Mechanisms of Damage to DNA Labeled with Electrophilic

Review Article pubs.acs.org/JPCB

Mechanisms of Damage to DNA Labeled with Electrophilic Nucleobases Induced by Ionizing or UV Radiation Janusz Rak,* Lidia Chomicz, Justyna Wiczk, Kinga Westphal, Magdalena Zdrowowicz, Paweł Wityk, Michał Ż yndul, Samanta Makurat, and Łukasz Golon Faculty of Chemistry University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland ABSTRACT: Hypoxiaa hallmark of solid tumorsmakes hypoxic cells radioresistant. On the other hand, DNA, the main target of anticancer therapy, is not sensitive to the near UV photons and hydrated electrons, one of the major products of water radiolysis under hypoxic conditions. A possible way to overcome these obstacles to the efficient radio- and photodynamic therapy of cancer is to sensitize the cellular DNA to electrons and/or ultraviolet radiation. While incorporated into genomic DNA, modified nucleosides, 5-bromo-2′deoxyuridine in particular, sensitize cells to both near-ultraviolet photons and γ rays. It is believed that, in both sensitization modes, the reactive nucleobase radical is formed as a primary product which swiftly stabilizes, leading to serious DNA damage, like strand breaks or cross-links. However, despite the apparent similarity, such radio- and photosensitization of DNA seems to be ruled by fundamentally different mechanisms. In this review, we demonstrate that the most important factors deciding on radiodamage to the labeled DNA are (i) the electron affinity (EA) of modified nucleoside (mNZ), (ii) the local surroundings of the label that significantly influences the EA of mNZ, and (iii) the strength of the chemical bond holding together the substituent and a nucleobase. On the other hand, we show that the UV damage to sensitized DNA is governed by long-range photoinduced electron transfer, the efficiency of which is controlled by local DNA sequences. A critical review of the literature mechanisms concerning both types of damage to the labeled biopolymer is presented. Ultimately, the perspectives of studies on DNA sensitization in the context of cancer therapy are discussed. to necrosis, apoptosis, or senescence.5 Whereas many copies of crucial proteins exist in the cell, only a single copy of nuclear DNA is present. Moreover, while damaged proteins can be restored via biosynthesisthe biochemical process that employs genetic information encoded in DNAthe latter molecule cannot be repaired if damaged beyond a certain level. Thus, damage to DNA molecules constitutes the most efficient way to kill cells and, therefore, this biomolecule is the main target of radiotherapy.2 It is worth emphasizing that hypoxiaa hallmark of solid tumorsseems to be the enfant terrible of radiotherapy.6 Indeed, the so-called oxygen effect reduces the efficacy of a given dose of IR in hypoxic cells by 2- to 3-fold compared to oxygenated ones. On the one hand, this effect is directly related to the diminished level of oxygen, since the dissolved in cytoplasm O2 serves as a species which perpetuates the initial DNA damage induced by hydroxyl radicals.6 On the other hand, solvated electrons, inactive toward native DNA, are produced under hypoxia in an amount that is comparable to the •OH radicals.7 This situation implies the employment of radiosensitizers in an efficient radiotherapy of solid tumors. The fact that hydrated electrons are the second most abundant product of water radiolysis at low oxygen concentrations suggests the potential for using radio-

1. INTRODUCTION Cancer, the main cause of death in highly developed countries and the second most frequent reason for death in developing countries,1 is difficult to combat; despite extraordinary progress in medicine observed in recent years, cancer still cannot be considered a disease that is under our full control. Three modalities commonly applied against tumors are surgery, radiotherapy, and chemotherapy.2 Unfortunately, all of these methods induce serious side effects. The first two affect a tumor directly, with a surgeon’s scalpel or beam of high energy radiation, respectively, while the latter is a systemic approach in which a specific drug travels through the patient’s body before it reaches the targettumor cells.3 As a consequence, the selectivity of chemotherapy is usually the smallest among the three treatments mentioned above. A distinct form of progress over chemotherapy itself is its combination with radiotherapy within a single treatment called chemoradiotherapy.3 The latter modality is not just a simple sum of the features of the two techniques but constitutes a specific synergy between them. A kind of chemoradiotherapy sensitized radiotherapyis based on the delivery of a sensitizer into the tumor prior to irradiation, which greatly increases the required effects related to a given dose of ionizing radiation (IR).4 The direct and indirect effects of interaction between IR and cells lie at the heart of radiotherapy and come down to the nonrepairable damage to cellular structures which leads, in turn, © 2015 American Chemical Society

Received: April 24, 2015 Revised: June 9, 2015 Published: June 10, 2015 8227

DOI: 10.1021/acs.jpcb.5b03948 J. Phys. Chem. B 2015, 119, 8227−8238

Review Article

The Journal of Physical Chemistry B

thymidine kinase and for DNA replication.8,12 The level of incorporation of both analogues into DNA correlates with radiosensitization demonstrated in vitro and in animal models. Several clinical trials of 5-BrdU and 5-IdU have also been reportedthe utility of these compounds in the radiotherapy of head-and-neck cancer, malignant gliomas, soft tissue sarcomas, intrahepatic cancer, and cervical cancer has been described.13 5-FU (5-fluorouracil) and 5-FdU (5-fluoro-2′-deoxyuridine) are other halogenated pyrimidines which enhance the cellular response to ionizing radiation. These analogues are metabolized in cells to the monophosphate form (5-FdUMP) and act as inhibitors of thymidylate synthase. 5-FdUMP can be converted to the triphosphate as well and is incorporated into DNA or RNA in this form. The radiosensitizing action of 5-FU has been investigated in numerous clinical trials.14 The most promising results have been reported in the case of patients with anal, pancreatic, rectal, esophageal, and stomach cancer. Several reports have demonstrated that gemcitabine (2′,2′difluoro-2′-deoxycitidine), a well-known chemotherapeutic agent, can increase the cytotoxic effect of irradiation in cancer cells. The role of gemcitabine in combination with radiation was examined in many clinical applications.15 Modified nucleosides also have DNA photosensitizing properties, meaning that they can be used in photodynamic anticancer therapies. The halogen derivatives of nucleobases are among the most widely studied group of photosensitizing compounds. Some reports concerning the combined use of PDT and 5fluorouracil are described in treatment of porokeratosis of Mibelli or actinic keratosis.16,17 To this end, it is worth mentioning the S1 prodrug, a novel oral formulation of 5-FU. It has been proven that S-1 is well tolerated and that the combination of photodynamic treatment with S-1 results in considerable improvement in overall survival and progression-free survival compared with PDT alone in the case of patients with unresectable hilar cholangiocarcinoma.18 Both in vitro and in vivo studies suggest that 4-thiothymidine (S4TdR) is potentially a photochemotherapeutic drug. Substitution of the oxygen atom with a sulfur atom in position 4 of thymine causes a bathochromic shift of λmax from 260 to 335 nm (UVA). This phenomenon enables only the labeled DNA to be sensitized to the UVA radiation. In addition, S4TdR is efficiently incorporated into the DNA of replicating cells and shows no cytotoxicity alone. Importantly, the described compound shows selectivity toward cancer tissue. The survival of normal cells, irradiated with UVA and incubated with S4TdR, is high, which is in contrast to the very low survival of cancer cells treated with S4TdR prior to UVA irradiation. Thus, S4TdR shows great potential as a PDT photosensitizer, but the limited penetrating power of UVA radiation can raise doubts with regard to its practical usefulness in clinical applications. However, it has been proven that the concentration of the drug and the intensity of UVA radiation are sufficient to cause efficient death by apoptosis of malignant skin cells with a negligible effect on normal skin.19,20 In view of the unquestionable advantages of modified nucleosides as sensitizers, new compounds that can sensitize cancer cells to UV or ionizing radiation are still being sought. A combination of theoretical studies with negative ion photoelectron spectroscopy experiments demonstrates that 5thiocyanato- and 5-cyanatouracil possess properties required for efficient radiosensitizers. Recent reports indicate that also pyrimidine nucleosides with a halogen atom in position 6 should be considered candidates for radiosensitizers.21−23

sensitizing molecules which could make electrons reactive toward DNA. Modified nucleosides accepted by human kinases8 and polymerases,9 with high electron affinity and undergoing dissociative electron attachment (DEA) that ultimately produces reactive radicals inside DNA, are ideal sensitizers of this kind. Interestingly, such electrophilic nucleosides lead not only to radiosensitivity but also to photosensitivity of the biomolecule.10 Indeed, nucleosides with increased EA and prone to DEA are excellent photooxidants. If guanine, a nucleobase of the lowest ionization potential is separated from the modified nucleoside in double-stranded DNA (dsDNA) by a bridge enabling a longdistance electron transfer (ET) and an electron may migrate from the guanine to the modified nucleoside after its electronic excitation.10 Such an ET reaction initiates similar processes that are triggered in the system by the solvated electrons generated by IR. These spectacular features of the electrophilic nucleosides also open a route to their usage in the photodynamic therapy (PDT) of cancer that dramatically differs from a classical PDT approach (which is based on the photoinduced formation of singlet oxygen)11 but is devoid, as every PDT should be, of the adverse effects of chemotherapy or IR treatment. The current review is focused on radio- and photosensitization developed by the modified nucleosides while incorporated into DNA. First, we briefly outline the literature information concerning such radio- and photosensitizing nucleosides. Then, various mechanisms addressing the electron-attachmentinduced formation of strand breaks and cross-links in the labeled DNA will be discussed. Next, the pathways regarding the damage mentioned above but produced by near UV photons in the labeled DNA will be considered. Finally, the perspectives of such a Trojan horse (after incorporation into DNA and irradiation, some modified nucleosides become a deadly threat to the cell’s life cycle) anticancer therapy will be briefly discussed.

