Mechanisms of free radical-induced damage to DNA

Abstract

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5′-cyclopurine-2′-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.     

DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems.

When cells are exposed to oxidative stress, DNA damage frequently occurs. The molecular mechanisms causing this damage may include activation of nucleases and direct reaction of hydroxyl radicals with the DNA. Several oxygen-derived species can attack DNA, producing distinctive patterns of chemical modification. Observation of these patterns and measurement of some of the products formed has been used to determine the role of different oxygen-derived species in DNA cleavage reactions, to assess the extent of oxidative damage to DNA in vivo and to investigate the mechanism of DNA damage by ionizing radiation and chemical carcinogens.     

The effect of reductant levels on the formation of damage lesions derived from a 2-deoxyribose radical in ssDNA

Thiols play a major role in the outcome of oxidative damage to DNA when it is initiated through cellular exposure to ionizing radiation. DNA radicals formed under aerobic conditions are converted to peroxyl radicals through trapping by oxygen at a diffusion-controlled rate. As a primary source of cellular reductant, thiols are responsible for the conversion of these DNA-derived peroxyl radicals to their corresponding hydrogen peroxides and subsequent strand breaks. Through the use of modified nucleotides, which act as precursors to nucleic acid radicals, we have investigated the effect of varying amounts of the cellular thiol glutathione (GSH) on the distribution of damage products produced from a 2-deoxyribose radical in DNA: the C3'-thymidinyl radical. The C3'-thymidinyl radical results from the abstraction of a hydrogen atom from the C3'-position of DNA oligomers at a thymidine residue, and is known to deliver several DNA damage lesions including the 3'-phosphoglycolaldehyde, 3'-phosphoglycolate and a 5'-aldehyde. Here we show that the level of GSH present has an impact on the level of production of these C3'-thymidinyl radical derived damage products.     

鸟嘌呤碱基与羟基自由基反应的密度泛函理论

羟基自由基 (·OH)进攻嘌呤碱基是破坏核酸造成DNA断链损伤的重要原因之一 .采用密度泛函 (DFT)理论中B3LYP方法在 6— 31G基组水平上对鸟嘌呤 (G)受羟基自由基进攻形成的各种可能产物自由基进行几何全优化 .根据总能量、键长和自旋密度的计算结果 ,从理论上确认了C 5和C 8位加成机制 .得产物自由基G5OH·、G8OH· ,且G5OH·易与N 11位H脱水得一个更稳定的产物自由基 ,而G8OH·不易发生开环反应 ,得到与实验一致的结论 .这些稳定自由基的形成造成DNA断链损伤     

Measurement of oxidatively induced DNA damage and its repair,by mass spectrometric techniques

Abstract

Oxidatively induced damage caused by free radicals and other DNA-damaging agents generate a plethora of products in the DNA of living organisms. There is mounting evidence for the involvement of this type of damage in the etiology of numerous diseases including carcinogenesis. For a thorough understanding of the mechanisms, cellular repair, and biological consequences of DNA damage, accurate measurement of resulting products must be achieved. There are various analytical techniques, with their own advantages and drawbacks, which can be used for this purpose. Mass spectrometric techniques with isotope dilution, which include gas chromatography (GC) and liquid chromatography (LC), provide structural elucidation of products and ascertain accurate quantification, which are absolutely necessary for reliable measurement. Both gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), in single or tandem versions, have been used for the measurement of numerous DNA products such as sugar and base lesions, 8,5’-cyclopurine-2’-deoxynucleosides, base-base tandem lesions, and DNA-protein crosslinks, in vitro and in vivo. This article reviews these techniques and their applications in the measurement of oxidatively induced DNA damage and its repair.     

