Photochemistry and Photobiology, 2015, 91: 140–155

Invited Review Oxidatively Generated Damage to Cellular DNA by UVB and UVA Radiation†,‡ Jean Cadet*1,2,3, Thierry Douki4,5 and Jean-Luc Ravanat4,5 1

University Grenoble Alpes, INAC, Grenoble, France CEA, INAC, Grenoble, France 3
partement de Me
decine Nucle
aire et Radiobiologie, Faculte
de Me
decine et des Sciences de la Sante
, Universite
de De Sherbrooke, Sherbrooke, QC, Canada 4 University Grenoble Alpes, INAC/SCIB, Grenoble, France 5 CEA, INAC/SCIB, Grenoble, France 2

Received 6 September 2014, accepted 9 October 2014, DOI: 10.1111/php.12368

ABSTRACT

A causative relationship has been established between UVB exposure and nonmelanoma skin cancers (1,2) that often exhibit a characteristic mutation signature consisting of cytosine (C) ? thymine (T) transitions at dipyrimidine sites and CC ? TT tandem base substitutions in their p53 genes (4–8). This is mostly accounted for by the formation of cyclobutane pyrimidine dimers (CPDs) which on the average are more slowly repaired than pyrimidine (6-4) pyrimidone (6-4PPs) photoproducts (9–11), the second major class of UVB-induced bipyrimidine lesions (12–14). Epidemiological evidence is also available showing that prolonged and repeated exposures of humans to artificial UVA light from either sun lamps or tanning beds constitute a major risk factor for melanoma induction (15–17). It was recently shown that oxidatively generated damage to DNA through sensitized reaction by melanin to UVA radiation is involved in melanomagenesis. (18) Major efforts have been devoted during the last two decades to the delineation of the molecular mode of action of solar radiation on skin. In that respect a major role is played by the wavelength of the incident photons on the photoinduced reactions to DNA which is the major cellular target for the carcinogenic and aging effects. Thus, UVB radiation is able through direct excitation of pyrimidine nucleobases to induce in an oxygen-independent manner two main classes of bipyrimidine photoproducts including CPDs and 6-4PPs in the DNA of fibroblasts, keratinocytes and human skin (19–22). Bipyrimidine photoproducts consisting mostly of thymine containing CPDs have been detected in nuclear DNA by high-performance liquid chromatography coupled through electrospray ionization to tandem mass spectrometry (HPLC-ESI-MS/ MS) upon UVA irradiation of isolated cells and human skin (19– 22). Evidence has been provided for a direct excitation mechanism (23) implicating a charge-transfer state that explains the lack of UVA-induced 6-4PPs (24). In addition UVA photons have been shown to be more efficient in converting initially UVB generated 6-4PPs within cellular DNA into related Dewar valence isomers (25). UVA radiation that is only weakly absorbed by DNA bases may also damage DNA through photodynamic effects that involve the participation of singlet oxygen (1O2) and to a lesser extent of hydroxyl radical (•OH) in cellular DNA and skin (12–14). UVB radiation is also able to induce oxidative degradation pathways,

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

INTRODUCTION It is well documented that both the UVB (290–320 nm) and UVA (320–400 nm) components of terrestrial solar radiation are strongly implicated in the etiology of most skin cancers that consist of basal cell carcinoma (BCCs), squamous cell carcinoma (SCCs) and cutaneous malignant melanoma (CMMs) (1–3). *Corresponding author: email: [email protected] (Jean Cadet) †This paper is part of the Special Issue commemorating the 65th birthday of Dr. Craig A. Elmets. ‡The content of the article was presented in part on the occasion of the 16th International Congress of Photobiology that was held in Cordoba, Argentina, September 8–12, 2014. © 2014 The American Society of Photobiology

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Photochemistry and Photobiology, 2015, 91 however, as a minor process, in cellular DNA (14,26). The review article provides a detailed and critical survey of the main available information on the oxidative reactions to cellular DNA that are initiated by exposure to UVB and UVA radiations. It may be pointed out that in most cases the latter degradation pathways are mediated by endogenous photosensitizers and eventually cellular responses that are discussed in terms of potential sources of reactive oxygen and nitrogen species. Mechanistic insights into the photoinduced formation of oxidatively generated base and sugar damage to nuclear DNA are provided using relevant data from detailed model studies involving 1O2, •OH and one-electron oxidants (27–30). The last section of the article is more prospective with the proposal of a few characteristic DNA lesions that could be formed in the DNA of cells or skin as the result of one-electron oxidation of guanine and addition of breakdown products of lipid peroxides to nucleobases, respectively.

UV RADIATION AS INITIATOR OF OXIDATIVE SPECIES AND PATHWAYS UVA radiation and to a lesser extent the UVB component of solar light are able to induce oxidative pathways in skin (31,32). This may be the result of photochemical reactions and/ or biochemical responses as further discussed below. UVB radiation is a poor generator of photooxidation reactions to DNA since ionization of guanine, the most favorable photoreaction involving DNA, is at best a minor process. In contrast, UVA through excitation of suitable endogenous or exogenous photosensitizers has the capability of giving rise to several reactive oxygen species (ROS) and/or promoting one-electron oxidation pathways (Scheme 1). Both UVB and UVA radiations are able to trigger oxidative responses that may persist after the irradiation is stopped. Thus, ROS and reactive nitrogen species (RNS) may be generated upon photoactivation of chromophores, induction of cellular responses such as bystander effects in nonirradiated cells and inflammation in skin. However, there is still a lack of relevant information on the latter delayed effects in term of oxidatively generated damage to DNA. It may, however, be pointed out that accumulation of damage in mitochondrial DNA has been proposed as a biomarker of aging in UVB-irradiated skin (32).