2. RADIO- AND PHOTOSENSITIZING NUCLEOSIDES Labeled nucleosides appear to be particularly well suited for radiation-induced cell killing because of a number of exceptional advantages. The most unique property of these compounds is the fact that they can serve (in the form of triphosphates) as substrates for nucleic acid synthesis because of their structural similarity to native nucleosides. Their incorporation into DNA during replication results in their additional selectivity toward cancer cells because the population of normal cells in the S phase is, in contrast to cancer cells, rather small. Moreover, if the structural modification introduced into nucleosides does not inhibit the activity of enzymes involved in cell metabolism, they are only marginally cytotoxic. In other words, DNA containing the modified nucleosides possesses the same structural and functional properties as the native biopolymer and only interactions between ionizing or UV radiation and nucleoside analogues incorporated into DNA produce lethal effects, which are mainly limited to the labeled cells. The above-described properties of the nucleoside analogues suggest that they could be employed as potent sensitizers in radio- or photodynamic therapy. Nevertheless, the number of modified nucleosides used in clinical practice is surprisingly small.3 Halogenated pyrimidines such as 5-BrdU (5-bromo-2′deoxyuridine) and 5-IdU (5-iodo-2′-deoxyuridine) are in a group of the most thoroughly studied nucleosides of this type; they have been used to increase radiation sensitivity in proliferating cancer cells. These compounds require phosphorylation for radiosensitizing activity. It has been shown that both 5-BrdU and 5-IdU are very good substrates for mammalian 8228


Review Article


Table 1. Adiabatic Electron Affinity (AEA) Values of Native NBs, 5-Brominated Pyrimidines, and 8-Brominated Purines, in eV native NBs

gas phase PCM (water) AIMD gas phase PCM (water)

brominated NBsf

U

T

C

A

G

0.024a 1.912c

−0.14−0.20b 1.85−2.06b 1.2d

−0.56−0.06b 1.89−2.01b ∼1.0e 0.27 2.07

−1.47−0.12b 1.44−1.53b ∼0.75e 0.01 1.87

−1.79 to −0.01b 1.27−1.33b ∼1.0e 0.02 1.36

0.49 2.27

a

W1BD.30 bVarious methods.29 cB3LYP/aug-cc-pVTZ.31 dAIMD, PBE0/6-311++G(d,p), extrapolated to an infinite number of water molecules.32 AIMD, PBE0/6-311++G(d,p), 15 water molecules.32 fB3LYP/6-31++G(d,p), methylated derivatives.33

e

3.2. Intramolecular DNA Radical Generation. Solvated electrons, upon reaching a DNA strand, may attach to its parts with the highest electron affinity, the so-called electron traps, producing the anion radical forms of DNA (DNA•−, eq 7). A useful quantity, allowing the assessment of how likely a molecule is to accept an excess electron, is adiabatic electron affinity (AEA), which is defined as the difference between the energy of a given molecule and its electron adduct in their respective equilibrium geometries. Experimental and theoretical studies on the electron attachment to the native nucleobases (NBs) were comprehensively discussed by the Sevilla group28 and more recently by Leszczynski et al.29 Since that time, new benchmark studies regarding uracil electron affinity both in the gas phase30 and in water solution have been reported.31 Furthermore, Smyth and Kohanoff32 analyzed the electron attachment process to native NBs surrounded by explicit water molecules at the ab initio molecular dynamics (AIMD) level. The electron affinity values from those reports are summarized in Table 1. Pyrimidines accept an excess electron more willingly than purines, becoming electron traps in the native DNA strand. Electron adducts of NBs are stabilized by polar environments; therefore, the AEAs increase in water compared with the respective gas phase values. Electrophilic properties of nucleobases can be increased by their substitution with a bromine atom.33 Indeed, the AEAs of 5bromo-substituted pyrimidines are larger, even by 0.47 eV (U, gas phase), compared with their native counterparts. However, the AEA values of 8-bromo-substituted purines, i.e., 1.87 eV for 8-bromoadenine (8-BrA) and 1.36 eV for 8-bromoguanine (8BrG), are still smaller than the AEAs of native pyrimidines (∼2 eV, PCM). It is in accordance with the experimental findings34 that 8-BrG is not a competitive electron trap in brominated oligonucleotides. The DFT study on 5-halogenouracils (5-YU, where Y stands for F, Cl, or Br)35 demonstrated that their aqueous electron affinity increases with the mass of halogen (AEAs calculated with the PCM model of water): 5-FU (2.21 eV) < 5-ClU (2.26 eV) < 5-BrU (2.44 eV). Similarly, the juxtaposition of AEAs (in free enthalpy scale; PCM model of water)23 for various 5-substituted uracils (5-YU, where Y = SH, N3, Br, OCN, SCN, NCS, CN, NO2) leads to the following series: −SH (2.26 eV) < −N3 (2.38 eV) < −Br (2.48 eV) < −OCN (2.62 eV) < −SCN (2.70 eV) < −NCS (2.73 eV) < −CN (2.83 eV) < −NO2 (3.55 eV). In summary, one can imagine a number of modifications able to increase the electron affinity of native uracil and, probably, that of other NBs. However, the ability to bind excess electrons is only a prerequisite rather than a sufficient condition to increase the sensitivity of labeled DNA to solvated electrons. The key property of modified nucleosides that should increase DNA radiosensitivity is their susceptibility to DEA, which generates reactive NB radicals. Already in the late 1960s, in their pulse radiolysis study on the oxygen-free solutions of 5-BrU, Zimbrick et al.36 suggested that the attachment of eaq− to 5-BrU

3. MECHANISM OF ELECTRON-INDUCED DAMAGE TO DNA LABELED WITH MODIFIED NUCLEOSIDES 3.1. Radiation-Induced Generation of Reactive Species in the Modified DNA. Ionizing radiation can interact with DNA in living cells by direct effects, being absorbed directly by the molecule of DNA and leading to DNA cation radicals via electron abstraction (DNA•+) or to DNA excitation (DNA*, eq 1). On the other hand, the radiation can act indirectly, through interactions with water (especially) and other atoms or molecules that are present in the cell, producing radicals (eq 2) that are capable of damaging the biopolymer.7,24 DNA + ionizing radiation → DNA•+ + e− + DNA*

(1)

H 2O + ionizing radiation → OH•, e−, H•, and H+, H 2O2 , H 2

(2)

Ionizing photons are absorbed by the components of a given mixture proportionally to their weights, which means that, when an aqueous DNA solution containing 500 mg/dm3 of DNA is irradiated with γ-rays, about 99.5% of its energy will give rise to indirect effects.7 In the cellular environment, where free-radical scavengers are present, the situation is more complex; however, it has been shown that, for radiation used in radiotherapy, the biological damage from free-radical action far exceeds that of the direct action of IR.7,24 Indeed, 65% of the cell death is caused by the hydroxyl radical, OH•, only.25 Hydroxyl radicals, especially in the presence of oxygen, are thought to be the most important DNA damaging species (eqs 3 and 4), causing abstraction of the hydrogen atom from the corresponding deoxyribose, which then leads to strand breakage.7 OH• + DNA → DNA( +OH)•

(3)

OH• + DNA → DNA( −H) + H 2O

(4)

The situation changes in hypoxic cancer cells, where a hydrated electron (eaq−) instead of OH• is added to base units. Solvated electrons do not form in the oxygenated cell, whereas the yield of OH• under hypoxic conditions is just the same as that of eaq−.7 In pure water, a solvated electron has a lifetime in the order of microseconds, has a high rate of diffusion, and is highly reactive.26 An electron released from water by a high energy photon forms a prehydrated intermediate (epre−) which becomes a hydrated electron in femtoseconds after a water ionization event (eqs 5 and 6).24,27 H 2O + ionizing radiation → H 2O•+ + e− −

