Mechanisms of photosensitized DNA cleavage

203

J. Photochem. Photobiol. B: Biol., 20 (1993) 203-209

Mechanisms Nicole

Paillous+

of photosensitized and Patricia

DNA cleavage

Vicendo

Laboratoire des IMRCP, URA 470 au CNRS, Universite’ Paul Sabatier, I18 route de Narbonne, 31062 Toulouse Cedex (France) (Received

May 19, 1993; accepted

June 2, 1993)

Abstract Photosensitization may promote DNA damages such as nucleic acid oxidation or single strand breaks via three main pathways: hydroxyi radicals attack, electron transfer process or oxidation by singlet oxygen. While direct production of OH’ by photosensitization is rarely observed, the mechanism of DNA attack by OH’ is now well established on the basis of informations provided by water radiolysis experiments. Some dyes may also induce single strand breaks via an electron transfer occurring from a nucleobase to the sensitizer in the excited state. This process generates base radical cations identical to those arising from DNA photoionisation. These radicals may undergo deprotonation or dehydration to form the same neutral radicals as those produced by OH’ but with a slightly different pattern. In contrast, while many sensitizers produce singlet oxygen, the mechanism of DNA damages induced by this way is still unclear. In this case the guanine moiety in nucleosides or in DNA is selectively altered leading to the formation of 8 oxoG or 8 oxodG and FapyGua. The mechanism of single strand breaks formation by singlet oxygen is discussed in this overview.

Key words: Hydroxyl

radicals;

Singlet

oxygen;

Electron

Numerous studies on the mechanisms of photosensitized DNA damage have been carried out for a better understanding of problems related to phototoxicity and phototherapy, either to prevent cell damaging and degenerative skin diseases or to enhance the cytotoxicity of anticancer drugs. The effect of UV-visible light mediated by endogenous or exogenous sensitizers leads to the formation of a wide range of DNA modifications. Among these lesions, strand breaks are one of the major damaging processes. Our aim in this short overview is to gather the different pathways that may promote DNA cleavage. According to the nature of the sensitizers, strand breaks may result from type I or type II mechanisms including attack by hydroxyl radicals, the electron transfer process and oxidation by singlet oxygen.

to whom correspondence

loll-1344/93/$6.00

DNA

damage;

8-oxoguanine;

2. Hydroxyl radicals

1. Introduction

‘Author

transfer;

should be addressed.

FapyGua

attack

The hydroxyl radical is one of the more noxious reactive oxygen species towards DNA. Few photosensitizers generate hydroxyl radicals directly by photodegradation. However, some of them may undergo electron transfer to an oxygen molecule to give hydroxyl radicals by a subsequent Fenton reaction. Electron transfer between OH- and metal complexes such as Co(II1) and Ru(II1) salts [l-3] is an alternative pathway to produce these radicals. As hydroxyl radicals are also commonly produced by water radiolysis (indirect effects of ionizing radiations), their reactions with DNA are now well understood and have been extensively reviewed [4-g]. It was shown that OH’ attack is a very efficient process with no particular selectivity for the different bases [lo]. In a first step, OHmay add to nucleobases producing base radicals. In a second step, these intermediate radicals may abstract hydrogen from the adjacent deoxyribose, producing sugar radicals that lead to a DNA backbone cleavage, as reported by SchulteFrohlinde and coworkers [4,11,12]. The oxidation reactions involved in the attack of nucleic acids by OH’ are extremely complex. However, many 0 1993 - Elsevier

Sequoia. All rights reserved

204

N. Paillous, P. ficendo I DNA cleavage mechanisms

oxidation products have been isolated and now most base radicals and intermediates have been characterized following difficult analytical work. Pyrimidine modifications have been largely studied. It is well established that hydroxyl radicals cause damage to pyrimidines such as thymine by binding to the C,C, double bond of the ring mainly at the C, (60%) and to a lesser extent at the C, (35%) positions [5]. In addition, OH’ may abstract hydrogen from the methyl group, although this process is incidental (5%) (Fig. 1) [13]. In aerobic conditions, these nucleobase radicals react rapidly with oxygen to produce hydroperoxides (50%) as main products after a reduction step. Now, most of these peroxides have been isolated and identified [14,15]. Peroxyl intermediates may also lead to the formation of cyclic or ring-opened by-products [16,17]. Reactions between OH‘ and purines produce lower yields of oxidation products and are thus not so readily studied. This lack of reactivity does not stem from the rate constant values of OH’ with purines which are in fact higher than those with their pyrimidine homologues [9]. The low reactivity of purine radicals is accounted for by the fact that they are less oxidizable and so tend to revert to the starting nucleobase [15]. The oxidation of purines by OH’ results essentially from the addition of OH’ at the C, or Cs positions of the imidazole ring [9,18]. As shown by Vieira and Steenken, 40H and 80H radical adducts may undergo dehydration and ring-opening reactions [19,20] (Fig. 2). Hydroxylation of the C, carbon may also occur [18], but it is always a minor process. Both 50H and 40H radical adducts give rise to the same final product after dehydration PO1 * For 2’-deoxyadenosine, the predominant modification observed is an addition of OH- at the C, position of the imidazole ring giving the C, OH radical adduct which can be converted into 8-oxo-7,8-dihydro-2’-deoxyadenosine [6] and the corresponding formamidopyrimidine (Fapy). In