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UVB radiation Occurrence of oxidative stress in human skin upon exposure to UVB radiation has been demonstrated by measuring 8-isoprostane, a relevant degradation product of lipid peroxides using an immunohistochemical assay (33). More specific information concerning the involvement of superoxide anion radical (O2
 ) in the UVB response of an human skin equivalent model was gained from real-time chemiluminescence imaging measurements with a timedependent increase within the few minutes following the end of the irradiation (34,35). Evidence has been provided for UVBinduced generation of hydrogen peroxide (H2O2) in the epidermis using noninvasive Fourier-Transform Raman spectroscopy analysis (36). ROS, in fact most likely •OH, detected using the nonspecific 20 ,70 -dicholorodihydrofluorescein diacetate (H2DCF-DA) fluorophore probe (37) were found to be generated in UVB-irradiated keratinocytes (38,39) that are the major targets of the latter radiation. A delayed time-course formation of ROS was observed in normal (40) and Colo-16 human keratinocytes (39) providing clear evidence for indirect photoinduced oxidative stress. It was also found that UVB radiation is a more efficient generator of H2O2 than UVC photons (41). It has been proposed that UVB activation of catalase (42), cyclooxygenase and NADPH oxidase (38,43) was a major source of ROS. In addition, UVB radiation has been shown to promote inflammatory responses and leukocyte infiltration in both human and animal skins (44–47), the process having been shown to be more efficient in obese mice as inferred from the increased formation of H2O2 and nitric oxide (NO•) (45). It was reported that UVB radiation is able to stimulate expression of inducible nitric oxide synthase (iNOX) in vessel endothelia of normal human skin and cultured endothelial cells (48) giving rise to NO• and potentially peroxynitrite (ONOO), if O2
 is formed in the vicinity (Scheme 1). Further indirect support for the UVBinduced oxidative stress in keratinocytes was provided by the observation of a protective action against deleterious biological effects by various antioxidant agents and enzymes (39,49–51) and the induction of antioxidant expression (52). It is likely that at least part of the ROS thus generated by UVB exposure, are involved in cell signaling transduction (53). Formation of delayed mutations in the hypoxanthine phosphoribosyl transferase (hprt) gene has been observed in Chinese hamster fibroblasts following exposure to either UVB or UVA radiation. UVB photons are five-fold more efficient than UVA radiation in inducing the mutations that were suggested to occur as the result of bystander effects (54,55). The latter effects that were found to be mediated in part by gap junctional intercellular communication and to persist for several cell generations are expected to contribute to UV genomic instability. Indirect support for the implication of oxidation reactions in the formation of delayed mutations was provided by the observation of protective effects exerted by several antioxidants (54). However, the ability for UVB radiation to trigger bystander effects in human HaCaT keratinocytes and MRC5 fibroblasts has been questioned (56). In contrast, it was concluded in a recent study that UVB was able to induce bystander effect in nontarget-neighboring fibroblast cells with an increase in the intracellular levels of ROS (57).

UVA RADIATION Scheme 1. Delayed generation of ROS and NOS with subsequent formation of peroxynitrite and carbonate radical anion through NADPH oxidase activation, inflammation, bystander effect.

It is well documented that the cytotoxic action of UVA radiation on mammalian cells is strongly oxygen dependent involving photosensitization reactions leading to the so-called “photodynamic

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effects” (58). We may distinguish direct oxidation reactions mediated by UVA-excited photosensitizers from the delayed and/or indirect generation of ROS and RNS that reflects onset of various biological responses. UVA radiation has been shown to be carcinogenic for skin mice through oxidative processes (59). The UVA photons that are only partly absorbed by the upper layers of skin are able to penetrate in deepness into the dermis by contrast with UVB radiation which is mostly absorbed by several major cutaneous targets in the epidermis. UVA is thus involved in the two main cutaneous effects of excessive exposure to sunlight: cancer and aging. Photosensitized reactions Photodynamic effects are mediated by endogenous photosensitizers, possibly cytochromes, flavin, heme, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and porphyrins that may act upon UVA excitation according to two main mechanisms (12,60–62). The photosensitizer, likely in a long-lived triplet excited state may be involved in a charge-transfer reaction with a vicinal target molecule, the so-called type I photosensitization mechanism. This, in most cases, implicates an electron abstraction from the target molecule, leading to the formation of a pair of charged radicals (Scheme 2). The radical cation thus generated from the target molecule may undergo in an aqueous environment either deprotonation and/or hydration reactions which in both cases give rise to neutral radicals. In subsequent steps the latter transient species react with molecular oxygen or eventually with O2
 when the radicals are highly oxidizing as it is the case for those deriving from the deprotonation of the radical cations of guanine, tryptophan or tyrosine (63–68). The resulting peroxyl radicals and/or hydroperoxides constitute important intermediates that through further reactions generate final oxidation products which for DNA, lipids and proteins are expected to exhibit deleterious biological properties. The photosensitizer radical anions (PS•) that arise from electron capture are usually oxidized back to the starting molecules by O2 leading to the release of O2
 (Scheme 2). The second major photosensitization mechanism (type II) deals mostly with energy transfer to molecular oxygen giving rise to oxygen in the first excited 1Dg state (1O2) which specifically reacts with biomolecules exhibiting double bonds rich in electrons including guanine, tryptophan and histidine (69–71). The highly dienophile 1O2 may react according to three main mechanisms including [4 + 2] Diels-Alder cycloaddition, [2 + 2] cycloaddition and the ene-reaction giving rise to endoperoxides, dioxetanes and hydroperoxides, respectively (72,73). A side reaction of the latter type II photosensitization mechanism which at best is a

Scheme 2. Generation of reactive oxygen species through type I and type II photosensitization reactions.