−

e + nH 2O → e pre → eaq e− + DNA → DNA•−

−

(5) (6) (7) 8229


Review Article

The Journal of Physical Chemistry B lead to the 5-BrU•− anion radical (not observed directly). As this is quite unstable, it releases Br−, which results in the uracil-5-yl radical (U•)a highly reactive species, that is willing to react with any H source to generate uracil. Some years later, with the use of a low temperature (77 K) electron spin resonance (ESR) experiment, Sevilla et al.37 observed both the π*-type 5-BrU•− anion in the neutral glass containing 5-BrU and the U• radical produced after warming the system to 155 K. Two other uracil derivatives, 5-ClU and 5-FU, analyzed in the ESR experiment did not produce U• but only the stable π*-type anion radicals. Those experimental observations were supported by negative ion photoelectron spectroscopy38 and DFT calculations.39 Both papers concerned the electron-induced dehalogenation of 5-BrU, 5-ClU, and 5-FU and showed that this reaction was only possible in the case of 5-BrU and 5-ClU, via transforming the primarily generated π*-type anion radical into the dissociative σ*-type anion radical. The sensitivity of the studied halouracils to electron-induced dehalogenation decreases in the following order: 5-BrU ≈ 5-ClU > 5-FU; this is in agreement with previous experimental observations.37 Not only uracil derivatives were studied against their sensitivity to DEA. For instance, a rapid debromination of 8-BrA40 and 8BrG41 was proven in the pulse radiolysis studies. The comparison of computational susceptibility of the brominated NBs (BrX) to electron-induced dissociation producing a reactive NB• radical was carried out at the DFT level and in the PCM model of water33 and later on in the explicit water environment, with the use of ab initio molecular dynamics.42 All BrXs undergo electroninduced debromination, with a low activation barrier for bromopyrimidines or even barrier-free in the case of the bromopurine anion radicals. Electron-induced debromination was also shown for an aqueous solution of the brominated nucleotides of purines43,44 or pyrimidines22,23 as well as for those nucleotides incorporated into DNA.45 In the case of pyrimidines, not only 5-bromo- but also 6-bromo-substituted derivatives were confirmed to produce nucleobase radicals after debromination. In summary, all considered brominated nucleobases/nucleotides were shown to have radiosensitizing potential, as they are able to generate reactive radicals. Treating the high AEA and potential to produce reactive radicals as the basic characteristics of deciding on the required capabilities of a modified nucleoside, a simple computational protocol has been proposed that enables the radiosensitizing properties of a given species to be examined.23 This protocol, used against 5-substituted uracils, resulted in two derivatives, 5thiocyanatouracil and 5-cyanatouracil, which have not been previously considered radiosensitizers. 3.3. From Radical Reactions to Single Strand Breaks. Among the pathways leading from nucleobase radicals to strand breaks (SBs), the simplest case of a primary break can be realized in two steps: first, the base radical undergoes a hydrogen atom transfer from the 2′-deoxyribose residue to the NB, and then, the dissociation of the phosphodiester bond occurs in this secondary radical. For the radicals localized on the NB carbon atom next to the N-glycosidic nitrogen (C6 for the substituted pyrimidines/ C8 for the substituted purines), hydrogen atom abstraction from the 2′-, 3′-, or 5′-position is feasible (Scheme 1a).46 On the other hand, in the 5-substituted pyrimidine nucleotides, the NB radical is generated on the C5 carbon and steric reasons prevent abstracting a hydrogen from its own sugar (kinetic barrier at least 40 kcal/mol, see Table 2). Despite this, the radiosensitizing properties of 5-BrU, for instance, are wellknown. A possible radiosensitization mechanism explaining this

Scheme 1. Targets of Possible Hydrogen Atom Abstraction for Uracil Radicals: (A) 6-Radical (Radical next to Glycosidic Bond); (B) 5-Radical

Table 2. Thermodynamic Stimuli (ΔG) and Kinetic Barriers (ΔG*) for 3′- and 5′-Hydrogen Abstraction Reactionsa 3′ substrate radical 22

5-uracilyl 6-uracilyl22 5-cytosinyl21 6-cytosinyl21 8-adeninyl43 8-guaninyl44

5′

ΔG

ΔG*

ΔG

ΔG*

−23.0 −16.6 −20.0 −15.8 −19.5 −20.3

75.5 11.4 67.1 13.7 12.9 12.4

−21.8 −15.4 −23.8 −19.6 −18.9 −20.8

43.9 8.6 53.1 8.2 4.3 3.2

a

All values in kcal/mol, calculated in aqueous solution at the B3LYP/ 6-31++G(d,p) level.

apparent contradiction is the hydrogen atom transfer from the 2′deoxyribose residue on the 5′ side, most probably from 1′- and 2′-positions (Scheme 1b).47 The uracil-5-yl radical has also been shown to react with a water molecule (Scheme 1b).48 The resulting hydroxyl radical can either react with the uracil ring, forming a 5,6-dihydro-5-hydroxyuracil-6-yl radical, which could act similarly to the uracil-6-yl radical, or could abstract a hydrogen atom from the sugar residue, forming a sugar radical. The kinetic barriers calculated for hydrogen abstraction by 8230


Review Article


Scheme 2. Mechanisms of Primary SBs Resulting from DNA Sugar Radicals: (a) 3′-Radical; (b) 5′-Radical; (c) 4′-Radical

observed experimentally by the Hunting group.56 They found that the type of damage depends on the DNA helix type (see Figure 1).57 In the case of the native B-helical DNA, ICLs were

hydroxyl radical from 2′-deoxyadenosine imply that 5′-hydrogens are the most favorable target.49 In the case of mismatched fragments (forming bulges) in double-stranded DNA, the uracil5-yl can also abstract a hydrogen atom from the opposite strand (Scheme1b), causing breakage in that strand.50 When the radical is centered on the C3′ or C5′ carbon atom of the 2′-deoxyribose residue, the only possible one-step pathway leading to SB is OP bond breakage. The alternative pathway of CO bond break is impossible because the products would be a high energy (more than 50 kcal/mol higher than the substrate) open shell singlet, localized on the 2′-deoxyribose residue and a phosphate radical.46 For the OP bond break, the products are a much more stable carbonyl compound and a phosphine radical (Scheme 2a,b). The C4′-centered radicals can undergo a heterolytic C3′-site CO bond break, leaving cation radicals centered on the sugar residue, which can then react with water to reform the C4′-radical and heterolytically cleave the CO bond at the C5′-site (Scheme 2c).51 The C2′-radical could, in principle, undergo β-fragmentation, leading to the C2′C3′ double bond and C3′O bond breakage, although there is no evidence of this particular mechanism working in DNAit has only been observed in RNA to date.52 Sugar radicals can undergo base release, forming abasic sites, such as 2′-deoxyribonolactone, which may also be converted to SBs under cellular conditions.53 3.4. Other Types of DNA Radiodamage. The main type of lesions induced by high energy radiation in DNA labeled with electrophilic nucleosides seems to be SBs; however, other types of radiodamage can also be observed. To this end, one should mention cyclopurine mutants: 5′,8-cyclo-2′-deoxyadenosine (cyclo-dA) and 5′,8-cyclo-2′-deoxyguanosine (cyclo-dG). Their chemical and biological significance was comprehensively discussed by the Chatgilialoglu group.54 This kind of DNA damage can be generated during electron-induced degradation of brominated purines, as was recently shown in computational studies by Chomicz et al.43,44 Cyclopurine mutations which induce helix-distortions and block DNA replication and transcription54 are still under undiminished interest. For instance, Karwowski,55 using the QM/MM approach, recently demonstrated that cyclo-dA formed in double-stranded DNA (dsDNA) leads to helix distortion which may impair repair processes or polymerase activity. The most cytotoxic type of radiation-induced damage to labeled DNA is the formation of interstrand cross-links (ICLs)

Figure 1. Main degradation pathways for 5-BrdU-labeled singlestranded, double-stranded, and mismatched DNA. Adapted with permission from ref 57. Copyright 2009 American Chemical Society.

mainly found for mismatched DNA. Later on, Hunting et al.58 proposed a mechanism of ICL formation in mismatched DNA based on the specific zipper-like DNA structure. They found, moreover, that 5-BrU incorporated into mismatched DNA labeled with 5-BrU is more likely reduced by hydrated electrons.

4. UV-INDUCED DAMAGE TO DNA LABELED WITH THE ELECTROPHILIC NUCLEOSIDES 4.1. Formation of Strand Breaks. 5-Halouracils such as 5bromouracil (5-BrUBrU) and 5-iodouracil (5-IUIU), wellknown DNA radiosensitizers, are also photoreactive analogues of thymine, which are widely used in biological59 and chemical60 research. Thymine can be substituted with 5-BrU or 5-IU, and this modified DNA remains functional in vivo.61 The substitution of thymine in DNA with 5-halouracil increases the photosensitivity of the cell with DNA strand breakage (single strand breaks (SSBs) and double strand breaks (DSBs)) and alkali-labile sites being the most abundant type of photodamage.62,63 The photoreaction of 5-halouracil has been extensively studied using DNA fragments with defined sequences. Attempts to comprehend mechanisms behind the experimentally observed strand breaks were undertaken at the beginning of the 1990s by the Saito group.64 They reported that UV-induced damage in short duplex DNA containing 5-halouracil was highly dependent on the identity of the nucleotide bonded to the C5′ site of 5-halo2′-deoxyuridine. These authors64 demonstrated that UV irradiation of short oligomers, d(GCAxUGC)2, where x = Br or 8231


Review Article


Scheme 3. Putative Mechanism of Photoinduced Formation of Ribonolactone (1) and Erythrose (2) in DNA Duplex Labeled with Br U or IUa

a

Adapted with permission from ref 68. Copyright 1996 Elsevier.

