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This review article is aimed at providing updated information on the contribution of immediate and delayed oxidative reactions to the photo-induced damage to cellular DNA/skin under exposure to UVB/UVA radiations and visible light. Low-intensity UVC and UVB radiations that operate predominantly through direct excitation of the nucleobases are very poor oxidizing agents giving rise to very low amounts of 8-oxo-7,8-dihydroguanine and DNA strand breaks with respect to the overwhelming bipyrimidine dimeric photoproducts. The importance of these two classes of oxidatively generated damage to DNA significantly increases together with a smaller contribution of oxidized pyrimidine bases upon UVA irradiation. This is rationalized in terms of sensitized photooxidation reactions predominantly mediated by singlet oxygen together with a small contribution of hydroxyl radical that appear to also be implicated in the photodynamic effects of the blue light component of visible light. Chemiexcitation-mediated formation of "dark" cyclobutane pyrimidine dimers in UVA-irradiated melanocytes is a recent major discovery that implicates in the initial stage, a delayed generation of reactive oxygen and nitrogen species giving rise to triplet excited carbonyl intermediate and possibly singlet oxygen. High-intensity UVC nanosecond laser radiation constitutes a suitable source of light to generate pyrimidine and purine radical cations in cellular DNA via efficient biphotonic ionization., (© 2024 American Society for Photobiology.)

Molecular oxygen sensitizes DNA to damage induced by ionizing radiation, Fenton-like reactions, and other free radical-mediated reactions. It rapidly converts carbon-centered radicals within DNA into peroxyl radicals, giving rise to a plethora of oxidized products consisting of nucleobase and 2-deoxyribose modifications, strand breaks and abasic sites. The mechanism of formation of single oxidation products has been extensively studied and reviewed. However, much evidence shows that reactive peroxyl radicals can propagate damage to vicinal components in DNA strands. These intramolecular reactions lead to the dual alteration of two adjacent nucleotides, designated as tandem or double lesions. Herein, current knowledge about the formation and biological implications of oxidatively generated DNA tandem lesions is reviewed. Thus far, most reported tandem lesions have been shown to arise from peroxyl radicals initially generated at pyrimidine bases, notably thymine, followed by reaction with 5'-flanking bases, especially guanine, although contiguous thymine lesions have also been characterized. Proper biomolecular processing is impaired by several tandem lesions making them refractory to base excision repair and potentially more mutagenic., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Several classes of oxidatively generated DNA damage including oxidized purine and pyrimidine bases, interstrand base crosslinks and DNA-protein crosslinks have been previously shown to be generated in both isolated DNA and cellular DNA upon exposure to either 266-nm laser irradiation or one-electron oxidants. In this study, we provide evidence that biphotonic ionization of guanine bases by UVC laser irradiation of double-stranded deoxyoligonucleotides in aerated aqueous solutions induces the formation of interstrand crosslinks (ICLs). This is supported by various experiments including sequencing gel analyses of formed photoproducts and effects of UVC laser intensity on their formation. This constitutes a novel example of the diversity of reactions of guanine radical cation that can be generated by various one-electron oxidants including UVC laser biphotonic ionization, direct effect of ionization radiation and type I photosensitizers. However, the exact structure of the interstrand base adducts that is a challenging analytical issue remains to be further established. Examples of relevant biochemical/structural applications of biphotonic induction of ICLs in DNA samples by high-intensity UVC laser pulses are provided., (© 2021 American Society for Photobiology.)

UV-induced DNA damage plays a key role in the initiation phase of skin cancer. When left unrepaired or when damaged cells are not eliminated by apoptosis, DNA lesions express their mutagneic properties, leading to the activation of proto-oncogene or the inactivation of tumor suppression genes. The chemical nature and the amount of DNA damage strongly depend on the wavelength of the incident photons. The most energetic part of the solar spectrum at the Earth's surface (UVB, 280-320 nm) leads to the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (64PPs). Less energetic but 20-times more intense UVA (320-400 nm) also induces the formation of CPDs together with a wide variety of oxidatively generated lesions such as single strand breaks and oxidized bases. Among those, 8-oxo-7,8-dihydroguanine (8-oxoGua) is the most frequent since it can be produced by several mechanisms. Data available on the respective yield of DNA photoproducts in cells and skin show that exposure to sunlight mostly induces pyrimidine dimers, which explains the mutational signature found in skin tumors, with lower amounts of 8-oxoGua and strand breaks. The present review aims at describing the basic photochemistry of DNA and discussing the quantitative formation of the different UV-induced DNA lesions reported in the literature. Additional information on mutagenesis, repair and photoprotection is briefly provided.

In this review article, emphasis is placed on the critical survey of available data concerning modified nucleobase and 2-deoxyribose products that have been identified in cellular DNA following exposure to a wide variety of oxidizing species and agents including, hydroxyl radical, one-electron oxidants, singlet oxygen, hypochlorous acid and ten-eleven translocation enzymes. In addition, information is provided about the generation of secondary oxidation products of 8-oxo-7,8-dihydroguanine and nucleobase addition products with reactive aldehydes arising from the decomposition of lipid peroxides. It is worth noting that the different classes of oxidatively generated DNA damage that consist of single lesions, intra- and interstrand cross-links were unambiguously assigned and quantitatively detected on the basis of accurate measurements involving in most cases high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The reported data clearly show that the frequency of DNA lesions generated upon severe oxidizing conditions, including exposure to ionizing radiation is low, at best a few modifications per 10 6 normal bases. Application of accurate analytical measurement methods has also allowed the determination of repair kinetics of several well-defined lesions in cellular DNA that however concerns so far only a restricted number of cases., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

Genomic DNA is prone to a large number of insults by a myriad of endogenous and exogenous agents. The base excision repair (BER) is the major mechanism used by cells for the removal of various DNA lesions spontaneously or environmentally induced and the maintenance of genome integrity. The presence of persistent DNA damage is not compatible with life, since abrogation of BER leads to early embryonic lethality in mice. There are several lines of evidences showing existence of a link between deficient BER, cancer proneness and ageing, thus illustrating the importance of this DNA repair pathway in human health. Although the enzymology of BER mechanisms has been largely elucidated using chemically defined DNA damage substrates and purified proteins, the complex interplay of BER with another vital process like transcription or when DNA is in its natural state (i.e. wrapped in nucleosome and assembled in chromatin fiber is largely unexplored. Cells use chromatin remodeling factors to overcome the general repression associated with the nucleosomal organization. It is broadly accepted that energy-dependent nucleosome remodeling factors disrupt histones-DNA interactions at the expense of ATP hydrolysis to favor transcription as well as DNA repair. Importantly, unlike transcription, BER is not part of a regulated developmental process but represents a maintenance system that should be efficient anytime and anywhere in the genome. In this review we will discuss how BER can deal with chromatin organization to maintain genetic information. Emphasis will be placed on the following challenging question: how BER is initiated within chromatin?, (Copyright © 2017 Elsevier Inc. All rights reserved.)