minor process involves charge transfer with oxygen leading to the formation of O2
 . The latter radical is quite unreactive toward most biomolecules at the exception of a few oxidizing radicals as already mentioned above. However, it may undergo either spontaneous or enzymatic dismutation into H2O2, another low-reactive ROS which may migrate through the cell and promote in the presence of reduced transition metals such as ferrous ion the occurrence of Fenton reactions. The resulting highly reactive •OH is able to react at the site where it is generated by adding to double bonds and/or abstracting hydrogen atoms (67). Both reactions give rise to carbon-centered radicals, the likely precursors of peroxyl radicals through subsequent efficient O2 addition. Further confirmation for the occurrence of the latter sensitized reactions to UVA radiation as the result of excitation of endogenous chromophores was gained from the noninvasive detection of 1O2 and O2
 in UVA exposed skin of mice (74,75). This was achieved using a specific and a sensitive chemiluminescence probe and ultralowlight imaging device with a CCD camera. It may be added that O2
 has been also shown to be produced in UVA-irradiated HaCat keratinocytes by direct electrochemical monitoring (76). It is also likely that O
2 is generated at least partly from delayed biochemical responses as further discussed below. Both deleterious and signaling effects of type II photosensitization-mediated 1O2 are now well recognized (76–84). Further support for the major role played by 1O2 in the UVA induction of cell death was provided by the observation of the modulation of the photodynamic effects of UVA radiation. Thus, D2O was able to further enhance UVA-induced biological responses (85,86) by extending the 1O2 lifetime whereas either sodium azide or a-tocopherol showed a protective effect as the result of their ability to scavenge 1O2. Delayed generation of ROS and NOS Similar to what was already discussed for UVB radiation, it is now well documented that exposure of skin and cells to UVA photons leads to the generation of ROS and RNS as delayed biochemical processes (Scheme 1). Evidence is being accumulated showing that UVA-induced inflammation reactions in the skin are mediated by initially generated ROS that are likely to initiate signal transduction processes (87). This is accompanied by the release of inflammation mediators such as isoprostanes (88) and prostaglandin E2 (PGE2) that is produced by cyclooxygenase 2mediated degradation of arachidonic acid (33). As a consequence, cytokines that are able to modulate endothelial cell functions during inflammation processes were shown to enhance the expression of inducible nitric oxide synthase, an efficient generator of NO• (89). Evidence has been subsequently provided for another UVAinduced source of NO• that involves the photolysis of NO stores (90–92). The latter radical that has been found to inhibit propagative lipid peroxidation by scavenging transient peroxyl radicals (93) was found to protect UVA-irradiated endothelial cells against necrosis and apoptosis up to 2 h following the UVA exposure (94,95). UVA irradiation of human keratinocytes led to the induction of a rapid generation of ROS and RNS that may consist of H2O2, peroxynitrite and peroxides (87) as detected by using the rather poorly informative carboxy-H2DCF-DA fluorescence probe. The release of ROS and/or RNS that were found to be mediated by mitochondria and NADPH oxidase upon activation of the Nox1 catalytic subunit showed a maximum 16 min after the end of UVA irradiation while persisting for 1 h. Evidence was also

Photochemistry and Photobiology, 2015, 91 provided that the released reactive oxygen and/or nitrogen species that were previously shown to be involved in UVA-induced responses (96) were implicated in the production of PGE2. The formation of ROS and/RNS was found to be more efficient in keratinocytes in which the membrane content in 7-dehydrocholesterol was increased at the expense of cholesterol as it is the case in the UVA sensitive patients who suffer from the Smith–Lemli– Optiz syndrome (96). Induction of delayed mutations in the hprt gene of UVA-irradiated Chinese hamster ovary (CHO) cells that are indicative of genomic instability was accounted for by a bystander effect (54,55). The latter biological response that is far less important than that induced by UVB photons has been suggested to be mediated by ROS. This, however, is not in agreement with the results of another study showing that a bystander effect is triggered by UVA irradiation in human keratinocytes and fibroblasts whereas UVB light is totally inefficient (56). It has also been observed that melanocytes which are more resistant than keratinocytes and fibroblasts to the direct effects of UVA radiation are more vulnerable that the two other skin cells to bystander oxidative signaling (97). Evidence for the occurrence of another type of bystander effect has been recently gained: thus UVA excitation of dermal extracellular matrix protein chromophores gives rise to diffusible ROS that may subsequently hit neighboring cells (62). The UVA-induced release of chelatable/labile iron in human skin cells as the result of unregulated degradation of ferritin (98) represents another potential source of oxidative stress and is likely to affect cell homeostasis (99,100). This is particularly of concern since UVA-mediated generation of ROS including H2O2 that is the oxidizing precursor of the Fenton reaction is well documented (101). The release of labile irons that is sustained for at least 2 h following UVA exposure is several-fold more intense in human fibroblasts than in keratinocytes showing an apparent positive correlation with the incidence of necrosis (102). Interestingly, supplementation of human fibroblasts with epicatechin was found to suppress the UVA-induced release of labile irons by maintaining the lysosomal integrity (103). Another indication of the major oxidative response induced by UVA radiation-sensitized formation of 1O2 (104) in fibroblasts and mammalian skins is the strong up-regulation of heme oxygenase-1 (105,106), the heme-catabolizing enzyme that exhibits both antioxidant and anti-inflammatory properties (107). However, it should be noted that the activation of heme oxygenase leads to transient oxidative stress through the release of labile iron from free heme (108).

UVB RADIATION-MEDIATED OXIDATIVE REACTION TO CELLULAR DNA Oxidatively generated damage Evidence has been provided by using the alkaline elution technique and its modified version that involves a preincubation step with DNA repair enzymes that several classes of photoproducts were generated in the DNA of AS52 CHO cells upon exposure to UVB radiation (109,110). CPDs that are known to constitute the predominant class of UVB-induced DNA lesions were revealed as enzymatically generated strand breaks by incubation with T4 endonuclease V whereas oxidized pyrimidine and modified purine bases were detected as endonuclease III (endo III)and formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites, respectively. Thus, 150 CPDs were found to be generated per