Hydrogen peroxide-induced base damage in deoxyribonucleic acid

Aqueous solutions of calf thymus deoxyribonucleic acid (DNA) were exposed to hydrogen peroxide in the presence of air. Base products formed in DNA were identified and quantitated following acid hydrolysis and trimethylsilylation using gas chromatography-mass spectrometry. The yields of these products were dependent upon the hydrogen peroxide concentration, and increased in the following order: 8-hydroxyadenine, cytosine glycol, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine, thymine glycol, and 4,6-diamino-5-formamidopyrimidine. Previous studies have shown that these compounds are typically formed in DNA in aqueous solution by hydroxyl radicals generated by ionizing radiation. Hydrogen peroxide is thought to participate in a Fenton-like reaction with transition metals, which are readily bound to DNA in trace quantities, resulting in the production of hydroxyl radicals close to the DNA. This proposed mechanism was examined by exposing DNA to hydrogen peroxide either in the presence of a hydroxyl radical scavenger or following pretreatment of DNA with metal-ion chelators. The results indicate that trace quantities of transition metal ions can react readily with hydrogen peroxide to produce radical species. The production of radical species was monitored by determining the altered bases that resulted from the reaction between radicals and DNA. The yields of the base products were reduced by 40 to 60% with 10 mmol dm-3 of dimethyl sulfoxide. A 100-fold increase in the concentration of dimethyl sulfoxide did not result in a further reduction in hydrogen peroxide-induced base damage. DNA which was freed from bound metal ions by pretreatment with metal ion chelators followed by exhaustive dialysis was found to be an ineffective substrate for hydrogen peroxide. The yields of base products measured in this DNA were at background levels. These results support the role of metal ions bound to DNA in the site-specific formation of highly reactive radical species, most likely hydroxyl radicals, in hydrogen peroxide-induced damage to the bases in DNA.     

Myeloperoxidase-induced Genomic DNA-centered Radicals

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.     

Carcinogenic nitrosamines: Free radical aspects of their action

NDMA and other nitrosamines may be activated into DNA binding intermediates by a cytochrome P450-dependent formation of -nitrosamino radicals or photochemically. Within the catalytic site of cytochrome P450, these radical intermediates either combine with HO· to form -hydroxynitrosamines or decompose into nitric oxide and N-methylformaldimine. In the presence of phosphate, mutagenic -phosphonooxy derivatives are formed from radicals generated chemically/photochemically. Studies on lipid peroxidation, in vivo and in vitro, have further suggested that radicals are formed as intermediates from N-nitrosodialkylamines. The level of nitrosamine-induced lipid peroxidation parallels hepatocartgenicity in rats. These data, although preliminary, provide further evidence that free radical damage and DNA alkylation are involved in carcinogenesis induced by nitrosamines.     

The role of reactive N-bromo species and radical intermediates in hypobromous acid-induced protein oxidation

Activated eosinophils, and hypobromous acid (HOBr) generated by these cells, have been implicated in the tissue injury in asthma, allergic reactions, and some infections. Proteins are major targets for this oxidant, but limited information is available on the mechanisms of damage and intermediates formed. Reaction of HOBr with proteins is shown to result in the formation of bromamines and bromamides, from side-chain and backbone amines and amides, and 3-bromo- and 3,5-dibromo-Tyr, from Tyr residues; these materials account for ca. 70% of the oxidant consumed. Protein carbonyls, dityrosine, and 3,4-dihydroxyphenylalanine are also formed, though these are minor products (<5% of HOBr added). With BSA, extensive (selective and nonspecific) protein fragmentation and limited aggregation are also observed. The bromamines/bromamides are unstable and induce further oxidation and free radical formation as detected by EPR spin trapping. Evidence was obtained for the generation of nitrogen-centered radicals on side-chain and backbone amide groups of amino acids, peptides, and proteins. These radicals readily undergo rearrangement reactions to give carbon-centered radicals. With proteins, alpha-carbon (backbone) radicals are detected, which may play a role in protein fragmentation. A novel damage transfer pathway from Gln side-chain amide groups to backbone sites was also observed.     