Scheme 4. Strand Scission Produced in DNA Duplex Containing 5′-dABrdUa

a

Adapted with permission from ref 69. Copyright 2000 American Chemical Society.

the C2′α by the uracil-5-yl radical, was shown by photochemical studies on the d(GCAxUGC)2 hexamer to involve stereospecifically deuterated 2′-deoxyadenine. The kinetic isotope effect, kH/ kD, for the formation of the erythrose residue in the strands labeled with 5-BrU or 5-IU was as high as 7.5 and 7.2, respectively.68 The efficient formation of a deoxyribonolactone and erythrose residue due to photoirradiation was only observed when the duplex hexamers contained the 5′-ABrU sequence. Interestingly, hexanucleotides comprising the 5′-GBrU instead of 5′-ABrU fragment had very poor photoreactivity.64,65 A similar result of sequence-specific photoreactivity was achieved by Greenberg’s group.69 All of these findings remain in contradiction to the actual ionization potentials (IPs) of nucleobases. Indeed, it was assumed that this photochemical reaction begins from photoinduced single electron transfer (PSET) involving the 5′adjacent nucleobase and the photoexcited 5-BrU. To this end, one should remember that the IP of guanine is smaller than that

I, produces 2′-deoxyribonolactone (Scheme 3, 1) and erythrose (Scheme 3, 2) with the concomitant release of free adenine.65 The observed photoreaction products were explained by a mechanism involving the C1′ or C2′ hydrogen atom abstraction at the 5′ side of the xU residue by the photochemically generated uracil-5-yl radical. As the C1′ and C2′ oxidation pathways are competitive mechanisms, H-abstraction occurs even in singlestranded oligonucleotides. It was pointed out that the ratio of 1 to 2 (Scheme 3) was highly dependent on the reaction conditions, particularly on the concentration of dissolved oxygen.64,66 Using oligonucleotides labeled with the C1′ deuterated 2′-deoxyadenine (a kinetic isotope effect (kH/kD) of 1.7), Fujimoto et al. showed that the formation of ribonolactone in DNA involved abstraction of the C1′ H from the 5′-adjacent adenosine.67 On the other hand, mass spectrometry of ribonolactone-containing oligonucleotides indicated that the 1′ oxygen atom of ribonolactone originated from water. The suggested mechanism for the creation of erythrose, following the H abstraction from 8232


Review Article

The Journal of Physical Chemistry B of adenine. Thus, in order to explain this “contrathermodynamic” effect, Greenberg et al.69 assumed that the photoreaction outcome is the result of not only the primary electron transfer (kET, Scheme 4) but also several other processes like the back electron transfer (kBET, Scheme 4) and hole migration (kHolMig, Scheme 4) and base stacking.69 This problem was, in turn, analyzed by Sanche et al.70 They suggested, similarly to Greenberg’s group, that the UV irradiation of labeled DNA leads to SSBs (the damage was detected by denaturing electrophoresis). Furthermore, Sugiyama’s group observed specific breaks at the 5′-(G/C)AAxUxU-3′ and 5′-(G/ C)AxUxU-3′ sequences only after a heat treatment (x = Br or I). The HPLC product analysis indicated that 2′-deoxyribonolactone was effectively generated in these sequences.71 These inconsistent findings were resolved by Rak’s group.72 In those studies, the system reported by Sanche at al.70 was reassessed with totally denaturing HPLC (DHPLC). The analysis showed the formation of an abasic site comprising 2′-deoxyribonolactone as a result of UV irradiation rather than a direct (frank) strand break. As a matter of fact, ribonolactone gives rise to SSBs only at elevated temperatures, since, in double-stranded DNA, it is a thermally unstable species (with a half-life at 37 °C of 32−54 h73) decomposing into a DNA fragment with phosphate termini and methylenefuranone. The photoreaction of 5-bromouracil has been extensively studied using various oligonucleotides possessing the 5′-ABrUT3′ sequence. It was found that not only the presence of proximate A but also distant G is important for these photoreactions.75,74 On the basis of those results, the putative electron donor, G, was replaced with various modified purine bases that differed in oxidation potential. It was observed that the amount of ribonolactone-containing products increased with decreasing ionization potential of the purine base in the oligonucleotide.75 Therefore, a mechanism was proposed, where an initial electron transfer occurs from G (an electron donor) to the electronically excited 5-BrU (an electron acceptor), through the stacked As present between the donor and acceptor. The intervening A bases between the donor and acceptor played the role of a bridge making possible electron transfer from G to 5-BrU and simultaneously preventing back electron transfer. The yield of ribonolactone formation increased with the number (n) of the complementary (A/T)n pairs in the bridge, reaching the maximum for n = 2, and then gradually decreased. This observation was explained by the competition between the forward and back electron transfers. 75 For n = 0, no ribonolactone was found, which indicates that back electron transfer is faster than the release of the bromide ion from the anion radical of 5-BrU. It is also worth noting that G in the complementary strand may take part in efficient electron transfer, leading to the formation of ribonolactone as well.71 In contrast to the 5-BrU photoreactivity, 5-IU was reactive even when the number of As in the bridge was zero or more than five. These facts suggest the coexistence of two mechanisms involving: (i) the heterolytic C−I bond dissociation (as in the 5BrU case) and (ii) a distance-independent homolytic C−I bond cleavage. The rates of (i) and (ii) are highly sequence dependent. In a situation where no electron donor is located in the proximity of 5-IU, efficient generation of the uracil-5-yl radical is realized via the homolytic pathway (see Scheme 5).76 4.2. Formation of Cross-Link Lesions. Investigations of the UV-induced formation of intrastrand cross-link lesions within short single- and double-stranded DNA fragments has been the goal of many research groups. In 2004, Zheng and

Scheme 5. Heterolytic and Homolytic Pathways from the C−I Cleavagea

a

Adapted with permission from ref 76. Copyright 2008 Elsevier.

Wang77 demonstrated easily formed intrastrand cross-link products between cytosine and guanine. They showed that the UV irradiation of the dinucleoside monophosphate resulted in the formation of d(C[5−8]G) (Figure 2a).78 The interaction between guanine and 5-bromocytosine leads to the formation of a cation radical of guanine and an anion radical of 5bromocytosine. The latter eliminates a bromide ion (Br−), giving the C5 radical of cytosine and binding to the guanine cation radical. The newly created product deprotonates to yield the cross-linked lesion.77 They also irradiated a 12-mer (dATGGCGBrCGCTAT/dATAGCGCGCCAT) duplex and observed three cross-linked products: C[5−8]G, C[5−N2]G, and G[8−5]C. The major product was the lesion involving the C8 carbon atom of guanine at position 6 and the C5 carbon of cytosine at position 7. Furthermore, the d(A[8−5]U) (Figure 2b) and d(A[2−5]U) cross-links (Figure 2c) were generated during the photolysis of d(ABrU).79 Moreover, Hong and Wang80 observed covalently bound products between cytosine and adenine (irradiation of d(BrCA). They confirmed the presence of the three types of intrastrand cross-link lesions, d(C[5−N6]A) (Figure 2d), d(C[5−2]A) (Figure 2e), and d(C[5−8]A) (Figure 2f). The irradiation of d(ABrCA) resulted, in turn, in the formation of two other products: d(A[2−5]C) and d(A[8−5]C). All five cross-linked products were observed due to the irradiation of a double-stranded dodecamer containing the 5′-ABrCA-3′ motif (d(ATGGCABrCACTAT/d(ATAGTGTGCCAT)). The most abundant cross-link was the d(A[8−5]C) dimer. Its prevalence was explained by the shortest distance between the sites involved in the studied reaction. The observation described above led to the following putative mechanism: (i) UV irradiation brings about the transfer of an electron from adenine to its adjacent 5-bromocytosine, (ii) the ensuing 5-bromocytosine anion radical eliminates the bromide ion and gives the cytosine-5-yl radical, (iii) the latter radical may interact with the C2 or C8 carbon atom of the adjacent adenine, and (iv) then the dimer can deprotonate and give a cross-linked product where the C5 of cytosine is covalently bound to the C2 or C8 of adenine. The C−Br bond of 5-bromocytosine may also undergo homolytic cleavage,69 and the C5 centered radical can be formed. This radical has an ability to attack the C2 or C8 carbon atom of its adjacent adenine, which resulted in the formation of the corresponding products. The production of d(C[5−N6]A) proceeds according to a different mechanism. The 2′-deoxyadenosine cation radical can release the hydrogen atom and produce an exocyclic nitrogen-centered radical81 which further interacts with the C5 atom of adjacent 5′ cytosine. The resulting product eliminates a bromide ion to give d(C[5− 8233


Review Article


Figure 2. Intrastrand cross-link products generated during irradiation of dinucleoside monophosphate.