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106 nucleobases in the DNA of CHO cells that were exposed to a UVB dose of 1 kJ m2. This frequency has to be compared with the much smaller yields of DNA strand breaks, oxidized pyrimidine bases and purine modifications that were estimated to be 0.37, 0.36 and 0.62 per 106 nucleobases, respectively, under the same irradiation conditions. This clearly shows if one considers that the formation of the three latter classes of damage involves oxidative degradation pathways that the molecular effects of UVB radiation on cellular DNA are predominantly accounted for by oxygen-independent formation of pyrimidine dimers. The relative contribution of oxidative reactions to the overall photoproduct formation is slightly lower than 1% if the formation of 6-4PPs that arise from direct UVB excitation is also taken into consideration. Similar conclusions were made by measuring 8-oxo-7,8-dihydro-20 -deoxyguanosine (8oxodGuo), a relevant oxidation product of 20 -deoxyguanosine (27), and CPDs in the DNA of UVB-irradiated CHO cells by using an HPLC-electrochemical detection (ECD) method and an immunoassay, respectively (111). The relative yield of 8-oxodGuo versus CPDs was close to 1%, further confirming that UVB-mediated oxidation reactions of cellular DNA are rather of low importance at least in terms of quantitative importance. The UVB-induced formation of 8-oxodGuo has been observed, mostly on the basis of accurate HPLC-ECD measurements, in the DNA of hairless mouse epidermis (112), cultured adult rat liver epithelial cells (113), mouse keratinocytes (114) and human keratinocytes (115–117). It was shown that narrowband UVB was slightly less efficient in inducing 8-oxodGuo than a broadband UVB source for a same clinically effective radiation dose (116). The latter observation is conflicting with previous measurements of 8-oxodGuo that were performed by immunodetection on UVB-irradiated mouse and organ-cultured human skin showing an opposite trend (118). This could be explained by the low specificity of the N45.1 antibodies anti-8-oxodGuo (119) that would prevent accurate measurement as noted for the immunodetection of single-oxidized nucleobases that are in most cases poorly antigenic (120). It may be added that the latter antibodies have been used to monitor the formation of 8-oxodGuo in the epidermis of hairless mice following exposure to UVB radiation (121,122). Detection of UVB-induced 8-oxodGuo was also achieved in JB6 mouse epidermal cells (123) through the binding of avidin-fluorescent isothiocyanate probe according to a slightly modified version of a mimicking immunoassay (124) whose specificity is, however, still open to debate (125). A time-course study of UVB radiation-induced formation of human 8-oxoguanine DNA glycosylase (hOGG1)-sensitive sites that are expected to predominantly consist of 8-oxo-7,8-dihydroguanine (8-oxoGua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) shows a maximum 1 h postirradiation in the DNA of human keratinocytes, thus suggesting occurrence of delayed oxidation reactions (126). Indirect support for the biological significance of UVBinduced 8-oxodGuo was provided by the increased susceptibility of OGG1 knockout mice to UV skin cancer (122). In a subsequent study it was shown that versican, an inflammatory response pathway gene, was up-regulated following UVB irradiation as a likely mediator of skin carcinogenesis (127). Another support for a putative deleterious role played by 8-oxodGuo was provided by the observed protecting effect of the topical application of a formulation of liposome-encapsulated OGG1 enzyme against the progression of UVB-induced tumor (128).

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Information on the formation of specific UVB-induced oxidation pyrimidine products in cellular DNA is still lacking although evidence has been provided for the generation of endonuclease III-sensitive sites in a slightly smaller yield that oxidized purine bases (109,110). Along likely candidates one may mentioned several oxidized bases that have been identified in the DNA of c-irradiated cultured cells as •OH oxidation products of thymine, cytosine and 5-methylcytosine (129–132). These include 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydroxy-5,6-dihydrocytosine, 5-hydroxy-5-methylhydantoin, 5-hydroxyhydantoin and 1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine. It may be pointed out that another class of UVB-induced DNA damage that is an excellent substrate for endo III consists of 6-hydroxy-5,6-dihydrocytosine the so-called “cytosine photohydrate” (134) that arises from the photohydration reaction of cytosine through a singlet excited state according to an oxygen-independent process (12). It was recently shown using a suitable HPLC-MS/MS assay that the 20 -deoxycytidine “photohydrates” that were measured as the related deamination products, namely the 6R and 6S diastereomers of 6-hydroxy-5,6-dihydro-20 -deoxyuridine, are formed in the DNA of UVB-irradiated human monocytes (135). However, the yield of the latter photoproducts that is very low, about 0.1% of the CPDs, is likely to represent no more than 10% of the overall UVB-induced endonuclease III-sensitive sites (136). Further support for the UVB-mediated formation of oxidatively generated damage to DNA was gained from the observed high photosensitivity of the xthA deficient strain of E. coli that can be, however, prevented by the chelation of ferrous ions with 2,20 -bipyridine (137). The direct formation of UVB-induced DNA double-strand breaks (DSBs) was ruled out using CPD photolyase transgenic mouse dermal fibroblasts as target cells (138). It was shown by transcriptome analysis that the UVB-induced accumulation of cH2AX foci resulted from the collapse of replication forks at unrepaired CPDs leading to the generation of single- and double-strand DNA breaks. Evidence was provided that the phosphorylation of the variant histone c-H2AX occurs within the nucleus during the G1 phase of the cell cycle in UVC-irradiated human fibroblasts was dependent on the nucleotide excision repair. This contrasts with the generation of c-H2AX foci in gamma-irradiated cells that is triggered by the initial formation of DSBs (139). It was recently demonstrated that phosphorylated c-H2AX do not constitute specific biomarkers of DSBs (140,141).

O

H2N

As discussed above UVB radiation is able to induce as a minor process the formation of 8-oxodGuo in the DNA of skin, prokaryotic and eukaryotic cells. Several oxidative pathways including those mediated by •OH, one-electron oxidants and 1O2 are available for explaining the generation of 8-oxodGuo that could be considered as a ubiquitous DNA oxidation product (27,28,133). However, the mechanism(s) involved in the UVBinduced generation of 8-oxoGua in cellular DNA remain open to debate. Several lines of evidence would suggest that •OH is implicated in the oxidation reactions of the guanine base. One is based on the protecting effects exerted by mannitol against 8oxodGuo formation in human fibroblast and keratinocytes even if scavenging of highly reactive •OH in cell is challenging (115). Another fact is the UVB-induced formation of DNA singlestrand breaks (SSBs) including alkali-labile sites (109,111,142) that is mostly explained in terms of •OH-mediated hydrogen abstraction at C3, C4 and C5 of the 2-deoxyribose moiety (67,143–145). Therefore, the mechanism of 8-oxoGua generation may be rationalized by the addition of •OH radical to the C8 of the guanine moiety giving rise to the reducing 8-hydroxy-7,8dihydroguanyl radical (Fig. 1). Subsequently, one-electron oxidation of the purine radical leads to the formation of 8-oxoGua whereas its competitive one-electron reduction generated FapyGua through the opening of the imidazole ring (27,133,146). It would be of interest in forthcoming studies to search for an increase in the steady-state level of FapyGua upon UVB-irradiation of cellular DNA. It may added that the same guanine photoproduct distribution is expected from one-electron oxidation reactions (Fig. 1) that may be initiated by a charge-transfer process upon guanine photoexcitation giving rise to the transient 8-hydroxy-7,8-dihydroguanine radical through hydration of the initially generated guanine radical cation (Gua+•) (27,147–149). However, this reaction that does not lead to the formation of DNA single-strand breaks (SSBs) appears to be at best of little importance for UVB radiation. It may be reminded that evidence for oxidation, likely through ionization, of the base of isolated 20 -deoxyguanosine has been provided in aqueous solutions upon exposure to more energetic UVC photons (150). A third possibility that may be proposed from the results of model studies would involve oxidation by 1O2 that specifically gives rise to 8-oxoGua in both isolated and cellular DNA

O

°

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Mechanistic aspects

N

N

-e-

OH H

H2N

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HN H2N

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O N

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°+

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Figure 1. Common degradation pathways of guanine by addition of
OH at C8 and one-electron oxidation.