This introductory article should be viewed as a prologue to the Free Radical Biology & Medicine Special Issue devoted to the important topic of Oxidatively Damaged DNA and its Repair. This special issue is dedicated to Professor Tomas Lindahl, co-winner of the 2015 Nobel Prize in Chemistry for his seminal discoveries in the area repair of oxidatively damaged DNA. In the past several years it has become abundantly clear that DNA oxidation is a major consequence of life in an oxygen-rich environment. Concomitantly, survival in the presence of oxygen, with the constant threat of deleterious DNA mutations and deletions, has largely been made possible through the evolution of a vast array of DNA repair enzymes. The articles in this Oxidatively Damaged DNA & Repair special issue detail the reactions by which intracellular DNA is oxidatively damaged, and the enzymatic reactions and pathways by which living organisms survive such assaults by repair processes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

UV-induced formation of pyrimidine dimers in DNA is a major deleterious event in both eukaryotic and prokaryotic cells. Accumulation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts can lead to cell death or be at the origin of mutations. In skin, UV induction of DNA damage is a major initiating event in tumorigenesis. To counteract these deleterious effects, all cell types possess DNA repair machinery, such as nucleotide excision repair and, in some cell types, direct reversion. Different analytical approaches were used to assess the efficiency of repair and decipher the enzymatic mechanisms. We presently review the information provided by chromatographic methods, which are complementary to biochemical assays, such as immunological detection and electrophoresis-based techniques. Chromatographic assays are interesting in their ability to provide quantitative data on a wide range of damage and are also valuable tools for the identification of repair intermediates., (© 2016 The American Society of Photobiology.)

Purpose: The main aim of the present study is to gain mechanistic insights into the modulating effect of molecular hydrogen on the γ-radiation-induced alteration pathways of DNA nucleobases., Materials and Methods: Aerated aqueous solutions of calf thymus DNA were exposed to a (60)Co source at doses ranging from 0 to 55 Gy under normoxic conditions, in the presence or not of 0.7 MPa hydrogen or helium. The measurement of several modified bases was performed using HPLC associated with electrospray ionization tandem pass spectrometry (HPLC-ESI-MS/MS). Bleaching of aqueous solutions of p-nitrosodimethylaniline (p-NDA) solutions was also used to allow the quantification of hydroxyl radical (•OH) formation., Results: pNDA bleaching was significantly reduced in the presence of hyperbaric hydrogen. This is undoubtedly due to (•)OH scavenging by H2 since, under the same conditions, He had no effect. Similarly, base alterations were significantly reduced in the presence of hydrogen, as compared to controls under normal atmosphere or in the presence of helium. The relative proportions of modified nucleobases were not changed, showing that the only effect of H2 is to scavenge (•)OH without exhibiting reducing properties., Conclusions: Our findings demonstrate that H2 exerts a significant protection against radiation-induced DNA base damage in aqueous solutions, (•)OH scavenging being the only mechanism involved.

This review chapter presents a critical survey of the main available information on the UVB and UVA bipyrimidine photoproducts which constitute the predominant recipient classes of photo-induced DNA damage. Evidence is provided that UVB irradiation of isolated DNA in aqueous solutions and in cells gives rise to the predominant generation of cis-syn cyclobutane pyrimidine dimers (CPDs) and, to a lesser extent, of pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), the importance of which is strongly primary sequence dependent. A notable change in the photoproduct distribution is observed when DNA either in the dry or in desiccated microorganisms is exposed to UVC or UVB photons with an overwhelming formation of 5-(α-thymidyl)-5,6-dihydrothymidine, also called spore photoproduct (dSP), at the expense of CPDs and 6-4PPs. UVA irradiation of isolated and cellular DNA gives rise predominantly to bipyrimidine photoproducts with the overwhelming formation of thymine-containing cyclobutane pyrimidine dimers at the exclusion of 6-4PPs. UVA photons have been shown to modulate the distribution of UVB dimeric pyrimidine photoproducts by triggering isomerization of the 6-4PPs into related Dewar valence isomers. Mechanistic aspects of the formation of bipyrimidine photoproducts are discussed in the light of recent photophysical and theoretical studies.

Hydroxyl radical (OH) and one-electron oxidants that may be endogenously formed through oxidative metabolism, phagocytosis, inflammation and pathological conditions constitute the main sources of oxidatively generated damage to cellular DNA. It is worth mentioning that exposure of cells to exogenous physical agents (UV light, high intensity UV laser, ionizing radiation) and chemicals may also induce oxidatively generated damage to DNA. Emphasis is placed in this short review article on the mechanistic aspects of OH and one-electron oxidant-mediated formation of single and more complex damage (tandem lesions, intra- and interstrand cross-links, DNA-protein cross-links) in cellular DNA arising from one radical hit. This concerns DNA modifications that have been accurately measured using suitable analytical methods such as high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Evidence is provided that OH and one-electron oxidants after generating neutral radicals and base radical cations respectively may partly induce common degradation pathways. In addition, selective oxidative reactions giving rise to specific degradation products of OH and one-electron oxidation reactions that can be used as representative biomarkers of these oxidants have been identified., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA., Conclusion: There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation.