Substrate specificities and excision kinetics of DNA glycosylases involved in base-excision repair of oxidative DNA damage

Reactive oxygen-derived species such as free radicals are formed in living cells by normal metabolism and exogenous sources, and cause a variety of types of DNA damage such as base and sugar damage, strand breaks and DNA-protein cross-links. Living organisms possess repair systems that repair DNA damage. Oxidative DNA damage caused by free radicals and other oxidizing agents is mainly repaired by base-excision repair (BER), which involves DNA glycosylases in the first step of the repair process. These enzymes remove modified bases from DNA by hydrolyzing the glycosidic bond between the modified base and the sugar moiety, generating an apurinic/apyrimidinic (AP) site. Some also possess AP lyase activity that subsequently cleaves DNA at AP sites. Many DNA glycosylases have been discovered and isolated, and their reaction mechanisms and substrate specificities have been elucidated. Most of the known products of oxidative damage to DNA are substrates of DNA glycosylases with broad or narrow substrate specificities. Some possess cross-activity and remove both pyrimidine- and purine-derived lesions. Overlapping activities between enzymes also exist. Studies of substrate specificities have been performed using either oligodeoxynucleotides with a single modified base embedded at a specific position or damaged DNA substrates containing a multiplicity of pyrimidine- and purine-derived lesions. This paper reviews the substrate specificities and excision kinetics of DNA glycosylases that have been investigated with the use of gas chromatography/mass spectrometry and DNA substrates with multiple lesions.     

Chemical and molecular mechanisms of antioxidants: experimental approaches and model systems

Free radicals derived from oxygen, nitrogen and sulphur molecules in the biological system are highly active to react with other molecules due to their unpaired electrons. These radicals are important part of groups of molecules called reactive oxygen/nitrogen species (ROS/RNS), which are produced during cellular metabolism and functional activities and have important roles in cell signalling, apoptosis, gene expression and ion transportation. However, excessive ROS attack bases in nucleic acids, amino acid side chains in proteins and double bonds in unsaturated fatty acids, and cause oxidative stress, which can damage DNA, RNA, proteins and lipids resulting in an increased risk for cardiovascular disease, cancer, autism and other diseases. Intracellular antioxidant enzymes and intake of dietary antioxidants may help to maintain an adequate antioxidant status in the body. In the past decades, new molecular techniques, cell cultures and animal models have been established to study the effects and mechanisms of antioxidants on ROS. The chemical and molecular approaches have been used to study the mechanism and kinetics of antioxidants and to identify new potent antioxidants. Antioxidants can decrease the oxidative damage directly via reacting with free radicals or indirectly by inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes. The new chemical and cell-free biological system has been applied in dissecting the molecular action of antioxidants. This review focuses on the research approaches that have been used to study oxidative stress and antioxidants in lipid peroxidation, DNA damage, protein modification as well as enzyme activity, with emphasis on the chemical and cell-free biological system.     

New Aspects in the Free-Radical Chemistry of Pyrimidine Nucleobases

The contribution will cover three aspects:

i) It has been known for some time that OH radicals and H atoms react with the pyrimidines by adding to the C(5)-C(6) double bond, but only the u.v.-spectra of the sum of these radicals have been reported so far. It will be shown how to arrive at the individual spectra of the C(5) and the C(6) adduct radicals.

ii) α-Hydroxyalkyl radicals are known to inactivate biologically active DNA. In contrast to the electrophilic radicals H and OH they are nucleophilic and the hydroxymethyl radicals add exclusively at the C(6) position of 1,3-dimethyluracil (k ～ 10⁴dm³ mol^?1 s^?1). In the corresponding thymine derivative this reaction also occurs, but one third of the hydroxymethyl radicals abstract an H-atom from the C(5)-methyl group thereby forming an allylic radical. In the course of these reactions pyrimidines with an exocyclic double bond are formed. These products react much more rapidly with hydroxymethyl radicals than the starting material leading to highly hydroxymethylated material at very low doses.

iii) The direct effect of ionizing radiation which would produce a pyrimidine base radical cation can be mimicked by reacting the pyrimidine with SO₄^?, a very good electron acceptor. In water, the radical cation of 1,3-dimethyluracil is rapidly (t_1/2 2μs) converted into the C(5) OH adduct radical. In the presence of peroxodisulphate a chain reaction sets in which leads to the cis-glycol.