Photochemistry and Photobiology, 2015, 91

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without generating any FapyGua lesions and strand breaks (151,152). It has also been shown that 1O2 is generated with a low efficiency by the reaction of molecular oxygen with triplet excited purine and pyrimidine bases (153,154). However, no evidence has been provided so far for the generation of 1O2 in either isolated or cellular DNA upon exposure to UVB radiation.

Table 1. Oxidatively generated damage to DNA of human THP1 monocytes upon exposure to UVA and ionizing radiations. The frequency of the lesions is expressed in number of modifications per 109 bases and per either kJ m2 (dose range 0–150 kJ m2) or per Gy of ionizing radiation (dose range 0–40 Gy)–. Lesions

UVA radiation (per kJ m2)

Gamma rays (per Gy)

UVA RADIATION-MEDIATED OXIDATION REACTIONS TO CELLULAR DNA

8-OxodGuo* FapyGua† Endo III-sensitive sites§ Fpg-sensitive sites§ Strand breaks‡

0.98 Not determined 0.3 1.9 0.9

11 27 53 48 130

Major efforts have been made during the last two decades to gain insights into the molecular effects of UVA radiation on cellular DNA with emphasis on the delineation of the putative role of ROS that are generated by endogenous photosensitizers and biochemical responses (vide supra). This interest is explained, at least, partly by the likely implication of UVA radiation in cutaneous carcinogenesis (155,156) with a possible role being played by oxidative stress processes (157). A causative effect of UVA light was proposed for the occurrence of skin melanoma in heavily pigmented hybrids of Xiphophorus fish that would mostly involve oxidative reactions (158,159). It may be noted, however, that no evidence was provided in these studies for a UVA-mediated enhancement of defined oxidatively DNA damage. Furthermore, it was recently shown that the initiation of melanogenesis in the latter fish models is essentially triggered by UVB irradiation with no implication of oxidative reactions (160). However, this does not preclude a putative role played by UVA generated ROS in tumor progression (161). Oxidatively induced damage to DNA During the last 15 years relevant information has been gained on the nature of several UVA-induced oxidation lesions to cellular DNA and skin that were mostly detected using either DNA repair enzyme-based assays or HPLC methods. As for the damage generated by exposure to UVB radiation three main types of DNA modifications including modified purine bases, oxidized pyrimidine bases and strand breaks were identified as either classes of lesions or as single 8-oxoGua. Thus, it was shown using a modified alkaline elution assay that the formation in DNA of Fpg-sensitive sites, mostly 8-oxoGua, following UVA irradiation of CHO cells is higher than that of SSBs and endo III-sensitive sites (19,109). More precisely the relative yields of the three classes of oxidatively generated DNA damage decrease in the following order: 1 Fpg site > 0.45 SSB > 0.40 endo III site (19). A similar trend was noted for UVA-irradiated human monocytes whose DNA damage was analyzed using the alkaline comet assay that included a preincubation step with DNA repair glycosylases: Fpg-sensitive sites were still predominant over SSBs and oxidized pyrimidine bases (Table 1) (162). It was also found that the action spectrum for the formation of Fpg-sensitive sites exhibits a maximum around 430 nm within the 330–490 nm range of UVA–visible light that was emitted from a xenon arc lamp (109). Evidence was also provided for the fast repair of Fpg-sensitivesites in the DNA of UVA-irradiated mouse embryonic fibroblasts as inferred from alkaline agarose gel electrophoresis analysis; interestingly nuclear DNA was totally restored within 30 min following the UVA irradiation (11). A somewhat slower repair of UVA-induced Fpg-sensitive sites was observed in XP-A and MRC5 cells on the basis of alkaline assay measurement (163).

*HPLC-ECD. †HPLC/GC-MS. ‡Comet assay (single-strand breaks, double-strand breaks and alkali-labile sites). §Modified comet assay. ¶From Ref. (162).

Additional support for the UVA-mediated oxidation of cellular DNA is provided by the specific measurement of 8-oxodGuo in the DNA of mammalian cells that was achieved by either HPLC-ECD (19,20,111,113,162,164–169) or HPLC-ESI-MS/MS (170–172). The formation of 8-oxodGuo that was shown to be linear with the applied doses of UVA radiation within the 0–200 J cm2 dose range is more efficient in human fibroblasts than in keratinocytes. Thus, the respective yields of 8-oxodGuo that were inferred for the linear regression analysis of the slopes are 25.8  5.8 and 15.1  5.3 per 106 normal nucleosides and per kJ cm2 (20). It is worth noting that in both cases the frequency of UVA-induced 8-oxodGuo is much lower than that of cyclobutane dimer thymine (TT) as reported in Table 2. This was also observed in human melanocytes that were exposed to UVA radiation (172). Interestingly the yield of 8-oxodGuo that was measured by HPLC-MS/MS was twice higher in the DNA of melanocytes than in keratinocytes. This contrasted with the levels of SSBs and CPDs that were, respectively, similar in both cell types (172). Indirect support for the UVA-induced formation of 8-oxodGuo was provided from the observed relocalization of DNA repair N-glycosylase hOOG1 into nuclear foci of UVAirradiated HeLa cells that is likely to be triggered by the generation of ROS and not as the result of the recognition of 8-oxodGuo by the repair enzyme (173). The repair of UVB- and UVA-induced 8-oxodGuo was affected by the decrease in human 8-oxoguanine DNA glycosylase I as the result of downregulation of Cockaine syndrome B protein in human keratinocytes (126). Exposure of human skin explants to UVA radiation was found to lead to an increase in the level of 8-oxodGuo in the DNA extracted from biopsies as inferred from HPLC-ECD measurement. As an additional piece of information on the ability for UVA radiation to trigger oxidative reactions in whole organisms, evidence was provided for an increase in the level of 8-oxodGuo

Table 2. UVA-induced formation yield of TT and 8-oxodGuo in the DNA of fibroblasts, keratinocytes and human expressed as number of lesions per 106 normal bases and per kJ m2. Lesions TT 8-OxodGuo TT/8-oxodGuo

Fibroblasts*

Keratinocytes*

Human skin†

16.41  3.7 2.58  0.58 6.3

4.92  0.36 1.51  0.53 3.2

6.0  2.5 0.71  0.25 9.4

*From Ref. (20). †From Ref. (21).