The one-electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation produces cross-links between guanine and thymine bases (G*-T*), characterized by a covalent bond between C8 guanine (G*) and N3 thymine (T*) atoms. The DNA lesions were quantified by isotope dilution LC-MS/MS methods in the multiple reaction-monitoring mode using isotopically labeled [(15)N, (13)C]-nucleotides as internal standards. Among several known pyrimidine and 8-oxo-7,8-dihydroguanine lesions, the G*-T* cross-linked lesions were detected at levels of ~0.21 and 1.19 d(G*-T*) lesions per 10(6) DNA bases at laser intensities of 50 and 280 mJ/cm(2)/pulse, respectively.

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.

There is an increasing interest for oxidatively generated complex lesions that are potentially more detrimental than single oxidized nucleobases. In this survey, the recently available information on the formation and processing of several classes of complex DNA damage formed upon one radical hit including mostly hydroxyl radical and one-electron oxidants is critically reviewed. The modifications include tandem base lesions, DNA-protein cross-links and intrastrand (purine 5',8-cyclonucleosides, adjacent base cross-links) and interstrand cross-links. Information is also provided on clustered lesions produced essentially by exposure of cells to ionizing radiation and high energetic heavy ions through the involvement of multiple radical events that induce several lesions DNA in a close spatial vicinity. These consist mainly of double strand breaks (DSBs) and non-DSB clustered lesions that are referred as to oxidatively generated clustered DNA lesions (OCDLs)., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Although the carcinogenic potential of ultraviolet radiation is well-known, UV light may interact with DNA by direct absorption or through photosensitization by endogenous or exogenous chromophores. These chromophores can extend the "active" fraction of the solar spectrum to the UVA region and beyond, which means that photosensitizers increase the probability of developing skin cancer upon exposure to sunlight. Therefore researchers would like to understand the mechanisms involved in photosensitized DNA damage both to anticipate possible photobiological risks and to design tailor-made photoprotection strategies. In this context, photosensitized DNA damage can occur through a variety of processes including electron transfer, hydrogen abstraction, triplet-triplet energy transfer, or generation of reactive oxygen species. In this Account, we have chosen benzophenone (BP) as a classical and paradigmatic chromophore to illustrate the different lesions that photosensitization may prompt in nucleosides, in oligonucleotides, or in DNA. Thus, we discuss in detail the accumulated mechanistic evidence of the BP-photosensitized reactions of DNA or its building blocks obtained by our group and others. We also include ketoprofen (KP), a BP-derivative that possesses a chiral center, to highlight the stereodifferentiation in the key photochemical events, revealed through the dynamics of the reactive triplet excited state ((3)KP*). Our results show that irradiation of the BP chromophore in the presence of DNA or its components leads to nucleobase oxidations, cyclobutane pyrimidine dimer formation, single strand breaks, DNA-protein cross-links, or abasic sites. We attribute the manifold photoreactivity of BP to its well established photophysical properties: (i) it absorbs UV light, up to 360 nm; (ii) its intersystem crossing quantum yield (ϕ(ISC)) is almost 1; (iii) the energy of its nπ* lowest triplet excited state (E(T)) is ca. 290 kJ mol(-1); (iv) it produces singlet oxygen ((1)O(2)) with a quantum yield (ϕ(Δ)) of ca. 0.3. For electron transfer and singlet oxygen reactions, we focused on guanine, the nucleobase with the lowest oxidation potential. Among the possible oxidative processes, electron transfer predominates. Conversely, triplet-triplet energy transfer occurs mainly from (3)BP* to thymine, the base with the lowest lying triplet state in DNA. This process results in the formation of cyclobutane pyrimidine dimers, but it also competes with the Paternò-Büchi reaction in nucleobases or nucleosides, giving rise to oxetanes as a result of crossed cycloadditions. Interestingly, we have found significant stereodifferentiation in the quenching of the KP triplet excited state by both 2'-deoxyguanosine and thymidine. Based on these results, this chromophore shows potential as a (chiral) probe for the investigation of electron and triplet energy transport in DNA.

The survey focuses on recent aspects of photochemical reactions to cellular DNA that are implicated through the predominant formation of mostly bipyrimidine photoproducts in deleterious effects of human exposure to sunlight. Recent developments in analytical methods have allowed accurate and quantitative measurements of the main DNA photoproducts in cells and human skin. Highly mutagenic CC and CT bipyrimidine photoproducts, including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are generated in low yields with respect to TT and TC photoproducts. Another striking finding deals with the formation of Dewar valence isomers, the third class of bipyrimidine photoproducts that is accounted for by UVA-mediated isomerization of initially UVB generated 6-4PPs. Cyclobutadithymine (T<>T) has been unambiguously shown to be involved in the genotoxicity of UVA radiation. Thus, T<>T is formed in UVA-irradiated cellular DNA according to a direct excitation mechanism with a higher efficiency than oxidatively generated DNA damage that arises mostly through the Type II photosensitization mechanism. C<>C and C<>T are repaired at rates intermediate between those of T<>T and 6-4TT. Evidence has been also provided for the occurrence of photosensitized reactions mediated by exogenous agents that act either in an independent way or through photodynamic effects., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

High-Altitude Andean Lakes (HAAL) of the South American Andes are almost unexplored ecosystems of shallow lakes. The HAAL are recognized by a remarkably high UV exposure, strong changes in temperature and salinity, and a high content of toxic elements, especially arsenic. Being exposed to remarkably extreme conditions, they have been classified as model systems for the study of life on other planets. Particularly, Acinetobacter strains isolated from the HAAL were studied for their survival competence under strong UV-B irradiation. Clinical isolates, Acinetobacter baumannii and Acinetobacter johnsonii, served as reference material. Whereas the reference strains rapidly lost viability under UV-B irradiation, most HAAL-derived strains readily survived this exposure and showed less change in cell number after the treatment. Controls for DNA repair activity, comparing dark repair (DR) or photo repair (PR), gave evidence for the involvement of photolyases in the DNA repair. Comparative measurements by HPLC-mass spectrometry detected the number of photoproducts: bipyrimidine dimers under both PR and DR treatments were more efficiently repaired in the HAAL strains (up to 85 % PR and 38 % DR) than in the controls (31 % PR and zero DR ability). Analysis of cosmid-cloned total genomic DNA from the most effective DNA-photorepair strain (Ver3) yielded a gene (HQ443199) encoding a protein with clear photolyase signatures belonging to class I CPD-photolyases. Despite the relatively low sequence similarity of 41 % between the enzymes from Ver3 and from E. coli (PDB 1DNPA), a model-building approach revealed a high structural homology to the CPD-photolyase of E. coli.