N6]A). It is also possible that the anion radical of 5bromocytosine is able to eliminate a bromide anion and produce the C5-centered radical of cytosine, which can bond to the N6 radical of adenine, giving d(C[5−N6]A). The photoirradiation of dinucleotides d(BrUU) and d(UBrU) and hexanucleotides containing either of these sequences involved the formation of a product between modified and native uridine. In both cases, the debromination of the starting dinucleotide is involved in the concomitant addition of a hydroxyl group followed by its further photochemical and thermal degradation to a 5,5′-diuridynyl product.82 The comparison of photoproducts originating from d(BrUG) and d(GBrU) revealed that d(U[5−8]G) (Figure 2g) is formed from d(BrUG) but d(G[8−5]U) (Figure 2h) and d(G[N2−5]U) (Figure 2i) are generated from d(GBrU). It is also worth mentioning that d(G[N2−5]U) is not observed due to the irradiation of dsDNA containing the d(GBrUG) motif.79 The results obtained for longer sequences revealed that the photoirradiation of 40 bp length duplex DNA containing the 5′GAAAABrUA-3′ sequence resulted in the formation of the d(A[2−5]U) dimer, in which the C5 carbon atom of dU and the C2 carbon atom of dA were covalently bonded,83 while UV irradiation of dsDNA (80 bp length) containing all thymines substituted with 5-BrdU led to the formation of two intrastrand cross-linked products: d(U[5−5]C) and d(U[5−5]U). These dimers were produced by the electronic excitation of BrdU followed by photocycloaddition. After photochemical and thermal degradation, the intrastrand cross-linked dimer was detected.84

Considering the sequence-dependent formation of intrastrand cross-linked lesions, stacking energies between the halogenated nucleobases and the adjoining purine base should be considered. For example, for both 5-BrdC- and 5-BrdU-containing oligonucleotides, the stacking preference follows the 5′dG > 5′dA order. It is worth mentioning that the 5′-purine:pyrimidine3′ complex stacks more efficiently than the 5′-pyrimidine:purine3′ one,79,85 which suggests that better stacking favors crosslinked products.77 Finally, in contrast to strand break formation, ICLs are preferentially formed when the flanking 5′-nucleobase is not adenine but guanine.77 Photoinduced cross-linked lesions may also form between the opposite strands of DNA. Kypr et al. demonstrated that UVC light leads to such interstrand cross-links (inter-CLs) without external chemical agents.86 They found that the yield of interCLs depends on the dose of UV radiation as well as on the length of the irradiated DNA fragment. Moreover, the production of UV-induced CLs between complementary strands increased with the (AT) content of DNA.87 On the other hand, in 2005, Hunting’s group reported the creation of inter-CLs that was strongly dependent on DNA conformation.70 In two dsDNA sequences labeled with 5-BrdU, a photoinduced inter-CL was observed only in the sequence containing a bulge in the labeled region. Furthermore, Skalski’s group88 observed an inter-CL lesion in dsDNA comprising a d(AFSUA) motif (where FSU stands for 5-fluoro-4-thio-2′-O-methyluridine), while Fujimoto et al.89 reported a novel interstrand photo-cross-linked product in oligonucleotides containing the p-carbamoylvinyl phenol derivative of nucleoside. Undoubtedly, the interstrand cross-link formation in DNA appeals to a number of research groups, since 8234


Review Article

The Journal of Physical Chemistry B Biographies

this kind of damage belongs to the most cytotoxic ones and control over it cannot be overestimated as far as anticancer therapy is concerned. However, the mechanism responsible for inter-CL formation is still not clear and requires further studies in order to propose a protocol that will enable modified nucleosides to be designed rationally.

Janusz Rak received his MSc (1986; physical chemistry) and PhD (1992; physical and theoretical chemistry) with honors from the University of Gdańsk. In 2000, he got his habilitation in theoretical and physical chemistry, and in 2007, he obtained a professor title from the President of Poland. In 1996/97, he worked at Oakland University (MI, USA) as a Fulbright Foundation Fellow and then in 1997/98 at the Technical University of Munich as a Humboldt Foundation Fellow. For two months in 2006 and 2008, he worked, as a visiting professor at Jackson State University (Mississippi, USA). Presently, he leads Biological Sensitizers Laboratory at the Faculty of Chemistry, University of Gdańsk (UG). His scientific interests concern quantum chemical investigations of electron-induced degradation of DNA, long-range electron transfer in this biopolymer, and theoretically experimental studies on DNA sensitization to UV photons and ionizing radiation.

5. SUMMARY Electrophilic nucleosides, especially brominated ones, make labeled DNA sensitive to solvated electrons and UV photons, i.e., to physical factors to which the native DNA is insensitive. These findings open a route by which to employ such electrophilic nucleosides in radiotherapy and the photodynamic therapy of solid cancer. In this Mini-Review Article, the current status of studies concerning the reactivity of modified nucleosides in DNA context toward excess electrons and near UV photons is summarized. Although the mechanisms responsible for the ionizing radiation-induced and photoinduced damage to DNA are quite different, the ultimate outcome of the γ and photo irradiation of labeled DNA is similar. Namely, the major lesions observed are strand breaks, cross-links, and abasic sites. The similarity of ultimate products triggered by electrons and photons is related to the formation of radicals localized to nucleobases in both reaction pathways. These neutral radicals originate from nucleobase radical anions easily releasing an anionic substituent. The radical anions of modified nucleobases occurring in the radiolysis form as a result of electron attachment to the modified nucleosides which play the role of an electron trap in DNA. Therefore, the yield of this process should be correlated with the ease by which mNZ undergoes DEA and its electron affinity, which depends on the local DNA sequence. On the other hand, photoinduced strand breaks, which belong to the most efficient type of photodamage, are governed by long-range electron transfer from a distant guanine and, therefore, are also sequence-dependent. The present rather deep comprehension of the mechanisms lying behind the radiation and photoinduced damage to the DNA labeled with electrophilic nucleosides makes the rational design of new, more potent than 5-BrdU, modified nucleosides possible. Currently, we are studying the sensitizing features of 5SCNdU and 5-OCNdUmNZsthat we proposed quite recently23 using molecular modeling which employed the knowledge on DNA radiosensitization by the substituted uracil. A therapeutically valuable compound should not only be a potent DNA sensitizer but also has to be efficiently phosphorylated in the cell and then accepted by human polymerases during DNA biosynthesis. Therefore, before animal tests and clinical trials, any promising mNZ has to be checked in silico, both as a damaging agent and as a substrate for kinases and polymerases, then synthesized, tested in model radiationchemical and photochemical studies, introduced to cancer cells, and ultimately tested under the destined conditions. Only the mNZs that passed this interdisciplinary and quite complex evaluation procedure could be employed in future anticancer therapies.

■

Lidia Chomicz received her MSc degree in 2009 and defended her doctoral thesis with honors in 2015 at the Faculty of Chemistry, UG. She did two research stays: in Institute of Applied Radiation Chemistry, Technical University of Łódź (2 months, 2008), and Interdisciplinary Center for Nanotoxicity, Jackson State University (3 months, 2011). Since 2013, she has been working as a Research Assistant in Physical Chemistry Chair, Faculty of Chemistry, UG. She is engaged in studies on the mechanisms of modified DNA radiodegradation, with the use of quantum theory tools (DFT methods especially). Justyna Wiczk obtained her BSc (2009) and MSc (2011) from the Faculty of Chemistry, UG. Since that time, she has been doing her PhD in Biological Sensitizers Laboratory. In 2014, she took a scholarship at Jackson State University (MS, US), held under the People Program (Marie Curie Actions) of the European Union Seventh Framework Program“NanoBRIDGES”. She focuses on the assessment of the impact of the type of sensitizer and its local surroundings on photo- and radiodamage to DNA. In her research, she uses real-time PCR, denaturing HPLC, gel electrophoresis, and LC/MS techniques. Kinga Westphal completed her BSc and MSc at the Faculty of Chemistry, UG, in 2011 and 2013, respectively. Since then, she has been doing her PhD studies in Biological Sensitizers Laboratory. In 2014, she took an internship at Jackson State University (MS, US), held under the People Program (Marie Curie Actions) of the European Union Seventh Framework Program“NanoBRIDGES”. Her scientific interests concern the assessment of UV/radio-induced damage to double- or single-stranded DNA labeled with modified nucleobases. In her studies, she applies a number of experimental techniques including qPCR, HPLC, and LC/MS. Magdalena Zdrowowicz received her BSc (2010) and MSc (2012) degree at the Faculty of Chemistry, UG. Now she is doing the third year of her PhD studies, which is focused on the photo- and radiosensitivity of DNA labeled with nucleoside analogues. Currently, she is looking for a pathway to cellular DNA sensitization via UV and γ irradiation. Her main expertise is in the synthesis of modified oligonucleotides and analysis of DNA damage. Paweł Wityk received his Engineer’s degree in 2014 at Gdańsk University of Technology. Since 2014, he is doing his PhD studies (at UG) on photo- and radiodamage to the native or labeled DNA molecules alone or interacting with proteins/peptides. In his research, he uses theoretical (classic and ab initio MD, QM/MM methods) and experimental approaches, like CLICK reaction for covalently bound DNA/peptide conjugates and a wide range of analytical techniques (e.g., HPLC, CD, nano-DSC, microLC-MS). Michał Ż yndul received his BSc at the University of Białystok in 2010 and MSc in 2012 at the University of Gdańsk. Since 2014, he has been