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in the DNA of Drosophila larvae upon exposure to 364 nm laser light (169). Only a few attempts have been made so far to identify oxidized pyrimidine bases that are generated by UVA radiation. Thus, an efficient induction of 5-hydroxycytosine (5-OHCyt), an oxidation product of cytosine was noted in the DNA of normal and XP-A lymphoblasts after exposure to fluorescent light that mostly consisted of UVA and visible radiations (174). However, the yields of 5-OHCyt that were assessed using the questionable gas chromatography–mass spectrometry method were at least two orders of magnitude higher than those expected from the assessment of endo III-sensitive sites in UVA-irradiated mammalian cells (19,109,162). Further work using accurate analytical approaches such as the HPLC-ESI-MS/MS method is required to obtain more consistent information on the chemical structure of the UVA-induced pyrimidine oxidation products. The ability for UVA radiation to generate SSBs and/or alkalilabile lesions as minor classes of DNA damage has been demonstrated in Chinese ovary cells and human monocytes using the alkaline elution technique, agarose gel electrophoresis or the alkaline comet assay (19,109,162,163). Similar types of DNA lesions have been shown to be produced in adherent human keratinocytes (HaCaT) that have been exposed to UVA radiation (175) using an improved version of the alkaline comet (176). As an illustration of the low efficiency for UVA to cleave cellular DNA, it was found that an exposure to a dose of 100 kJ m2 of UVA radiation was able to increase the tail moment value from 1.91 up to 2.66 in relative units. Indirect evidence has been also provided for the UVA-induced formation of DNA DSBs in Chinese ovary cells from the observation of micronuclei induction and the significant increase in the photosensitivity of the CHO cell line mutant for XRCC5 which is deficient in nonhomologous end-joining (NHEF), the main repair pathway of DSBs (177). More direct support for the UVA-mediated generation of DSBs in HaCaT was gained from either immunohistochemical detection of c–H2AX foci (175,178) or application of the neutral comet assay (175). However, it has not been established whether these putative DSBs that may be implicated in the induction of micronuclei and chromosomal aberrations are generated directly or as a secondary biochemical process of initially produced SSB or base damages. In that respect it has been shown that either unrepaired single-strand breaks during G1 phase (179) or persistent CPDs (138) could be converted into DSBs during the S-phase of replication. More recently it was reported that physiological doses of UVA light in contrast to ionizing radiation were not able to trigger the activation of the recombination repair pathway in primary human skin fibroblasts, an observation that is suggestive of the absence of formation of DSBs (180). This received further confirmation from the absence of DNA double-strand breakage as inferred by neutral single-cell electrophoresis analysis of UVA-irradiated fibroblasts. Furthermore, no increase in the level of immunodetected c-H2AX foci was noted in fibroblast cells 60 min after being exposed to 400 kJ m2 of UVA radiation (180). In another recent study, the levels of c-H2AX foci were found to increase in a dose-dependent manner in HaCat keratinocytes upon exposure to UVA radiation that was delivered as either an acute dose or fractionated doses (181). Indication for DSB formation was also gained from neutral single cell gel electrophoresis. It was hypothesized that the suggested formation of DSBs would arise from the repair of oxidatively generated clustered DNA lesions

(OCDLs) and not through the replication of bulky types of DNA damage (181). However, the probability to have at least two proximal oxidized sites consisting of a modified base and a SSB on the two opposite strands within less than two DNA helix turns appears highly unlikely. How OCDLs that are hallmarks of the molecular effects of ionizing radiation (29,182) could be induced by independent radical hits likely mediated by •OH produced by Fenton reactions if one considers that the yield of isolated oxidatively induced lesions is less than 107 in UVAirradiated cells? The same remark applies as well for the formation of DSBs, another class of OCDLs, as discussed in a commentary article (183). Therefore, it is intriguing that the generation of independently induced SSBs in a close vicinity giving rise to DSBs was proposed to explain the observed phosphorylation of H2AvD, a homologue of H2AX, in Drosophila melanogaster larvae upon exposure to doses of LED-UVA higher than 600 kJ m2 (184). Furthermore, the yield of 8-oxodGuo did not significantly increase within the dose range 0– 1000 kJ m2 in the DNA of the larvae. This is not consistent with the proposed implication of UVA as an efficient inducer of SSBs via the generation of •OH. Further work is still required to further clarify the controversial issue concerning the rather unlikely induction of direct DSBs in UVA-irradiated cellular DNA. This should involve more direct methods than the immunodetection of c-H2AX that suffers from a lack of specificity as a measure of DSBs (140,141). Mechanistic aspects of UVA-mediated oxidation reactions to DNA DNA is one of the critical cellular targets of photosensitized reactions (26,152,185) that give rise as the main oxidative degradation pathways identified so far to 8-oxodGuo. The latter ubiquitous hallmark of oxidation reactions of cellular DNA is of little value in term of mechanistic diagnostic since it can be generated indifferently by •OH, one-electron oxidants, singlet oxygen and peroxynitrite (27,28). Therefore, there is a need of considering more specific degradation pathways that have been identified in model studies and in some cases validated in cellular DNA (27,28,69). Mechanistic insights into the UVA-mediated oxidation reactions were gained from a comparison of the effects of UVA light with those of radiation-induced •OH on human monocytes (186) in terms of three main classes of DNA damage including strand breaks and/or alkali-labile sites, Fpg-sensitive sites and oxidized pyrimidine bases that were revealed using a modified version of the alkaline comet assay. The main quantitative data thus obtained are reported in Table 1. This is strongly suggestive that the main oxidative degradation pathways of cellular DNA triggered by UVA radiation are different from those mediated by gamma rays (162). Under the latter conditions where •OH is the predominant reactive species, the yield of endo III- and Fpg-sensitive sites is similar, each of them being about three times lower than that of DNA strand breaks. This is still the case for the levels of endo III-sensitive sites and strand breaks formed in the DNA of UVA-irradiated cells. In contrast, the ratio between the yields of Fpg-sensitive sites and DNA strand breaks is six times higher upon UVA irradiation that after c-ray exposure (Table 1). Interpretation of the latter data requires consideration of the available information on the reactivity of DNA toward putative UVA-induced reactive species and processes that may involve •OH, one-electron oxidation and 1O2