A broad scientific community is involved in investigations aimed at delineating the mechanisms of formation and cellular processing of oxidatively generated damage to nucleic acids. Perhaps as a consequence of this breadth of research expertise, there are nomenclature problems for several of the oxidized bases including 8-oxo-7,8-dihydroguanine (8-oxoGua), a ubiquitous marker of almost every type of oxidative stress in cells. Efforts to standardize the nomenclature and abbreviations of the main DNA degradation products that arise from oxidative pathways are reported. Information is also provided on the main oxidative radicals, non-radical oxygen species, one-electron agents and enzymes involved in DNA degradation pathways as well in their targets and reactivity. A brief classification of oxidatively generated damage to DNA that may involve single modifications, tandem base modifications, intrastrand and interstrand cross-links together with DNA-protein cross-links and base adducts arising from the addition of lipid peroxides breakdown products is also included.

This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations., (2011 Elsevier B.V. All rights reserved.)

Exposure of cells and human skin to UVA radiation oxidatively induces damage to DNA that consists mostly of 8-oxo-7,8-dihydroguanine (8-oxoGua), together with relatively minor amounts of oxidized pyrimidine bases and oligonucleotide strand breaks. Singlet oxygen generated by the type II photosensitization mechanism forms 8-oxoGua, with a small contribution by hydroxyl radicals. The mechanisms of UVA-mediated formation of DNA oxidation products indicate the absence of induction of double-strand breaks.

Evaluation of the photoprotection provided by sunscreens is performed either through the induction of erythema and expressed as the sun protection factor (SPF), or by the UVA-mediated persistent pigment darkening (PPD). None of these two endpoints has a link with skin cancer, the most deleterious consequence of excess exposure to solar UV radiation. We thus set up a complementary approach to evaluate the protection provided by sunscreens to the genome of human skin. This is based on the quantification of the thymine cyclobutane dimer (TT-CPD), the main DNA lesion induced by both UVB and UVA radiations. Irradiations were performed ex vivo on human skin explants and the level of TT-CPD in DNA was determined by HPLC associated with tandem mass spectrometry. The technique was first optimized and validated with three standard sunscreens. The study was then extended to the evaluation of a commercial high SPF sunscreen exhibiting efficient UVA photoprotection. The DNA protecting factor was found to reflect the ratio between UVB and UVA photoprotection, although the absolute values of the genomic protection were, as a general trend, lower than either SPF or PPD. These data show the usefulness of the proposed approach for the evaluation of the genoprotection afforded by sunscreens., (© 2010 The Authors. Photochemistry and Photobiology © 2010 The American Society of Photobiology.)

The objective of this study was to investigate the photobiological responses of the haloalkaliphilic euryarchaeon Natronomonas pharaonis to environmentally relevant polychromatic UV radiation, simulating either the present UV radiation climate (lambda>290 nm) or that of the early Earth (lambda>220 nm), and to monochromatic UVC radiation (lambda=254 nm) for comparison with the literature data. UV-induced bipyrimidine DNA photoproducts were determined using a sensitive and accurate HPLC tandem mass spectrometry assay, allowing to identify and quantify each type of photoproducts formed in the DNA of a UV-irradiated halophilic archaeon. The thymine cytosine (TC) pyrimidine (6-4) pyrimidone photoproduct and the TC cyclobutane pyrimidine dimer accounted for almost 80% of the total induced DNA photolesions, regardless of the wavelength range tested. These prominent formation rates of TC photoproducts correlated with the genomic frequencies of TC dinucleotides in N. pharaonis.

The survey is aimed at critically reviewing information on the UVA-mediated oxidative reactions to cellular components with emphasis on DNA as the result of mostly photosensitized pathways. It appears clearly that UVA radiation is relatively much more efficient than UVB photons in inducing oxidative processes. The main UVA-induced oxidative degradation pathways of DNA are reported and discussed mechanistically. They are mostly rationalized in terms of a major contribution of singlet molecular oxygen ((1)O(2)) and to a lesser extent of hydroxyl radical ( OH), that in the latter case originates from Fenton-type reactions. This leads to the predominant formation of 8-oxo-7,8-dihydroguanine together with smaller amounts of oxidized pyrimidine bases and DNA strand breaks in UVA-irradiated cells.

The formation of deoxyribonucleic acid (DNA) adducts can have important and adverse consequences for cellular and whole organism function. Available methods for identification of DNA damage and quantification of adducts are reviewed. Analyses can be performed on various samples including tissues, isolated cells, and intact or hydrolyzed (digested) DNA from a variety of biological samples of interest for monitoring in humans. Sensitivity and specificity are considered key factors for selecting the type of method for assessing DNA perturbation. The amount of DNA needed for analysis is dependent upon the method and ranges widely, from <1 microg to 3 mg. The methods discussed include the Comet assay, the ligation-mediated polymerase reaction, histochemical and immunologic methods, radiolabeled ((14)C- and (3)H-) binding, (32)P-postlabeling, and methods dependent on gas chromatography (GC) or high-performance liquid chromatography (HPLC) with detection by electron capture, electrochemical detection, single or tandem mass spectrometry, or accelerator mass spectrometry. Sensitivity is ranked, and ranges from approximately 1 adduct in 10(4) to 10(12) nucleotides. A brief overview of oxidatively generated DNA damage is also presented. Assay limitations are discussed along with issues that may have impact on the reliability of results, such as sample collection, processing, and storage. Although certain methodologies are mature, improving technology will continue to enhance the specificity and sensitivity of adduct analysis. Because limited guidance and recommendations exist for adduct analysis, this effort supports the HESI Committee goal of developing a framework for use of DNA adduct data in risk assessment.