Deoxythymidine

a-r

3 5 %

6 0 %

e-

70 %

< 1%

5 % 30%

Fig. 1. Thymidinyl radicals resulting from the reaction of deoxythymidine with hydroxyl radicals or from the transformation of the cation radicals issuing from an electron transfer process.

8 OH-adduct

Fig. 2. Hydroxyl radical adducts resulting deoxyguanosine with hydroxyl radicals.

FapyGua

from the reaction

of

contrast, 2’-deoxyguanosine is mainly hydroxylated at the C, and C, positions leading to 2,2-diamino [(2-/3-D-deoxyerythropentofuranosyl)-4-amino]-5oxazolone and its 2-amino [(2-P-D-deoxyerythropentofuranosyl)-5-amino]-4-imidazolone precursor [21]. C, hydroxylation can also take place, producing 8-oxo-7,8-dihydro-2’-deoxyguanosine (8oxodG) and its corresponding FapyGua, although this oxidation pathway is relatively minor [S] (Fig. 2). In DNA, hydroxyl radicals produce single-strand breaks (SSB) and apurinic and alkali-labile sites [22]. It has been established that DNA mod& cations arise from the same reactions as those described above for nucleoside model compounds. As previously mentioned, the radicals formed by hydroxylation of nucleobases lead to a sugar radical by abstraction of a hydrogen atom from the adjacent deoxyribose at Cr4 of the ring. Then the cleavage of DNA backbone results from a /3 elimination of the C’3 ester phosphate on the sugar radical so formed [4,11,12]. In an alternative pathway (20%), hydroxyl radicals may abstract a hydrogen atom from the sugar at the C’4 position [23]. The yield of SSB depends on the ability of hydroxyl radical adducts to abstract hydrogen from the deoxyribose, which is the determining step for DNA breakage. The reactivities of the different purines and pyrimidines differ considerably [24,25] and may be modulated by steric factors inherent in the structure’ of DNA. Hydroxyl radicals may also cause double-strand breaks. This process requires two single-strand breaks located on opposite strands [26,27]. The yield of DNA photocleavage depends on the DNA environment. DNA bases in mammalian chromatin, in aqueous suspension, exposed to ion-

N. Paillous, P. Eendo

205

I DNA cleavage mechanisms

izing radiation-generated free radicals give rise to fewer modified bases than calf thymus DNA irradiated in aqueous solution under similar conditions [28,29]. This discrepancy is attributed to nucleosome structure, scavenging OH’ by histones and to the fact that base radicals may not only induce strand breaks but also generate DNAprotein cross-linkings [28]. 3. Electron transfer process Photosensitizers can damage DNA via an electron transfer, which occurs generally from the ground state of the base to an excited state of the dye. This process yields purine and pyrimidine radical cations similar to those obtained by photoionization [5,9,30]. Electron transfer depends on the oxidation potential of the ground state of the base and the reduction potential and energy of the excited state of the dye. Upon irradiation, organometallic complexes such as Ru(II1) complexes, investigated by Kelly et aE. [31] for instance, may promote DNA strand breaks via an electron transfer. On the basis of lifetime fluorescence measurements, Kirsch-De Mesmaeker et al. [32] have shown that these complexes may initiate the formation of guanine radical cations if their excited state reduction potential is above 1.1 V (21s.SCE). The guanine which has the lowest oxidation potential [33,34] is selectively oxidized. If the reduction potential of the complex exceeds 1.4 V (~~1s.SCE) an electron transfer from adenine to the metal complex may also occur [32]. The determination of the cleavage site by sequencing experiments supports these hypotheses [35]. The base radical cations may react via different competitive pathways (deprotonation, dehydration) to form the same neutral radicals as those produced by hydroxyl radicals [8,36,37], but in different patterns [8]. These radicals may also induce SSB by p elimination of the Cf3 ester phosphate of deoxyribose [4,12,30,38]. 4. Oxidation by singlet oxygen Until recently, DNA breakage induced by IO2 has been a subject of controversy, partly because true single-strand breaks do not exceed 5% of the total DNA damage [39,40] and are thus difficult to observe. ‘O,-induced cleavage is a relatively minor cause of DNA damage, and its mechanism still remains unclear. It is now well established that singlet oxygen modifies deoxyguanosine se-