Photochemistry and Photobiology, 2015, 91 (14,27,152). It is well established that •OH is able to oxidize both purine and purine bases (28,30,133) whereas hydrogen abstraction within the sugar moiety leads in most cases to the formation of strand breaks (67,143–145). One-electron oxidation reactions give rise directly or indirectly after hole transfer within the DNA chain to the predominant formation of modified guanine lesions including 8-oxoGua and FapyGua without, however, DNA cleavage (27,147–149). Investigations using a suitable derivatized naphthalene endoperoxide as a chemical source of 1 O2 (186,187) have shown that the latter ROS reacts in a highly specific way with the guanine moiety of both isolated and cellular DNA to produce exclusively 8-oxoGua (69,152,187–189) according to a Diels-Alder [2 + 4] cycloaddition mechanism (Fig. 2) without inducing detectable amounts of strand breaks (151). It may be noted that the formation of SSBs in pBr322 plasmid DNA has been reported following exposure to 1O2 (190). This is likely to be explained by the generation of unstable secondary oxidation products of highly reactive 8-oxoGua (191) as precursors of abasic sites (192,193). However, the sequential two hit reaction of a guanine residue is unlikely to occur under biologically relevant 1O2 oxidation conditions It was also found that 1O2 is not able to act as a one-electron oxidant as inferred from the lack of formation of FapyGua which is also generated by the reaction of •OH with guanine (Fig. 1). It may be added that attempts to detect FapyGua in the DNA of UVAirradiated cells were unsuccessful (162), suggesting either the lack or more likely (vide infra) the low involvement of •OH and/or one-electron oxidation pathway in the formation of 8-oxoGua. The predominance of 8-oxoGua over DNA strand breaks and endo III-sensitive sites representing mostly oxidized pyrimidine bases, may be rationalized in terms of major participation of 1O2 (85%) together with a low contribution of •OH (15%). It is likely that the formation of the different classes of oxidatively generated damage to DNA would vary with the nature of the cells since they may contain different types of photosensitizers in variable amounts. One relevant example is given by melanocytes in which the presence of melanin is likely to explain the enhancement of the UVA-mediated formation of 8-oxodGuo (172). It clearly appears that 8-oxoGua, the main oxidatively generated base lesion identified so far in the DNA of UVA-irradiated cells is formed in very low amounts. In fact exposure of monocyte cells to a dose of UVA radiation up to 100 kJ m2 is required to double the level of steady-state level of 8-oxoGua (162) that arises mostly from oxidative metabolism in nonirradiated cells. This applies as well to human skin and related primary cultures of fibroblasts and keratinocytes (20,21). This strongly suggests that the contribution of 8-oxoGua to the overall biological deleterious effects of UVA radiation is low although one has to consider that oxidatively generated damage to DNA is likely to occur in the epidermis and therefore may exert more detrimental effects.

N

HN H2N

N

It remains to be determined whether other types of oxidatively generated lesions that may involve DNA and biomolecules such as proteins and unsaturated lipids are generated upon UVA irradiation. This is discussed in further details in the next section.

PUTATIVE UV-INDUCED OXIDATION REACTIONS TO CELLULAR DNA UVA and to a lesser extent UVB are able to oxidize cellular DNA mostly through the photosensitized generation of 1O2 with a smaller contribution of •OH, the likely product of Fenton reactions. It remains, however, in terms of •OH-mediated base degradation products to identify several oxidation products of adenine, guanine, thymine and cytosine as already pointed out. Main efforts should be made to search for other types of oxidatively generated DNA lesions that mainly arise from somewhat delayed biochemical and chemical processes. Base adducts with reactive breakdown products of lipid peroxides Lipid peroxidation has been shown to be induced by UVB photons and more efficiently by UVA radiation through the involvement of either •OH or 1O2 in cellular membranes. Electrophiles a,b-unsaturated aldehydes including malondialdehyde and 4hydroxy-2-nonenal that are released from the breakdown of unstable lipid peroxides are able to migrate within the cell and react with several amino acid residues in proteins and aminosubstituted bases in DNA (194–198). Guanine is the most reactive base target of the electrophilic addition of either MDA or HNE. The formation of pyrimido[1,2-a]purine-10(3H)one is explained by the initial Michael type addition of MDA at the 2exocyclic amino group of guanine followed by a cyclization reaction involving the N1-imino function and subsequent dehydration of the exocyclic diol thus formed (Fig. 3). A similar propanoadduct that is, however, not able to undergo dehydration is generated in the reaction of HNE with guanine (Fig. 3). So far no information is available on the generation of electrophilic reactive aldehyde adducts to cellular DNA upon UVB or UVA irradiation. It worth noting that sensitive and accurate HPLCESI-MS/MS methods are available for measuring a wide range of MDA and HNE exocyclic DNA adducts products including ethenobases (197,199–202). This should allow the search of at least the most abundant propano guanine adduct in the DNA of UV-irradiated cells. Lesions arising from one-electron oxidation of guanine Increasing evidence has been provided for the generation of NO• a mostly the result of biological responses including inflammation after UVB or UVA irradiation of cells and skin (45,48,90,92). It is now well documented that the efficient reac-

O

O

N

1O 2

O N

HN H2N

N

147

N O

H

H N

rearrangment HN

O

reduction

O

Figure 2. Singlet oxygen oxidation of guanine in DNA.