Nuclear DNA and other molecules in living systems are continuously exposed to endogenously generated oxygen species. Such species range from the unreactive superoxide radical (O2*-)the precursor of hydrogen peroxide (H2O2)to the highly reactive hydroxyl radical (*OH). Exogenous chemical and physical agents, such as ionizing radiation and the UVA component of solar light, can also oxidatively damage both the bases and the 2-deoxyribose moieties of cellular DNA. Over the last two decades, researchers have made major progress in understanding the oxidation degradation pathways of DNA that are most likely to occur from either oxidative metabolism or exposure to various exogenous agents. In the first part of this Account, we describe the mechanistic features of one-electron oxidation reactions of the guanine base in isolated DNA and related model compounds. These reactions illustrate the complexity of the various degradation pathways involved. Then, we briefly survey the analytical methods that can detect low amounts of oxidized bases and nucleosides in cells as they are formed. Recent data on the formation of oxidized guanine residues in cellular DNA following exposure to UVA light, ionizing radiation, and high-intensity UV pulses are also provided. We discuss these chemical reactions in the context of *OH radical, singlet oxygen, and two-quantum photoionization processes.

Bleomycin, a radiomimetic drug currently used in human cancer therapy, is a well known carcinogen. Its toxicity is mostly attributed to its potentiality to induce DNA double strand breaks likely arising from the formation of two vicinal DNA strand breaks, initiated by C4-hydrogen abstraction on the 2-deoxyribose moiety. In this work we demonstrate that such a hydrogen abstraction reaction is able to induce the formation of a clustered DNA lesion, involving a 3' strand break together with a modified sugar residue exhibiting a reactive alpha,beta-unsaturated aldehyde that further reacts with a proximate cytosine base. The lesion thus produced was detected as a mixture of four isomers by HPLC coupled to tandem mass spectrometry subsequent to DNA extraction and enzymatic digestion. The modified nucleosides that constitute new types of cytosine adducts were identified as the likely two pairs of diastereomers of 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-2-hydroxy-3(3-hydroxy-2-oxopropyl)-2,6-dihydroimidazo[1,2-c]-pyrimidin-5(3H)-one as inferred from mass spectrometry and NMR analyses of the chemically synthesized nucleosides. We demonstrate that bleomycin, and to a minor extent ionizing radiation, are able to induce significant amounts of the cytosine damage in cellular DNA. In addition, the repair kinetic of the lesion in a human lymphocyte cell line is rather slow, with a half-life of 10 h. The 2'-deoxycytidine adducts thus characterized that represent the first example of complex DNA lesions isolated and identified in cellular DNA upon one radical hit are likely to play an important role in the toxicity of bleomycin.

In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.

The spore photoproduct (SP) is the main DNA lesion after UV-C irradiation, and its repair is crucial for the resistance of spores to UV. The aims of the present study were to assess the formation and repair of bipyrimidine photoproducts in spore DNA of various Bacillus subtilis strains using a sensitive HPLC tandem mass spectrometry assay. Strains deficient in nucleotide excision repair, spore photoproduct lyase, homologous recombination (recA), and with wild-type repair capability were investigated. Additionally, one strain deficient in the formation of major small, acid-soluble spore proteins (SASPs) was tested. In all SASP wild-type strains, UV-C irradiation generated almost exclusively SP (>95 %) but also a few by-photoproducts. In the major SASP-deficient strain, SP and by-photoproducts were generated in equal quantities. The status time of 60 min, >75% of the SP was repaired in wild-type strains and in the SASP-deficient strain, while half of the photoinduced SP was removed in the recA-deficient strain. SP-lyase-deficient spores repaired 20% of the SP produced. Thus, SP lyase, with respect to nucleotide excision repair, has a remarkable impact on the removal of SP upon spore germination.

An intrastrand cross-link lesion, in which two neighboring nucleobases are covalently tethered, has been site-specifically synthesized into defined sequence oligonucleotides in order to perform in vitro replication studies using either bacterial replicative or translesional synthesis polymerases. The investigated tandem base lesion that involves a cross-link between the methylene group of thymine and the C8 of an adjacent guanine residue has been prepared by UV-photolysis under anaerobic condition of the photolabile precursor 5-(phenylthiomethyl)-2'-deoxyuridine that has been site-specifically incorporated into a 9-mer oligonucleotide. After ligation, the lesion-containing modified oligonucleotide was used as a DNA template in primer extension reactions catalyzed by several DNA polymerases including the fragment Klenow exo-(Kf-) of E. coli polymerase I, the Thermus aquaticus polymerase (Taq pol) and the E. coli translesional DNA polymerase Pol IV (dinB). It was found that the primer extension reaction was stopped after the incorporation of the correct nucleotide dAMP opposite the 3'-thymine residue of guanine(C8-CH2) thymine lesion by Kf- and Pol IV; however it was noted that the efficiency of the nucleotide incorporation was reduced. In contrast, the Taq polymerase was totally blocked at the nucleotide preceding the tandem lesion. These results are strongly suggestive that the present intrastrand cross-link lesion, if not repaired, would constitute a blocking lesion for prokaryotic DNA polymerases, being likely lethal for the cell.

Solar UV radiation is the most important environmental factor involved in the pathogenesis of skin cancers. The well known genotoxic properties of UVB radiation (290-320 nm) mostly involve bipyrimidine DNA photoproducts. In contrast, the contribution of more-abundant UVA radiation (320-400 nm) that are not directly absorbed by DNA remains poorly understood in skin. Using a highly accurate and quantitative assay based on HPLC coupled with tandem mass spectrometry, we determined the type and the yield of formation of DNA damage in whole human skin exposed to UVB or UVA. Cyclobutane pyrimidine dimers, a typical UVB-induced DNA damage, were found to be produced in significant yield also in whole human skin exposed to UVA through a mechanism different from that triggered by UVB. Moreover, the latter class of photoproducts is produced in a larger amount than 8-oxo-7,8-dihydro-2'-deoxyguanosine, the most common oxidatively generated lesion, in human skin. Strikingly, the rate of removal of UVA-generated cyclobutane pyrimidine dimers was lower than those produced by UVB irradiation of skin. Finally, we compared the formation yields of DNA damage in whole skin with those determined in primary cultures of keratinocytes isolated from the same donors. We thus showed that human skin efficiently protects against UVB-induced DNA lesions, whereas very weak protection is afforded against UVA. These observations emphasize the likely role played by the UVA-induced DNA damage in skin carcinogenesis and should have consequences for photoprotection strategies.