The relevance of these findings to radiobiological aspects of nucleic acid research will be discussed.     

Mechanisms of free radical-induced damage to DNA

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5'-cyclopurine-2'-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.     

Free radical-induced damage to DNA: mechanisms and measurement

Free radicals are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells, including DNA. The resulting damage to DNA, which is also called oxidative damage to DNA, is implicated in mutagenesis, carcinogenesis, and aging. Mechanisms of damage involve abstractions and addition reactions by free radicals leading to carbon-centered sugar radicals and OH- or H-adduct radicals of heterocyclic bases. Further reactions of these radicals yield numerous products. Various analytical techniques exist for the measurement of oxidative damage to DNA. Techniques that employ gas chromatography (GC) or liquid chromatography (LC) with mass spectrometry (MS) simultaneously measure numerous products, and provide positive identification and accurate quantification. The measurement of multiple products avoids misleading conclusions that might be drawn from the measurement of a single product, because product levels vary depending on reaction conditions and the redox status of cells. In the past, GC/MS was used for the measurement of modified sugar and bases, and DNA-protein cross-links. Recently, methodologies using LC/tandem MS (LC/MS/MS) and LC/MS techniques were introduced for the measurement of modified nucleosides. Artifacts might occur with the use of any of the measurement techniques. The use of proper experimental conditions might avoid artifactual formation of products in DNA. This article reviews mechanistic aspects of oxidative damage to DNA and recent developments in the measurement of this type of damage using chromatographic and mass spectrometric techniques.     

Formation of TEMPOL-hydroxylamine during reaction between TEMPOL and hydroxyl radical: HPLC/ECD study

Nitroxyl radicals are important antioxidants that have been used to protect animal tissues from oxidative damage. Their reaction with hydroxyl radical (^?OH) is generally accepted to be the mechanism of antioxidant function. However, the direct interaction of nitroxyl radicals with ^?OH does not always provide a satisfactory explanation in various pH, because the concentration of hydrogen ion may affect the generation of secondary ^?OH-derived radicals. In the present study, it was confirmed that the reaction between 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and ^?OH generated TEMPOL-hydroxylamine, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPON-hydroxylamine using HPLC coupled with electrochemical detection. In the absence of NADH, TEMPOL-H may be generated by the reaction with secondary ^?OH-derived radicals in acidic condition. In the presence of NADH, a large proportion of the non-paramagnetic products was TEMPOL-H. Finally, it was clarified that TEMPOL-H was generated during dopamine metabolism, which is believed to be one of the ^?OH sources in pathological processes such as Parkinson's disease.     

Formation of ring-opened and rearranged products of guanine: mechanisms and biological significance

DNA damage by endogenous and exogenous agents is a serious concern, as the damaged products can affect genome integrity severely. Damage to DNA may arise from various factors such as DNA base modifications, strand break, inter- and intrastrand crosslinks, and DNA-protein crosslinks. Among these factors, DNA base modification is a common and important form of DNA damage that has been implicated in mutagenesis, carcinogenesis, and many other pathological conditions. Among the four DNA bases, guanine (G) has the smallest oxidation potential, because of which it is frequently modified by reactive species, giving rise to a plethora of lethal lesions. Similarly, 8-oxo-7,8-dihydroguanine (8-oxoG), an oxidatively damaged guanine lesion, also undergoes various degradation reactions giving rise to several mutagenic species. The various products formed from reactions of G or 8-oxoG with different reactive species are mainly 2,6-diamino-4-oxo-5-formamidopyrimidine, 2,5-diamino-4H-imidazolone, 2,2,4-triamino-5-(2H)-oxazolone, 5-guanidino-4-nitroimidazole, guanidinohydantoin, spiroiminodihydantoin, cyanuric acid, parabanic acid, oxaluric acid, and urea, among others. These products are formed from either ring opening or ring opening and subsequent rearrangement. The main aim of this review is to provide a comprehensive overview of various possible reactions and the mechanisms involved, after which these ring-opened and rearranged products of guanine would be formed in DNA. The biological significance of oxidatively damaged products of G is also discussed.     