lectively, either as free nucleoside or in DNA [4143] (for review see refs. 44-46). Cadet et al. [47,48] were the first to isolate and identity the three main oxidation products arising from the reaction between singlet oxygen and 3’,5’-d&Oacetyl-2’-deoxyguanosine, namely the 4R* and 4S* diastereoisomers of 9-(3’,5’-di-0-acetyl)-4,8-dihydro-4-hydroxy-8-oxo-deoxyguanosine and the cyanuric acid derivative (Fig. 3). Cadet [47] and Matsuura [49] have suggested that 4,8,dihydro-4-hydroxy-8-oxo-deoxyguanosine may arise from a 14 cycloaddition of the ‘02 molecule to the imidazole ring leading to an unstable endoperoxide (Fig.4). This assumption was supported by the results of Ravanat et al. [21] showing incorporation of an oxygen molecule into deoxyguanosine, upon irradiation in the presence of ‘*OZ. The pathway to the cyanuric acid derivative is probably more complicated and a tentative mechanism involving two 2 + 2 cycloaddition of ‘02 has been proposed [471. In DNA, singlet oxygen also modifies guanine residues selectively. Floyd et al. [50] were the first to identity 8-oxodG as a photo-oxidation product using methylene blue. The question was to determine whether this compound was a result of DNA oxidation by singlet oxygen, as proposed, or as a result of a hydroxyl radical attack since methylene blue generates both ‘02 (55%) and OH’ (45%) [47]. However, the assumption that 0

“$4

b

“L oH:Ribosr N

4.0-dihydro-4-hydroxy8-oxo-deoxyguanosine “J&O

y kmsr k-$+O

Deoxyguanosine

‘dRibore 0 Cyanuric

acid

Fig. 3. Two main oxidation products resulting from the reaction of deoxyguanosine with singlet oxygen.

Deoxyguanosine

Deoxyguanosine 1.4.endoperoxyde

4.8.dihydro-4.hydroxy8-oxo-deoxyguanosine

Fig. 4. Mechanism of 4,8-dihydro+hydroxy-&oxo-deoxyguanosine formation from the reaction of deoxyguanosine with singlet oxygen.

206

N. Paillow

P. vicendo I DNA cleavage mechanisms

‘02 is able to promote the formation of 8-oxodG takes advantage of the fact that many photosensitizers such as rose bengal [51] and hematoporphyrin [42,52], which produce high yields of r02, also lead to the formation of 8-oxodG in DNA. The latest results of Devasagayam et al. 1531using a pure source of singlet oxygen (thermodissociation of 3,3’-(1,4naphthylidene) dipropionate or NPDOp [54]) clearly demonstrate that ‘02 generates 8-0xoG or 8-oxodG. The detail of these reactions is still unknown. It is possible that the reaction pathway between ‘02 and dG includes a reductive step. Several mechanisms, illustrated in Fig. 5, have been proposed. It has been postulated that the 4-hydroxy8-oxo-deoxyguanosine derivative may be an intermediate in 8-oxodG formation. For this transformation two reducing equivalents have to be supplied [53]. It has also been suggested that both compounds may derive from a common precursor that would be the unstable endoperoxide intermediate. A reducing step, leading to 8-oxodG, which may occur during the decomposition of the endoperoxide [21]. Addition of thiols such as dithiothreitol, cysteine, cysteamine, or glutathione, which are thought to play a role as reductive agents, significantly increase the ‘O,-induced formation of 8-oxodG derivative 1551.This observation is in agreement with the two formation pathways of 8-oxodG proposed in Fig. 5. Recently, in a study of photosensitization of DNA calf thymus by methylene blue, Boiteux et al. [56] detected the formation of FapyGua as a photoproduct of deoxyguanosine residues. They suggested that the formation of FapyGua and 8oxodG involves an electron transfer from the guanine to ‘02 [56]. This process should generate a superoxide radical anion and a guanine radical cation which could be converted into 8-oxodG and

4,s.dihydro-4-hydroxyB-oxo-deoxyguanosine

+Wl 7

1,4

endoperoxide

B-oxodG

Fig. 5. Different pathways proposed for the formation of 8-oxodG from the I,4 endoperoxyde of deoxyguanosine.

