet oxygen when bound in the minor groove of DNA AT sequences.     

CHEMISTRY OF SINGLET OXYGEN—48. ISOLATION and STRUCTURE OF THE PRIMARY PRODUCT OF PHOTOOXYGENATION OF 3,5-DI-t-BUTYL CATECHOL

The dye-sensitized photooxygenation of t -butyl substituted catechols has been investigated. The primary product from 3,5-di- t -butyl catechol has been isolated and shown to be a hydroperoxydienone by single crystal X-ray diffraction. The absence of sensitizer effects and the faster reaction rate in polar solvents suggest that the reaction proceeds with singlet oxygen as the primary oxygenating species. Charge-transfer or full electron-transfer from the catechol to singlet oxygen is probably involved. Substituent effects are in agreement with this mechanism. The products from thermal breakdown of the hydroperoxydienone are inconsistent with a Baeyer-Villiger mechanism.     

Secondary reactive oxygen species extend the range of photosensitization effects in cells: DNA damage produced via initial membrane photosensitization

The type-II photosensitization process is mediated by the formation of singlet oxygen (O2[1deltag]). The short lifetime of this species dictates that chemical reactions with biological substrates can only occur when O2(1deltag) is in very close proximity to the photosensitizer itself. In this study, deuteroporphyrin, a type-II, membrane-localized photosensitizer, was used to generate O2(1deltag) in human lymphoblast WTK-1 cells, and the range of influence was determined by a variety of biological assays. Surprisingly, the initial membrane-confined events were shown, by comet assay, to induce DNA damage in these cells. DNA damage was inhibited both by membrane-localized (alpha-tocopherol acetate) and by cytoplasmic (trolox) free radical scavengers. Comet formation also was inhibited by treatment at low temperature. DNA fragmentation was not influenced by treatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, showing that apoptosis was not responsible for fragmentation. Taken together, these results show that primary photosensitization reactions involving O2(1deltag), even when tightly confined in extranuclear locations, leads to the production of secondary reactive oxygen species, probably as a result of lipid peroxidation, that can act at greater distances from the photosensitizer itself. These experiments were carried out under conditions where cell survival was significant and raise questions regarding DNA damage and mutagenesis pathways, even when extranuclear O2(1deltag)-generating compounds are used.     

Synthesis and characterization of the oxidized dGTP lesions spiroiminodihydantoin-2'-deoxynucleoside-5'- triphosphate and guanidinohydantoin-2'-deoxynucleoside-5'- triphosphate

Two convenient synthetic routes to the oxidized guanosine triphosphate lesions spiroiminodihydantoin-2'-deoxynucleoside-5'-triphosphate (dSpTP) and guanidinohydantoin-2'-deoxynucleoside-5'-triphosphate (dGhTP) are reported. Both two-electron oxidation of 2'-deoxy-7,8-dihydro-8-oxoguanosine-5'-triphosphate (dOGTP) using SO4*- generated photolytically from K2S2O8 or four-electron oxidation of 2'-deoxyguanosine-5'-triphosphate (dGTP) from singlet oxygen provide either dSpTP or dGhTP at pH 8.0 or 4.4, respectively. Highly purified triphosphates are obtained by ion pair reversed-phase HPLC.