Identification by electron spin resonance spectroscopy of free radicals produced during autoxidative melanogenesis

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH⁻ substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.     

Radiation-activated nuclease activity of o,o'-Diphenyleneiodonium cations (DPI): a reductively initiated chain reaction involving the C1' chemistry

o,o'-Diphenyleneiodonium cations (DPI) convert relatively harmless radiation-produced electrons into efficient DNA cleaving agents. The cleavage products are unaltered DNA bases, 5-methylenefuranone (5-MF), and a complete set of 3' and 5'-phosphorylated DNA fragments. The production of alkali-labile sites is a minor factor in the process. Based on the production of 5-MF, it is concluded that DNA cleavage by DPI cations involves (but may not be limited to) the C1' chemistry. The loss of 3-aminoDPI (ADPI) cations bound to highly polymerized calf thymus DNA appears to be due to a short-chain reaction with an apparent length of up to 2.1 ADPI cations consumed for each radiation-produced electron. The suggested chain reaction mechanism includes the one-electron oxidation of DNA radicals (including the C1' sugar radical) by ADPI cations bound to the same duplex. The yields of DNA loss in complexes formed by ADPI with short synthetic duplexes indicate that there is more than a 60% probability of DNA damage after one-electron reduction of ADPI.     

Intrastrand G-U cross-links generated by the oxidation of guanine in 5'-d(GCU) and 5'-r(GCU)

It has been suggested that carbonate radical anions are biologically important because they may be produced during the inflammatory response. The carbonate radicals can selectively oxidize guanine in DNA and RNA by one-electron transfer mechanisms and the guanine radicals thus formed decay by diverse competing pathways with other free radicals or nucleophiles. Using a photochemical method to generate CO(3)(-) radicals in vitro, we compare the distributions of products initiated by the one-electron oxidation of guanine in the trinucleotides 5'-r(GpCpU) and 5'-d(GpCpU) in aqueous buffer solutions (pH 7.5). Similar distributions of stable end products identified by LC-MS/MS methods were found in both cases. The guanine oxidation products include the diastereomeric pair of spiroiminodihydantoin (Sp) and 2,5-diamino-4H-imidazolone (Iz). In addition, intrastrand cross-linked products involving covalent bonds between the G and the U bases (GCU) were also found, although with different relative yields in the 2'-deoxy- and the ribotrinucleotides. The positive-ion MS/MS spectra of the 5'-r(GpCpU) and 5'-d(GpCpU) products clearly indicate the presence of covalently linked G-U products that have a mass smaller by 2 Da than the sum of the G and U bases in both types of trinucleotides. The 5'-d(GCU) cross-linked product was further characterized by 1D and 2D NMR methods that confirm its cyclic structure in which the guanine C8 atom is covalently linked to the uracil N3 atom.     

Immunofluorescent detection of 8-oxoguanine DNA lesions in liver cells from aging OXYS rats, a strain prone to overproduction of free radicals

Production of free radicals in animals is accompanied with a number of pathologic conditions, some of which may be manifested through DNA damage. Studies of mechanisms of oxidative DNA damage by free radicals in vivo are hindered by the lack of good animal models with significant overgeneration of or increased sensitivity to free radicals. An inbred rat strain (OXYS) is characterized by inherited overgeneration of free radicals, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases. We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative age-dependent amounts and distribution of 8-oxoG in liver cells from OXYS and Wistar rats. 8-OxoG increased with age in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. Statistical analysis indicates that 8-oxoG does not uniformly accumulate in all cells with advancing age or increasing free radical load, but rather concentrates in a minor fraction of cells with a high damage level.