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 8235


Review Article


(14) Gaba, N. Halogenated Pyrimidines. In Radioprotectors and Radiosensitizers; LAP LAMBERT Academic Publishing GmbH & Co: Saarbrücken, Germany, 2011. (15) Shewach, D. S.; Hahn, T. M.; Chang, E.; Hertel, L. W.; Lawrence, T. S. Metabolism of 2′,2′-Difluoro-2′-deoxycytidine and Radiation Sensitization of Human Colon Carcinoma Cells. Cancer Res. 1994, 54, 3218−3223. (16) Marcus, L. Photodynamic Therapy for Actinic Keratosis Followed by 5-Fluorouracil Reaction. Dermatol. Surg. 2003, 29, 1061−1064. (17) Levitt, J.; Emer, J. J.; Emanuel, P. O. Treatment of Porokeratosis of Mibelli with Combined Use of Photodynamic Therapy and Fluorouracil Cream. Arch. Dermatol. 2010, 146, 371−373. (18) Park, D. H.; Lee, S. S.; Park, S. E.; Lee, J. L.; Choi, J. H.; Choi, H. J.; Jang, J. W.; Kim, H. J.; Eum, J. B.; Seo, D.-W.; et al. Randomized Phase II Trial of Photodynamic Therapy Plus Oral Fluoropyrimidine, S-1, Versus Photodynamic Therapy Alone for Unresectable Hilar Cholangiocarcinoma. Eur. J. Cancer 2014, 50, 1259−1268. (19) Gemenetzidis, E.; Shavorskaya, O.; Xu, Y.-Z.; Trigiante, G. Topical 4-Thiothymidine is a Viable Photosensitizer for the Photodynamic Therapy of Skin Malignancies. J. Dermatol. Treat. 2013, 24, 209−214. (20) Reelfs, O.; Karran, P.; Young, A. R. 4-Thiothymidine Sensitization of DNA to UVA Offers Potential for a Novel Photochemotherapy. Photochem. Photobiol. Sci. 2012, 11, 148−154. (21) Chomicz, L.; Golon, Ł; Rak, J. The Radiosensitivity of 5- and 6Bromocytidine Derivatives-Electron Induced DNA Degradation. Phys. Chem. Chem. Phys. 2014, 16, 19424−19428. (22) Golon, L.; Chomicz, L.; Rak, J. Electron-Induced Single Strand Break in the Nucleotide of 5- and 6-Bromouridine. A DFT Study. Chem. Phys. Lett. 2014, 612, 289−294. (23) Chomicz, L.; Zdrowowicz, M.; Kasprzykowski, F.; Rak, J.; Buonaugurio, A.; Wang, Y.; Bowen, K. H. How to Find out Whether a 5Substituted Uracil Could be a Potential DNA Radiosensitizer. J. Phys. Chem. Lett. 2013, 4, 2853−2857. (24) Lehnert, S. Biomolecular Action of Ionizing Radiation. Series in Medical Physics and Biomedical Engineering; Taylor & Francis: Boca Raton, FL, 2008. (25) Chapman, J.; Reuvers, A.; Borsa, J.; Greenstock, C. Chemical Radioprotection and Radiosensitization of Mammalian Cells Growing in Vitro. Radiat. Res. 1973, 56, 291−306. (26) Abel, B.; Buck, U.; Sobolewski, A.; Domcke, W. On the Nature and Signatures of the Solvated Electron in Water. Phys. Chem. Chem. Phys. 2012, 14, 22−34. (27) Nguyen, J.; Ma, Y.; Luo, T.; Bristow, R. G.; Jaffray, D. A.; Lu, Q.-B. Direct Observation of Ultrafast-Electron-Transfer Reactions Unravels High Effectiveness of Reductive DNA Damage. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 11778−11783. (28) Li, X.; Cai, Z.; Sevilla, M. D. DFT Calculations of the Electron Affinities of Nucleic Acid Bases: Dealing with Negative Electron Affinities. J. Phys. Chem. A 2002, 106, 1596−1603. (29) Gu, J.; Leszczynski, J.; Schaefer, H. F., III. Interactions of Electrons with Bare and Hydrated Biomolecules: From Nucleic Acid Bases to DNA Segments. Chem. Rev. 2012, 112, 5603−5640. (30) Gu, J.; Xie, Y.; Schaefer, H. F. Benchmarking the Electron Affinity of Uracil. J. Chem. Theory Comput. 2014, 10, 609−612. (31) Melicherčík, M.; Pašteka, L. F.; Neogrády, P.; Urban, M. Electron Affinities of Uracil: Microsolvation Effects and Polarizable Continuum Model. J. Phys. Chem. A 2012, 116, 2343−2351. (32) Smyth, M.; Kohanoff, J. Excess Electron Localization in Solvated DNA Bases. Phys. Rev. Lett. 2011, 106, 238108. (33) Chomicz, L.; Rak, J.; Storoniak, P. Electron-Induced Elimination of the Bromide Anion from Brominated Nucleobases. A Computational Study. J. Phys. Chem. B 2012, 116, 5612−5619. (34) Razskazovskii, Y.; Swarts, S. G.; Falcone, J. M.; Taylor, C.; Sevilla, M. D. Competitive Electron Scavenging by Chemically Modified Pyrimidine Bases in Bromine-Doped DNA: Relative Efficiencies and Relevance to Intrastrand Electron Migration Distances. J. Phys. Chem. B 1997, 101, 1460−1467.

doing his PhD studies at UG, where he focuses on the synthesis of modified nucleobases and nucleosides. He also carries out research on the photosensitivity of DNA fragments labeled with 5-bromo-2′deoxycytidine, using PCR, HPLC, and LC/MS techniques. Samanta Makurat received her BSc (2013) and MSc (2015) at the Faculty of Chemistry, UG. For her master’s thesis, she studied the effects of electron attachment to uracil analogues. Currently, she is focused on modeling new DNA sensitizers, with the use of hybrid (QM/MM) methods. She is one of the best students of Faculty of Chemistry, UG, regularly awarded for great academic performance. Łukasz Golon completed his BSc and MSc at the Faculty of Chemistry, UG, in 2011 and 2013, respectively. He completed a two month COST STSM research stay at Analytical Biochemistry Group, University of Groningen (Holland). His interests concern the DFT and hybrid QM/ MM methods. He works on describing the radiosensitizing properties of modified nucleosides (mNZs), focusing on modeling enzymatic reactions leading to the incorporation of mNZs into nuclear DNA.

■

ACKNOWLEDGMENTS This work was supported the Polish National Science Centre under Grant Nos. 2012/05/B/ST5/00368 (J.R.) and 2012/07/ N/ST5/01877 (M.Z.) as well as by the Polish Ministry of Science and Higher Education via Diamond Grant 0138/DIA/ 2014/43 (P.W.) and by the Foundation for Polish Science (L.C.).