5-Hydroxyuracil is a major oxidized nucleobase that can be generated by the action of (*)OH radical and one-electron oxidants. The latter modified base that exhibits a low ionization potential is highly susceptible to further degradation upon exposure to various oxidants. Emphasis was placed in this work on the formation and characterization of one-electron oxidation products of 5-hydroxyuracil within DNA fragments of defined sequence. For this purpose, 5-hydroxyuracil containing single- and double-stranded oligonucleotides of various lengths were synthesized and then exposed to the oxidizing action of iridium salts. Isodialuric acid was found to be formed almost quantitatively by a one-electron oxidation mechanism for which relevant information was inferred from a freeze-quenched ESR study. Information on the stability of isodialuric acid thus formed and its conversion products in aqueous solutions was also gained from experiments performed at acidic, neutral and alkali pH's. Moreover, biochemical features dealing with the substrate specificity of several bacterial and yeast base excision repair enzymes to remove isodialuric acid from site-specifically modified DNA fragments were determined.

Purpose: The deleterious processes triggered by heavy ions on DNA were studied through the determination of the yield of a series of oxidized bases. Emphasis was placed on the estimation of the respective contribution of direct ionization and indirect effects, mostly by comparison with low linear energy transfer (LET) gamma-rays., Material and Methods: DNA samples and human monocytes were exposed either to gamma-rays emitted by a (60)Co source or to (12)C(6+) or (36)Ar(18+) ions. The levels of thymidine and 2'-deoxyguanosine oxidation products were determined by liquid chromatography coupled to tandem mass spectrometry subsequently to DNA digestion into nucleosides., Results: The yields of thymidine lesions were similar to those of 8-oxo-7,8-dihydro-2'-deoxyguanosine within isolated DNA exposed either to gamma-rays or argon ions. Addition of spermine and Tris aimed at minimizing the indirect effect modified this ratio to the same extent with both types of radiation. In cells, the level of radiation-induced base damage was found to be correlated with the radiolytic yield of degrees OH that depends on the LET of the particle. In addition, radiation-induced thymidine and 2'-deoxyguanosine lesions were produced in similar amounts. In contrast, oxidation of 2'-deoxyguanosine was the main process when ionization was triggered in cellular DNA by ultraviolet laser-induced biphotonic processes., Conclusions: Predominant oxidation of 2'-deoxyguanosine is expected to be the hallmark of direct DNA ionization. The observation that thymidine and 2'-deoxyguanosine are equally damaged rules out a major contribution of the direct ionization in radiation-induced base damage to both isolated and cellular DNA by heavy ions. Dependence of the yield of lesions on the LET provides further support for this conclusion.

5-(Hydroxymethyl)uracil, 5-formyluracil or 5-carboxyuracil can be formed in DNA upon exposure to ionizing radiations. We report the effect of the latter oxidative lesions on the in vitro transcription by T7 RNA polymerase using a single-stranded short model template. Kinetic experiments showed that the transcription yield was increased with the modified template. Due to the unforeseen presence of a hairpin structure within the transcribed RNA, we postulated that we are in the presence of a T7 RNA polymerase pausing signal and that the lesions within this critical DNA structure could interfere with normal regulation of RNA transcription.

Emphasis is placed in this review article on recent aspects of the photochemistry of cellular DNA in which both the UVB and UVA components of solar radiation are implicated individually or synergistically. Interestingly, further mechanistic insights into the UV-induced formation of DNA photoproducts were gained from the application of new accurate and sensitive chromatographic and enzymic assays aimed at measuring base damage. Thus, each of the twelve possible dimeric photoproducts that are produced at the four main bipyrimidine sites can now be singled out as dinucleoside monophosphates that are enzymatically released from UV-irradiated DNA. This was achieved using a recently developed high-performance liquid chromatography-tandem mass spectrometry assay (HPLC-MS/MS) assay after DNA extraction and appropriate enzymic digestion. Interestingly, a similar photoproduct distribution pattern is observed in both isolated and cellular DNA upon exposure to low doses of either UVC or UVB radiation. This applies more specifically to the DNA of rodent and human cells, the cis-syn cyclobutadithymine being predominant over the two other main photolesions, namely thymine-cytosine pyrimidine (6-4) pyrimidone adduct and the related cyclobutyl dimer. UVA-irradiation was found to generate cyclobutane dimers at TT and to a lower extent at TC sites as a likely result of energy transfer mechanism involving still unknown photoexcited chromophore(s). Oxidative damage to DNA is also induced although less efficiently by UVA-mediated photosensitization processes that mostly involved 1O2 together with a smaller contribution of hydroxyl radical-mediated reactions through initially generated superoxide radicals.

In mammalian cells, the base excision repair (BER) pathway allows the remove of small DNA base lesions such as oxidized bases. It is initiated by glycosylases that removed the modified base leaving an abasic site that is subsequently processed by AP endonuclease activities. Measurement of BER activities in cell extracts is time consuming and hazardous when radioactive material is used. We report in this study, the parallelized fluorescent analysis of excision of several oxidation products of thymine by cell extracts. To conduct the study, 5-(hydroxymethyl)uracil, 5-formyluracil, 5-carboxyuracil and formylamine together with uracil and the control thymine, were incorporated into oligonucleotides of identical sequences and paired either with adenine or with guanine containing DNA fragments. The oligonucleotides were fixed by sandwich hybridization in wells of a microplate (OLISA technology). Excision by HeLa extracts of the six different DNA base lesions could be followed simultaneously in the same well. Our results showed that the extent of excision of the lesions was the same on support and in solution using classical PAGE analysis approach with modified (32)P-labeled oligonucleotides. We demonstrated that the simultaneous analysis on support is a successful approach to facilitate high-throughput screening of BER activities present in cell extracts. Moreover, extended study of 5-carboxyuracil revealed that this lesion displays similar biological properties as 5-formyluracil.

An original phosphoramidite building block of the thymidine glycol lesion has been prepared taking into account the additional diol function and the high lability of this oxidatively induced nucleobase damage. Then the modified nucleoside was site-specifically inserted into DNA fragments by solid support assembling followed by a "one-step" mild final deprotection treatment.

