Free radicals and other oxidants have gained importance in the field of biology due to their central role in various physiological conditions as well as their implication in a diverse range of diseases. The free radicals, both the reactive oxygen species (ROS) and reactive nitrogen species (RNS), are derived from both endogenous sources (mitochondria, peroxisomes, endoplasmic reticulum, phagocytic cells etc.) and exogenous sources (pollution, alcohol, tobacco smoke, heavy metals, transition metals, industrial solvents, pesticides, certain drugs like halothane, paracetamol, and radiation). Free radicals can adversely affect various important classes of biological molecules such as nucleic acids, lipids, and proteins, thereby altering the normal redox status leading to increased oxidative stress. The free radicals induced oxidative stress has been reported to be involved in several diseased conditions such as diabetes mellitus, neurodegenerative disorders (Parkinson’s disease-PD, Alzheimer’s disease-AD and Multiple sclerosis-MS), cardiovascular diseases (atherosclerosis and hypertension), respiratory diseases (asthma), cataract development, rheumatoid arthritis and in various cancers (colorectal, prostate, breast, lung, bladder cancers). This review deals with chemistry, formation and sources, and molecular targets of free radicals and it provides a brief overview on the pathogenesis of various diseased conditions caused by ROS/RNS.

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Free Radicals: Properties, Sources, Targets, and Their Implication in Various Diseases

Emphasis has been placed in this article dedicated to DNA damage on recent aspects of the formation and measurement of oxidatively generated damage in cellular DNA in order to provide a comprehensive and updated survey. This includes single pyrimidine and purine base lesions, intrastrand cross-links, purine 5',8-cyclonucleosides, DNA-protein adducts and interstrand cross-links formed by the reactions of either the nucleobases or the 2-deoxyribose moiety with the hydroxyl radical, one-electron oxidants, singlet oxygen, and hypochlorous acid. In addition, recent information concerning the mechanisms of formation, individual measurement, and repair-rate assessment of bipyrimidine photoproducts in isolated cells and human skin upon exposure to UVB radiation, UVA photons, or solar simulated light is critically reviewed.

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DNA Base Damage by Reactive Oxygen Species, Oxidizing Agents, and UV Radiation

Modified purine and pyrimidine bases constitute one of the major classes of hydroxyl-radical-mediated DNA damage together with oligonucleotide strand breaks, DNA-protein cross-links and abasic sites. A comprehensive survey of the main available data on both structural and mechanistic aspects of.OH-induced decomposition pathways of both purine and pyrimidine bases of isolated DNA and model compounds is presented. In this respect, detailed information is provided on both thymine and guanine whereas data are not as complete for adenine and cytosine. The second part of the overview is dedicated to the formation of.OH-induced base lesions within cellular DNA and in vivo situations. Before addressing this major point, the main available methods aimed at singling out.OH-mediated base modifications are critically reviewed. Unfortunately, it is clear that the bulk of the chemical and biochemical assays with the exception of the high performance liquid chromatographic-electrochemical detection (HPLC/ECD) method have suffered from major drawbacks. This explains why there are only a few available accurate data concerning both the qualitative and quantitative aspects of the.OH-induced formation of base damage within cellular DNA. Therefore, major efforts should be devoted to the reassessment of the level of oxidative base damage in cellular DNA using appropriate assays including suitable conditions of DNA extraction.

Hydroxyl radicals and DNA base damage

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 importan...

Mechanisms of free radical-induced damage to DNA

Oxidative DNA damage is a major cause of cell death and mutagenesis in all aerobic organisms, and several new oxidative base lesions have been identified in recent years. Improved chemistry for the synthesis of oligonucleotides with modified base residues at defined positions has allowed detailed studies of repair, replication, transcription and mutagenesis at specific lesions in vitro and in vivo. The aim of this review is to present the structure of all the various known oxidised DNA base lesions known to date and to summarise the present knowledge about the mutagenic and toxic effects of oxidised base modifications and their repair.

Mutagenicity, toxicity and repair of DNA base damage induced by oxidation.

Singlet oxygen, hydrogen peroxide, hydroxyl radical and hydrogen peroxide are the reactive oxygen species (ROS) considered most responsible for producing oxidative stress in cells and organisms. Singlet oxygen interacts preferentially with guanine to produce 8-oxo-7,8-dihydroguanine and spiroiminodihydantoin. DNA damage due to the latter lesion has not been detected directly in the DNA of cells exposed to singlet oxygen. In this study, the singlet oxygen-induced lesion was isolated from a short synthetic oligomer after exposure to UVA radiation in the presence of methylene blue. The lesion could be enzymatically excised from the oligomer in the form of a modified dinucleoside monophosphate. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the singlet oxygen lesion was detected in the form of modified dinucleoside monophosphates in double-stranded DNA and in the DNA of HeLa cells exposed to singlet oxygen. Pentamer containing the singlet oxygen-induced lesion and an isotopic label was synthesized as an internal standard for quantifying the lesion and served as well as for correcting for losses of product during sample preparation.

Singlet oxygen-induced DNA damage.

A new type of tandem base lesion has been observed in d(CpGpTpA) X-irradiated in aqueous solution. The lesion is attributed to the formation of a covalent bond between the C8 carbon atom of guanine and the methyl carbon atom of thymine. This tandem base lesion is formed in the absence of oxygen. It is the main product produced by ionizing radiation under these conditions.

Radiation-induced formation of a crosslink between base moieties of deoxyguanosine and thymidine in deoxygenated solutions of d(CpGpTpA).

Irradiation of the dinucleoside monophosphate d(GpT) in an oxygenated solution gives products characterized by damage on one or both guanine and thymine bases, the yields of which were proportional to radiation dose.

Vicinal lesions in X-irradiated DNA?

A DNA lesion which results from the breakdown of a pyrimidine base leaving a formamido remnant has been associated with oxidative stress This lesion is shown to be produced in keratinocytes irradiated in culture with UVB light The amount of formamido lesion produced is comparable to the amount of the 8-hydroxyguanine lesion The two lesions were measured by 32 P-postlabeling and electrochemical detection methods respectively

DNA damage in UVB-irradiated keratinocytes

Dinucleoside monophosphates in which the 5' nucleoside contained a radiation-modified base were tested as substrates to bovine spleen phosphodiesterase (SPD) and snake venom phosphodiesterase. The radiation-modified bases included thymine glycols, 5-hydroxymethyluracil, 8-hydroxyguanine, and a formamido remnant of thymine. The lesions had widely different effects on diesterase action, varying from little inhibition, as in the case of digestion of dT*pA by SPD, where T* is the hydroxymethyluracil modification, to severe inhibition, as in the case of digestion of dG*pC by SPD, where G* is the 8-hydroxyguanine modification.

Marianne S. Evans

Papers

Singlet oxygen-induced DNA damage.

Radiation-induced formation of a crosslink between base moieties of deoxyguanosine and thymidine in deoxygenated solutions of d(CpGpTpA).

Vicinal lesions in X-irradiated DNA?

DNA damage in UVB-irradiated keratinocytes

Enzymatic excision of radiation-induced lesions from DNA model compounds.