■

REFERENCES

(1) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. Global Cancer Statistics. Ca-Cancer J. Clin. 2011, 61, 69−90. (2) Joiner, M., van der Kogel, A., Eds. Basic Clinical Radiobiology; Hodder Arnold: London, 2009. (3) Choy, H. Chemoradiation in Cancer Therapy; Humana Press Inc.: Totowa, NJ, 2011. (4) Wardman, P. Chemical Radiosensitizers for Use in Radiotherapy. Clin. Oncol. 2007, 19, 397−417. (5) Kinsella, T. J. Update on Radiosensitization by Halogenated Thymidine Analogs-Molecular Mechanisms of Drug Processing and Cell Death Signaling. Cancer Biol. Ther. 2008, 7, 1567−1569. (6) Oronsky, B. T.; Knox, S. J.; Scicinski, J. Six Degrees of Separation: The Oxygen Effect in the Development of Radiosensitizers. Trans. Oncol. 2011, 4, 189−198. (7) von Sonntag, C. Free-Radical-Induced DNA Damage and Its Repair; Springer-Verlag: Heidelberg, Germany, 2006. (8) Lee, L.-S.; Cheng, Y.-c. Human Deoxythymidine Kinase. II: Substrate Specificity and Kinetic Behaviors of the Cytoplasmic and Mitochondrial Isozymes Derived from the Blast Cells of the Acute Myelocytic Leukemia. Biochemistry 1976, 15, 3686−3690. (9) Goz, B. The Effects of Incorporation of 5-Halogenated Deoxyuridines into the DNA of Eukaryotic Cells. Pharmacol. Rev. 1977, 29, 249−272. (10) Tashiro, R.; Sugiyama, H. Reactivity of 5-Halopyrimidines in Nucleic Acids. In Radical and Radical Ion Reactivity in Nucleic Acid Chemistry; Greenberg, M. M., Ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2009; pp 163−190. (11) Brown, S. B.; Brown, E. A.; Walker, I. The Present and Future Role of Photodynamic Therapy in Cancer Treatment. Lancet Oncol. 2004, 5, 497−508. (12) Prusoff, W. H.; Goz, B. Halogenated Pyrimidine Deoxyribonucleosides. Antineoplastic and Immunosuppressive Agents; Springer: Berlin, 1975. (13) Shewach, D. S.; Lawrence, T. S. Nucleoside Radiosensitizers. In Deoxynucleoside Analogs in Cancer Therapy; Godefridus, J. P., Ed.; Humana Press Inc.: Totowa, NJ, 2006. 8236


Review Article

The Journal of Physical Chemistry B (35) Li, X.; Sanche, L.; Sevilla, M. D. Dehalogenation of 5-Halouracils after Low Energy Electron Attachment: A Density Functional Theory Investigation. J. Phys. Chem. A 2002, 106, 11248−11253. (36) Zimbrick, J.; Ward, J.; Myers, L. Studies on the Chemical Basis of Cellular Radiosensitization by 5-Bromouracil Substitution in DNA: I. Pulse-and Steady-State Radiolysis of 5-Bromouracil and Thymine. Int. J. Radiat. Biol. 1969, 16, 505−523. (37) Sevilla, M.; Failor, R.; Zorman, G. Radicals Formed after Electron Attachment to 5-Halouracils in Aqueous Glasses. J. Phys. Chem. 1974, 78, 696−699. (38) Radisic, D.; Ko, Y. J.; Nilles, J. M.; Stokes, S. T.; Sevilla, M. D.; Rak, J.; Bowen, K. H. Photoelectron Spectroscopic Studies of 5-Halouracil Anions. J. Chem. Phys. 2011, 134, 015101. (39) Wetmore, S. D.; Boyd, R. J.; Eriksson, L. A. A Theoretical Study of 5-Halouracils: Electron Affinities, Ionization Potentials and Dissociation of the Related Anions. Chem. Phys. Lett. 2001, 343, 151−158. (40) Flyunt, R.; Bazzanini, R.; Chatgilialoglu, C.; Mulazzani, Q. G. Fate of the 2′-Deoxyadenosin-5-yl Radical under Anaerobic Conditions. J. Am. Chem. Soc. 2000, 122, 4225−4226. (41) Ioele, M.; Bazzanini, R.; Chatgilialoglu, C.; Mulazzani, Q. G. Chemical Radiation Studies of 8-Bromoguanosine in Aqueous Solutions. J. Am. Chem. Soc. 2000, 122, 1900−1907. (42) Wieczór, M.; Wityk, P.; Czub, J.; Chomicz, L.; Rak, J. A FirstPrinciples Study of Electron Attachment to the Fully Hydrated Bromonucleobases. Chem. Phys. Lett. 2014, 595−596, 133−137. (43) Chomicz, L.; Leszczynski, J.; Rak, J. Electron-Induced Degradation of 8-Bromo-2′-Deoxyadenosine 3′,5′-Diphosphate, a DNA Radiosensitizing Nucleotide. J. Phys. Chem. B 2013, 117, 8681− 8688. (44) Chomicz, L.; Furmanchuk, A.; Leszczynski, J.; Rak, J. ElectronInduced Single Strand Break and Cyclization: a DFT Study on the Radiosensitization Mechanism of the Nucleotide of 8-Bromoguanine. Phys. Chem. Chem. Phys. 2014, 16, 6568−6574. (45) Polska, K.; Rak, J.; Bass, A.; Cloutier, P.; Sanche, L. Electron Stimulated Desorption of Anions from Native and Brominated SingleStranded Oligonucleotide Trimers. J. Chem. Phys. 2012, 136, 075101. (46) Rak, J.; Kobyłecka, M.; Storoniak, P. Single Strand Break in DNA Coupled to the OP Bond Cleavage. J. Phys. Chem. B 2011, 115, 1911− 1917. (47) Schyman, P.; Zhang, R. b.; Eriksson, L. A.; Laaksonen, A. Hydrogen Abstraction from Deoxyribose by a Neighboring Uracil-5-yl Radical. Phys. Chem. Chem. Phys. 2007, 9, 5975−5979. (48) Chomicz, L.; Petrovici, A.; Archbold, I.; Adhikary, A.; Kumar, A.; Sevilla, M. D.; Rak, J. An ESR and DFT Study of Hydration of the 2′Deoxyuridine-5-yl Radical: A Possible Hydroxyl Radical Intermediate. Chem. Commun. 2014, 50, 14605−14608. (49) Shukla, P. K.; Kumar, N.; Mishra, P. C. Hydrogen Atom Abstraction Reactions of the Sugar Moiety of 2′-Deoxyguanosine with an OH Radical: A Quantum Chemical Study. Int. J. Quantum Chem. 2011, 111, 2160−2169. (50) Cecchini, S.; Girouard, S.; Huels, M. A.; Sanche, L.; Hunting, D. J. Single-Strand-Specific Radiosensitization of DNA by Bromodeoxyuridine. Radiat. Res. 2004, 162, 604−615. (51) Giese, B.; Dussy, A.; Meggers, E.; Petretta, M.; Schwitter, U. Conformation, Lifetime, and Repair of 4′-DNA Radicals. J. Am. Chem. Soc. 1997, 119, 11130−11131. (52) Resendiz, M. J. E.; Pottiboyina, V.; Sevilla, M. D.; Greenberg, M. M. Direct Strand Scission in Double-Stranded RNA via a C5-Pyrimidine Radical. J. Am. Chem. Soc. 2012, 134, 3917−3924. (53) Park, Y.; Polska, K.; Rak, J.; Wagner, J. R.; Sanche, L. Fundamental Mechanisms of DNA Radiosensitization: Damage Induced by LowEnergy Electrons in Brominated Oligonucleotide Trimers. J. Phys. Chem. B 2012, 116, 9676−9682. (54) Chatgilialoglu, C.; Ferreri, C.; Terzidis, M. A. Purine 5′,8Cyclonucleoside Lesions: Chemistry and Biology. Chem. Soc. Rev. 2011, 40, 1368−1382. (55) Karwowski, B. T. The Role of (5′R) and (5′S) 5′,8-Cyclo-2′deoxyadenosine in ds-DNA Structure. Comput. Theor. Chem. 2013, 1010, 38−44.