We are developing a technology based on the combined application of riboflavin (RB) and light for inactivating pathogens in blood products while retaining the biological functions of the treated cells and proteins. Virus and bacteria reduction measured by tissue culture infectivity or colony formation with UV light alone and in combination with RB yield equivalent results. The effects of RB as a sensitizing agent on DNA in white cells, bacteria and viruses in combination with UV light exposure have been evaluated. UV-mediated DNA degradation in Jurkat T cells and leukocytes in plasma as measured by the FlowTACS assay was significantly increased in the presence of RB. Agarose gel electrophoretic analysis of DNA in Escherichia coli and leukocytes in plasma demonstrated enhanced DNA degradation in the presence of RB. UV light in combination with RB prevented the reactivation of lambda phage compared with samples irradiated in the absence of RB. UV-mediated oxidative damage in calf thymus DNA was also enhanced in the presence of RB. These observations clearly demonstrate that the presence of RB and UV light selectively enhances damage to the guanine bases in DNA. These data also suggest that the type and extent of damage to DNA for virus in the presence of RB and light make it less likely to be repaired by normal repair pathways available in host cells.

4-Hydroxy-2-nonenal (HNE), one of the main aldehydic compounds released during lipid peroxidation, has been proposed to react with DNA bases in cells. Several classes of DNA lesions involving addition of either HNE or its 2,3-epoxide (epox-HNE) have been identified. In the present work, HPLC associated with tandem mass spectrometry was used to determine the pattern of HNE-induced DNA lesions. First, adducts were quantified within isolated DNA treated with HNE under peroxidizing conditions. The 1,N2-propano-2'-deoxyguanosine adduct of HNE (HNE-dGuo) was found to be the major lesion under all conditions studied. 1,N6-Ethenoadenine and 1,N2-ethenoguanine together with their (1,2-dihydroxyheptyl)-substituted derivatives, which all arise from the reaction of epox-HNE with DNA, were produced in significantly lower yields, even in the presence of 20 mM H2O2. The pyrimidopurinone malondialdehyde-2'-deoxyguanosine adduct was also found to be produced, although in very low yield. Similar results were obtained in cultured human monocytes incubated with HNE, because the HNE-dGuo adduct represented more than 95% of the overall adducts to DNA. In addition, the former lesion was poorly repaired, in contrast to 1,N2-ethenoguanine and, to a lesser extent, 1,N6-ethenoadenine. Altogether, these results suggest than HNE-dGuo may represent the best biomarker of the genotoxic effects of HNE., (Copyright 2004 Elsevier Inc.)

This study was undertaken with the aim to develop an optimised protocol for the evaluation of DNA damage in frozen whole blood. This was achieved by use of the single-cell gel electrophoresis (SCGE) or comet assay in its alkaline version. After collection of blood, the total blood sample was mixed with dimethyl sulfoxide (DMSO), a cryoprotectant commonly used for prevention of freezing-induced damage to living cells, and then stored at -80 degrees C. We observed no statistically significant differences in the level of DNA damage between fresh blood samples and frozen blood samples, as assessed by the comet assay. Considering the absence of effects of the freezing step, a frozen blood sample was included as a control sample in subsequent experiments. Thus the protocol was applied to blood samples of twenty healthy subjects including smokers and non-smokers. The comparative analysis indicated that the level of DNA damage was 56% higher in smokers than in non-smokers (P = 0.01). Altogether, this study strongly suggests that frozen whole blood could be utilised in association with the comet assay in human epidemiological bio-monitoring for the assessment of genetic damage in populations at risk.

The most commonly measured marker of oxidative DNA damage is 8-oxo-7,8-dihydroguanine (8-oxoGua) or its deoxyribonucleoside (8-oxodGuo). Published estimates of the concentration of 8-oxoGua/8-oxodGuo in DNA of normal human cells vary over a range of three orders of magnitude. Analysis by chromatographic methods (GC-MS, HPLC with electrochemical detection (ECD) or HPLC-MS/MS) is beset by the problem of adventitious oxidation of guanine during sample preparation. An alternative approach, based on the use of the DNA repair enzyme formamidopyrimidine DNA N-glycosylase (FPG) to make breaks in the DNA at sites of the oxidised base, gives much lower values. ESCODD, the European Standards Committee on Oxidative DNA Damage, has been testing the ability of different laboratories using a variety of methods to measure 8-oxoGua in standard samples of 8-oxodGuo, calf thymus DNA, pig liver, oligonucleotides, and HeLa cells, and in lymphocytes isolated from blood of volunteers. HPLC-ECD is capable of measuring 8-oxodGuo induced experimentally in calf thymus DNA or HeLa cells with high accuracy. However, there is no sign of consensus over the background level of this damage, suggesting that, even though standard extraction procedures were used, variable oxidation of Gua is still occurring. GC-MS failed to detect a dose response of induced 8-oxoGua and cannot be regarded as a reliable method for measuring low levels of damage. HPLC-MS/MS as yet has not proved capable of measuring low levels of oxidative DNA damage. FPG-based methods seem to be less prone to the artefact of additional oxidation. Although they can be used quantitatively, they require careful calibration and standardisation if they are to be used in human biomonitoring. The background level of DNA oxidation in normal human cells is likely to be around 0.3-4.2 8-oxoGua per 10(6) Gua. An effort should be made to develop alternative, validated methods for estimating oxidative DNA damage.

Mutagenic and carcinogenic UV-B radiation is known to damage DNA mostly through the formation of bipyrimidine photoproducts, including cyclobutane dimers (CPD) and (6-4) photoproducts ((6-4) PP). Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of thymine-thymine (TT) and thymine-cytosine (TC) CPD and (6-4) PP in the DNA of cultured human dermal fibroblasts. A major observation was that the rate of repair of the photoproducts did not depend on the identity of the modified pyrimidines. In addition, removal of CPD was found to significantly decrease with increasing applied UV-B dose, whereas (6-4) PP were efficiently repaired within less than 24 h, irrespective of the dose. As a result, a relatively large amount of CPD remained in the genome 48 h after the irradiation. Because the overall applied doses (<500 J m(-2)) were chosen to induce moderate cytotoxicity, fibroblasts could recover their proliferation capacities after transitory cell cycle arrest, as shown by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation and flow cytometry analysis. It could thus be concluded that UV-B-irradiated cultured primary human fibroblasts normally proliferate 48 h after irradiation despite the presence of high levels of CPD in their genome. These observations emphasize the role of CPD in the mutagenic effects of UV-B.