FapyGua via the formation of a C,-OH radical adduct as described above (for review see ref. 9). In addition to modification of guanines, apurinic sites, alkali and piperidine labile sites [57] and SSBs may also be observed [40,53,55,57,58], although singlet oxygen appears to promote formation of 8-oxodG rather than DNA strand breaks. For example, using NDPO*, Schneider et al. [59] have shown that single-strand nicking is 17-fold less frequent than the formation of 8-oxodG. The capacity of singlet oxygen to break DNA backbone has been largely contested. In previous works, Nieuwint et al. [60] and Lafleur et al. [61] did not detect any strand breaks using plasmid DNA as substrate and NDP02 as a source of ‘OZ. This point has been largely reinvestigated by different teams. Recent data of Di Mascio et al. [40] and Blazek et al. [57] have shown that ‘02 produced by different methods (thennodissociation of NDP02, microwave discharge and rose bengal irradiation) generates frank strand breaks on both single and double-stranded DNA. The effects of ‘02 quenchers and of DzO have confirmed the involvement of ‘02 in these breakages which now seems clearly established [40,55,57,58]. This apparent discrepancy may stem from differences in the experimental conditions such as ‘02 concentration and the ionic strength of the medium. Using high concentrations of NDPOZ, Di Mascio et al. [62] have also observed double-strand breaks of SV40 plasmid DNA. However, single-stranded DNA appears to be more sensitive to an attack of ‘02 than double-stranded DNA, while for hydroxyl radicals only a slight difference could be observed [63]. Since strand breaks occur exclusively at guanine residues [53,64,65] with no hot spot [53], the question currently at issue is whether guanine oxidation products, especially 8-oxo-deoxyguanine, are involved in true DNA strand breaks. Results of Schneider et al. 1591 reported above, showing that SSB occur less frequently than does formation of 8-oxodG, suggest that 8-oxodG is not at the origin of SSB. The possibility that 8-oxodG and SSB derived from the endoperoxide formed by reaction between guanine residues and ‘02 [53] is still a controversial matter [66]. This assumption is in agreement with kinetic data showing that the time courses of formation of both 8-0xodG and SSB are similar [53] and with the fact that thiols, when added to the medium, increase simultaneously these two ‘02-induced processes [53,55]. As proposed above, the enhancing effect of thiols on 8-oxodG formation may be explained by hydrogen donation to the endoperoxide. However, the en-

N. Paillous. P. Viiendo / DNA cleavage mechanisms

hancing effect of thiols on SSB production is more difficult to interpret. Regarding SSB formation, it should be noted that the enhancing effect of thiols with ‘0, contrasts to their chemical repair action observed in the case of a hydroxyl radicals attack [66]. The formation of thiyl radicals resulting from hydrogen donation or thiyl peroxyl radicals produced by oxidation under aerobic conditions is put forward to explain these protective or genotoxic effects of thiols [53,66], but the role of these radicals is quite unclear. The enhancement of lo,induced formation of both 8-oxodG and SSB resulting from the presence of thiols was recently discussed by Lafleur et al. [66]. Taking into account that the extent of these effects are highly inflenced by the nature of the thiols, they postulate that these DNA damages probably arise from different pathways. However, the debate remains open and new investigations are required to shed light on the mechanism of SSB formation.

5. Conclusion

In summary, among the various reactive oxygen species, hydroxyl radicals _appear to be the most efficient DNA breakers. Single strand breaks induced by OH’ are three times more widespread than those arising from radical cations [27,67] and far more frequently occurring than those formed by singlet oxygen [39,40]. Mechanisms of photosensitized DNA cleavage described in this report exhibit different base selectivity. Hydroxyl radicals attack the four nucleobases with no specificity while singlet oxygen only modifies the guanine residues. Similarly, guanine is also readily modified by electron transfer but this selectivity may be modulated by the reduction potential of the excited state of the sensitizer. Dyes acting by electron transfer appear to be promising site or sequence specific reagents for molecular biologists. Whatever the mechanism, guanine appears to be the major reactive component that leads mainly to the formation of 8-oxodG and FapyGua. These reaction products could be considered as a signature of an oxidative stress of DNA.

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