(56) Dextraze, M.-E.; Wagner, J. R.; Hunting, D. J. 5-Bromodeoxyuridine Radiosensitization: Conformation-Dependent DNA Damage. Biochemistry 2007, 46, 9089−9097. (57) Dextraze, M.-E.; Cecchini, S.; Bergeron, F.; Girouard, S.; Turcotte, K.; Wagner, J. R.; Hunting, D. J. Reaching for the Other Side: Generating Sequence-Dependent Interstrand Cross-Links with 5Bromodeoxyuridine and γ-rays. Biochemistry 2009, 48, 2005−2011. (58) Dextraze, M.-E.; Gantchev, T.; Girouard, S.; Hunting, D. DNA Interstrand Cross-Links Induced by Ionizing Radiation: an Unsung Lesion. Mutat. Res., Rev. Mutat. Res. 2010, 704, 101−107. (59) Hicke, B. J.; Willis, M. C.; Koch, T. H.; Cech, T. R. Telomeric Protein-DNA Point Contracts Identified by Photo-Crosslinking Using 5-Bromodeoxyuridine. Biochemistry 1994, 33, 3364−3373. (60) Hutchinson, F.; Köhnlein, W. The Photochemistry of 5Bromouracil and 5-Iodouracil. DNA Progress in Molecular and Subcellular Biology; Springer-Verlag Berlin and Heidelberg GmbH & Co. KG: Berlin, Heidelberg, Germany, 1980. (61) Matsutani, M.; Kohno, T.; Nagashima, T.; Nagayama, I.; Matsuda, T.; Hoshino, T.; Sano, K. Clinical Trial of Intravenous Infusion of Bromodeoxyuridine (BUdR) for Radiosensitization of Malignant Brain Tumors. Radiat. Med. 1988, 6, 33−39. (62) Howard-Flanders, P.; Boyce, R. P.; Theriot, L. Mechanism of Sensitization to Ultra-Violet Light of T1 Bacteriophage by the Incorporation of 5-Bromodeoxyuridine or by Pre-Irradiation of the Host Cell. Nature 1962, 195, 51−54. (63) Rupp, W. D.; Prusoff, W. H. Incorporation of 5-Iodo-2′deoxyuridine into Bacteriophage T1 as Related to Ultra-Violet Sensitization or Protection. Nature 1964, 202, 1288−1290. (64) Sugiyama, H.; Tsutsumi, Y.; Saito, I. Highly Sequence-Selective Photoreaction of 5-Bromouracil-Containing Deoxyhexanucleotides. J. Am. Chem. Soc. 1990, 112, 6720−6721. (65) Sugiyama, H.; Tsutsumi, Y.; Fujimoto, K.; Saito, I. Photoinduced Deoxyribose C2′ Oxidation in DNA. Alkali-Dependent Cleavage of Erythrose-Containing Sites via a Retroaldol Reaction. J. Am. Chem. Soc. 1993, 115, 4443−4448. (66) Sugiyama, H.; Fujimoto, K.; Saito, I. Stereospecific 1,2-Hydride Shift in Ribonolactone Formation in the Photoreaction of 2′Iododeoxyuridine. J. Am. Chem. Soc. 1995, 117, 2945−2946. (67) Fujimoto, K.; Ikeda, Y.; Ishihara, S.; Saito, I. Deoxyribonolactone Formation in Photoirradiation of 5-Bromouracil-Containing Oligonucleotides by Direct C1′ Hydrogen Abstraction. Tetrahedron Lett. 2002, 43, 2243−2245. (68) Sugiyama, H.; Fujimoto, K.; Saito, I.; Kawashima, E.; Sekine, T.; Ishido, Y. Evidence for Intrastrand C2′ Hydrogen Abstraction in Photoirradiation of 5-Halouracil-Containing Oligonucleotides by Using Stereospecifically C2′-Deuterated Deoxyadenosine. Tetrahedron Lett. 1996, 37, 1805−1808. (69) Chen, T.; Cook, G. P.; Koppisch, A. T.; Greenberg, M. M. Investigation of the Origin Of the Sequence Selectivity for the 5-Halo2′-Deoxyuridine Sensitization of DNA to Damage by UV-Irradiation. J. Am. Chem. Soc. 2000, 122, 3861−3866. (70) Cecchini, S.; Masson, C.; La Madeleine, C.; Huels, M. A.; Sanche, L.; Wagner, J. R.; Hunting, D. J. Interstrand Cross-Link Induction by UV Radiation in Bromodeoxyuridine-Substituted DNA: Dependence on DNA Conformation. Biochemistry 2005, 44, 16957−16966. (71) Watanabe, T.; Bando, T.; Xu, Y.; Tashiro, R.; Sugiyama, H. Efficient Generation of 2′-Deoxyuridin-5-yl at 5′-(G/C) AAXUXU3′(X= Br, I) Sequences in Duplex DNA under UV Irradiation. J. Am. Chem. Soc. 2005, 127, 44−45. (72) Wiczk, J.; Miloch, J.; Rak, J. UV-Induced Strand Breaks in DoubleStranded DNA Labeled with 5-Bromouracil: Frank or Secondary? J. Phys. Chem. Lett. 2013, 4, 4014−4018. (73) Zheng, Y.; Sheppard, T. L. Half-Life And DNA Strand Scission Products of 2-Deoxyribonolactone Oxidative DNA Damage Lesions. Chem. Res. Toxicol. 2004, 17, 197−207. (74) Cook, G. P.; Greenberg, M. M. A Novel Mechanism for the Formation of Direct Strand Breaks upon Anaerobic Photolysis of Duplex DNA Containing 5-Bromodeoxyuridine. J. Am. Chem. Soc. 1996, 118, 10025−10030. 8237


Review Article

The Journal of Physical Chemistry B (75) Watanabe, T.; Tashiro, R.; Sugiyama, H. Photoreaction at 5′-(G/ C)AA(Br)UT-3′ Sequence in Duplex DNA: Efficient Generation of Uracil-5-yl Radical by Charge Transfer. J. Am. Chem. Soc. 2007, 129, 8163−8168. (76) Tashiro, R.; Nakamura, K.; Sugiyama, H. Photoreaction of Iodouracil in DNA Duplex; C-I Bond is Cleaved via Two Different Pathways ‘Homolysis and Heterolysis’. Tetrahedron Lett. 2008, 49, 428− 431. (77) Zeng, Y.; Wang, Y. Facile Formation of an Intrastrand Cross-Link Lesion between Cytosine and Guanine upon Pyrex-filtered UV Light Irradiation of d((Br)CG) and Duplex DNA Containing 5-Bromocytosine. J. Am. Chem. Soc. 2004, 126, 6552−6553. (78) Woessner, R. D.; Loudy, D. E.; Wallace, C. D.; Montgomery, L. R.; Cross-Doersen, D. E.; Bush, T. L.; Lewis, M. T.; Prakash, N.; Bitonti, A. J.; Wright, P. S. Decreased Vascular Endothelial Growth Factor Expression Associated with Tumor Regression Induced by (E)-2′Deoxy-2′-(fluoromethylene)cytidine (MDL 101,731). Oncol. Res. 1997, 9, 543−552. (79) Zeng, Y.; Wang, Y. Sequence-Dependent Formation of Intrastrand Crosslink Products from the UVB Irradiation of Duplex DNA Containing a 5-Bromo-2′-deoxyuridine or 5-Bromo-2′-deoxycytidine. Nucleic Acids Res. 2006, 34, 6521−6529. (80) Hong, H.; Wang, Y. Formation of Intrastrand Cross-Link Products Between Cytosine and Adenine From UV Irradiation of d((Br)CA) and Duplex DNA Containing a 5-Bromocytosine. J. Am. Chem. Soc. 2005, 127, 13969−13977. (81) Wang, Y.; Liu, Z.; Dixon, C. Major Adenine Products from 2Methyl-1,4-naphthoquinone-sensitized Photoirradiation at 365 nm. Biochem. Biophys. Res. Commun. 2002, 291, 1252−1257. (82) Lepczyńska, J.; Komodziński, K.; Milecki, J.; Kierzek, R.; Gdaniec, Z.; Franzen, S.; Skalski, B. Photoaddition of 5-Bromouracil to Uracil in Oligonucleotides Leading to 5,5′-Bipyrimidine-type Adducts: Mechanism of the Photoreaction. J. Org. Chem. 2012, 77, 11362−11367. (83) Wiczk, J.; Miloch, J.; Rak, J. DHPLC and MS Studies of a Photoinduced Intrastrand Cross-Link in DNA Labeled with 5-Bromo2′-deoxyuridine. J. Photochem. Photobiol., B 2014, 130, 86−92. (84) Zdrowowicz, M.; Michalska, B.; Ż ylicz-Stachula, A.; Rak, J. Photoinduced Single Strand Breaks and Intrastrand Cross-Links in an Oligonucleotide Labeled With 5-Bromouracil. J. Phys. Chem. B 2014, 118, 5009−5016. (85) Ornstein, R. L.; Rein, R.; Breen, D. L.; Macelroy, R. D. An Optimized Potential Function for the Calculation of Nucleic Acid Interaction Energies I. Base Stacking. Biopolymers 1978, 17, 2341−2360. (86) Nejedlý, K.; Kittner, R.; Pospíšilová, Š.; Kypr, J. Crosslinking of the Complementary Strands of DNA by UV Light: Dependence on the Oligonucleotide Composition of the UV Irradiated DNA. Biochim. Biophys. Acta, Gene Struct. Expression 2001, 1517, 365−375. (87) Pospíšilová, Š.; Kypr, J. UV Light-Induced Crosslinking of the Complementary Strands of Plasmid pUC19 DNA Restriction Fragments. Photochem. Photobiol. 1997, 65, 945−948. (88) Nowak-Karnowska, J.; Chebib, Z.; Milecki, J.; Franzen, S.; Skalski, B. Highly Efficient Fluorescent Interstrand Photo-Crosslinking of DNA Duplexes Labeled with 5-Fluoro-4-thio-2′-O-methyluridine. ChemBioChem 2014, 15, 2045−2049. (89) Yoshimura, Y.; Ito, Y.; Fujimoto, K. Interstrand PhotoCrosslinking of DNA via p-Carbamoylvinyl Phenol Nucleoside. Bioorg. Med. Chem. Lett. 2005, 15, 1299−1301.

8238


Mechanisms of Damage to DNA Labeled with Electrophilic

Recommend Documents