High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) has been used to search for the formation of as yet unidentified radiation-induced DNA lesions. For that purpose, the characteristic fragmentation of most of 2'-deoxyribonucleosides that corresponds to the loss of the 2-deoxyribose moiety (loss of 116 mass units) has been utilized to specifically detect modified nucleosides. Aerated aqueous solutions of DNA were exposed to ionizing radiation, and subsequently DNA was digested to nucleosides with a cocktail of endo- and exonucleases. HPLC/ESI-MS/MS analysis of the resulting 2'-deoxyribonucleoside mixture allowed us to detect four novel DNA modifications. In a subsequent step, the sensitivity of the tandem mass spectrometer was used to search for the formation of the newly detected lesions in the DNA of gamma-irradiated cells. Thus, one of the four newly detected lesions was found to be significantly generated in cellular DNA upon exposure to ionizing radiation. In addition, the latter lesion was also shown to be present in untreated cells, indicating that the modified nucleoside could be formed endogenously., (Copyright 2004 John Wiley & Sons, Ltd.)

Most of the reactions induced by *OH radicals (indirect effects) and by one-electron oxidation (direct effects) as the result of exposure to ionizing radiation may be described for the four main DNA nucleobases. Relevant information is now available on the formation of single and tandem base lesions implicating guanine as the most susceptible DNA component to the deleterious effects of ionizing radiation. In contrast, there is still a paucity of information on the radiation-induced formation of base damage within cellular DNA. This is mostly a result of difficulties associated with the measurement of oxidized purine and pyrimidine bases that appear to be generated in very low yields. This is illustrated by the measurement of low amounts of E. coli formamidopyrimidine glycosylase- and endonuclease-III-sensitive sites in the DNA of neoplastic monocytes upon exposure to gamma rays (48 and 53 per 10(9) bases and per Gy, respectively) using a modified comet assay (the overall number of strand breaks and alkali-labile sites was estimated to be 130 per 10(9) bases and per Gy). More specifically, the level of several radiation-induced modified bases, including thymine glycols, 5-formyluracil, 5-(hydroxymethyl)uracil, 8-oxo-7,8-dihydroguanine, and 8-oxo-7,8-dihydroadenine, together with related formamidopyrimidine derivatives was assessed using the suitable HPLC-MS/MS method. Information is also provided on the substrate specificity of DNA repair enzymes and the mutagenic potential of base lesions using site-specific modified oligonucleotides as the probes.

The damage profile produced by the reaction of singlet molecular oxygen with cellular DNA was determined using the comet assay associated with DNA repair enzymes. Singlet oxygen was produced intracellularly by thermal decomposition of a water-soluble endoperoxide of a naphthalene derivative which is able to penetrate through the membrane into mammalian cells. We found that the DNA modifications produced by singlet oxygen were almost exclusively oxidised purines recognised by the formamidopyrimidine DNA N-glycosylase. In contrast, significant amounts of direct strand breaks and alkali-labile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced.

Emphasis is placed in the first part of this survey on mechanistic aspects of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) as the result of exposure to z.rad;OH radical, one-electron oxidants and singlet oxygen (1O(2)) oxidation. It was found that 8-oxoGua, which is generated by either hydration of the guanine radical cation or .OH addition at C8 of the imidazole ring, is a preferential target for further reactions with 1O(2) and one-electron oxidants, including the highly oxidizing oxyl-type guanine radical. Interestingly, tandem base lesions that involve 8-oxoGua and a vicinal formylamine residue were found to be generated within DNA as the result of a single .OH radical hit. The likely mechanism of formation of the latter lesions involves the transient generation of 5-(6)-peroxy-6-(5)-hydroxy-5,6-dihydropyrimidyl radicals that may add to the C8 of a vicinal guanine base before undergoing rearrangement. Another major topic which is addressed deals with recent developments in the measurement of oxidative base damage to cellular DNA. This was mostly achieved using the accurate and highly specific HPLC method coupled with the tandem mass spectrometry detection technique. Interestingly, optimized conditions of DNA extraction and subsequent work-up allow the accurate measurement of 11 modified nucleosides and bases within cellular DNA upon exposure to oxidizing agents including UVA and ionizing radiations. Finally, recently available data on the substrate specificity of DNA repair enzymes belonging to the base excision and nucleotide excision pathways are briefly reviewed. For this purpose modified oligonucleotides in which cyclopurine, and cyclopyrimidine nucleosides were site-specifically inserted were synthesized.

Exposure to solar UV radiation gives rise to mutations that may lead to skin cancer. UVA (320-340 nm) constitutes the large majority of solar UV radiation but is less effective than UVB (290-320 nm) at damaging DNA. Although UVA has been implicated in photocarcinogenesis, its contribution to sunlight mutagenesis has not been elucidated, and DNA damage produced by UVA remains poorly characterized. We employed HPLC-MS/MS and alkaline agarose gel electrophoresis in conjunction with the use of specific DNA repair proteins to determine the distribution of the various classes and types of DNA lesions, including bipyrimidine photoproducts, in Chinese hamster ovary cells exposed to pure UVA radiation, as well as UVB and simulated sunlight (lambda > 295 nm) for comparison. At UVA doses compatible with human exposure, oxidative DNA lesions are not the major type of damage induced by UVA. Indeed, single-strand breaks, oxidized pyrimidines, oxidized purines (essentially 8-oxo-7,8-dihydroguanine), and cyclobutane pyrimidine dimers (CPDs) are formed in a 1:1:3:10 ratio. In addition, we demonstrate that, in contrast to UVB and sunlight, UVA generates CPDs with a large predominance of TT CPDs, which strongly suggests that they are formed via a photosensitized triplet energy transfer. Moreover, UVA induces neither (6-4) photoproducts nor their Dewar isomers via direct absorption. We also show that UVA photons contained in sunlight, rather than UVB, are implicated in the photoisomerization of (6-4) photoproducts, a quickly repaired damage, into poorly repaired and highly mutagenic Dewar photoproducts. Altogether, our data shed new light on the deleterious effect of UVA.