H2N

N

N

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Jean Cadet et al. OH O

O

O

O

N

N

N

N

MDA HO

O

N

O

O

N

N

N

N

N

N

MDA adduct

N

HN H2N

N H

OH N

HN

O N

N

HNE O

N H

R

R

N

N

N H

R

N

N

HNE adduct (R = C6H13O) Figure 3. Main adducts of malondialdehyde (MDA) and 2-hydroxy-4-nonenal (HNE) to guanine.

DNA-DNA crosslinks

ine t os Cy O N

HN H2N

O

°+

N

O N

HN

Thymine

G*-T* adducts

N

H2N

N

N

N

O

N

RNH

2

O

O N

HN H2N

N

H N

R

(lysine)

R= NH

N NH2

R=

N H H N N H

(polyamines) NH2 NH2 H N

NH2

Figure 4. Nucleophilic addition nucleophiles to the guanine radical cation giving rise to intra- and interstrand crosslinks.

tion between O2
 whose formation is also enhanced after UV irradiation, with NO• gives rise to ONOO (203,204). It has also been shown that is able to react with CO2 giving rise to unstable nitrosoperoxycarbonate with subsequent release of highly reac
tive carbonate anion radical (CO3 ) and NO2
(204–206). Evidence for this sequence of reactions in cells has been provided by the measurement of 3-nitrotyrosine in tissues subject to inflammation (207). The formation of the modified amino acid is rationalized in terms of initial one-electron oxidation of the tyrosine residue followed by combination of the highly oxidizing tyrosyl radical thus formed with NO2
. Therefore, it may be expected that DNA could also be a target of the one-electron
oxidation mediated by CO3 with guanine as the predominant reactive base (208). Abundant information is now available on the reactions of the guanine radical cation (Gua•+), produced by one-electron oxidation, with several nucleophiles (27,29). The addition of water that leads to the formation of 8-oxoGua and FapyGua has already been mentioned (27,29). In addition, it has been shown that intrastrand crosslinks involving addition of thymine through its N3 positon to Gua+• (Fig. 4) are formed both in isolated (209,210) and cellular DNA (211). Other examples of relevant nucleophilic additions of Gua•+ were provided by the

formation of a covalent bond with either polyamines (212), small molecules found at mM concentration in the nucleus of eukaryotic cells, and a lysine residue of a tripeptide in model studies (213). Interestingly the formation of DNA-protein crosslinks has been detected in cells exposed to UVA radiation after the immuosuppressant 6-thioguanine (214), acting partly by one-electron oxidation, has been inserted into DNA (215). The formation of highly deleterious interstrand crosslinks (Fig. 4) has been also reported in 6-thioguanine containing cellular DNA upon UVA irradiation (216,217). The search for these specific one-electron oxidation products could be of interest for better delineating the putative role of the generation of NOS upon exposure of cells to UVB and UVA radiations.

CONCLUSION Information has been gained during the last two decades on the UV-induced oxidation reactions in cellular DNA and skin through mostly the detection of stable final degradation products and detailed mechanistic studies. UVB radiation appears to be a poor generator of oxidatively generated damage to DNA. Evidence has been provided that UVA radiation is more efficient

Photochemistry and Photobiology, 2015, 91 than shorter solar UV wavelengths in inducing 8-oxodGuo, the main photooxidation product as mostly the result of type II photosensitization involving the generation of 1O2. In agreement with the relatively low yield of 8-oxoGua, recent studies have confirmed that the UVA mutation spectra in several types of eukaryotic cells and skin epidermis of XP variant model mice is dominated by C to T transitions at 50 -TCG-30 sequences where C is possibly methylated. This characteristic “UV signature” (5) was observed at the exclusion of significant levels of T ? G transversions (6,218). It remains to be established the possible role of 8-oxoGua generated in dermis and the putative implication of delayed biochemical responses triggered by both UVB and UVA radiation in terms of genotoxic effects.

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42. 43.

44. 45. 46.

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AUTHOR BIOGRAPHIES Jean Cadet has obtained his PhD at Grenoble University in 1973 and has been research associate and visiting professor in several institutions including John Hopkins University (1977 and 1980) and Atomic Energy of Canada at Pinawa (1978) before creating the Laboratory “L
esions des Acides Nucl
eiques” at the French Atomic Energy Institute in Grenoble. He is since 2002 Scientific Adviser at CEA/Grenoble before his re-location at University of Sherbrooke by the end of 2014. His main research interests include various aspects of the chemistry and biochemistry of oxidatively generated and photoinduced damage to DNA (mechanisms of reactions, measurement in cells and assessment of biological features). He has so far coauthored 580 peer-reviewed articles and book chapters. Thierry Douki is a scientist at the Atomic Energy Commission in Grenoble, France. He was trained in organic synthesis. Most of his research activities focus on DNA damage produced by a wide variety of genotoxic agents including oxidative stress, chemicals and ionizing and ultraviolet radiations. In this last field, he is mostly involved in the characterization of photoproducts, the development of analytical tools for their quantification and the study of the formation and repair of UV-induced DNA damage in cells and skin. Thierry Douki is associate-editor of Photochemistry and Photobiology, member of the European and of the American Societies for Photobiology. He is also secretary of the French Society for Photobiology. He is coauthor of more than 160 original articles and a number of reviews and book chapters on various aspects of DNA damage.

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Jean-Luc Ravanat obtained a master in Biochemistry and a Ph. D. in Organic Chemistry from University of Montpellier and Grenoble (France), respectively. After two years as a postdoctoral fellow at Nestl
e Research Centre in Switzerland, in 1995 he became a staff scientist in the laboratory “L
esions des Acides Nucl
eiques”, CEA Grenoble, and since 2013 he is the team leader. Most of his research work focuses on DNA damage and repair induced by several genotoxic agents including mostly photosensitization reactions and ionizing radiations. In particular he devoted efforts to delineate the reactions of nucleic acids with singlet oxygen, hydroxyl radical and one-electron oxidants. In addition he has made a significant contribution in the development of sensitive and accurate assays to measure DNA lesions at the cellular level. More recently he concentrated his work to study the mechanisms of formation of radiationinduced complex DNA lesions. He is also a visiting scientist on the medical beam line of the European Synchrotron Research Facility in Grenoble where he works on the radiosensitization effects of heavy atoms (including nanoparticles) containing compounds for improving radiotherapeutic treatments. He is coauthor of about 140 research articles and several review articles.