DNA Damage by the Direct Effect of Ionizing Radiation: Products

Article pubs.acs.org/JPCA

DNA Damage by the Direct Effect of Ionizing Radiation: Products Produced by Two Sequential One-Electron Oxidations David M. Close* Department of Physics, East Tennessee State University, Johnson City, Tennessee 37614, United States

William H. Nelson Department of Physics and Astronomy, Georgia State University, Atlanta, Georgia 30303, United States

William A. Bernhard Department of Biochemistry and Biophysics, University of Rochester, Rochester, New York 14642, United States ABSTRACT: It has long been assumed that the population of radicals trapped in irradiated DNA (that is, the radicals escaping recombination) would quantitatively account for the lesions observed in DNA. Recent results indicate that this is not the case. The yield of DNA lesions exceed the yield of trappable radicals. To account for a portion of this shortfall, it is thought that some of the initially formed 2′-deoxyribose radicals undergo a second oxidation by nearby base cation radicals to form 2′-carbocations. The carbocations react to give strand breaks and free base release. Schemes are presented to account for the major oxidation products observed including 8-oxoGua, 8-oxoAde, 5-OHMeUra, and free base release. Theoretical calculations were performed to ascertain the likelihood of the second oxidation step in these reaction pathways actually occurring, and to account for base sequence dependence and various levels of hydration.

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INTRODUCTION Radiation damage to the 2′-deoxyribose moiety of DNA results in stable end products consisting of strand breaks, base damage, and free base release. For many years the mechanisms involved in producing this damage was thought to involve one-electron oxidation of the DNA backbone to produce a radical cation followed by deprotonation to yield a neutral carbon centered 2′-deoxyribose radical. In the presence of water these neutral radicals react to give strand breaks and free base release. A good presentation of these processes can be found in a review article by Bernhard and Close1 and a more recent review by Bernhard.2 The events that lead to DNA damage by ionizing radiation result from two different processes, the direct and indirect effects. Direct-type effects arise from the direct ionization of the DNA or from holes and electrons transferred to the DNA from its hydration shell. The indirect effect refers to ionization of the water producing the hydroxyl radical, HO•, which can interact with the 2′-deoxyribose by H-abstraction, or with the nucleobases by addition to an unsaturated bond. The indirect effect is the major effect in a dilute solution of DNA. The direct effect makes a significant contribution to DNA damage under the conditions in the nuclei of eukaryotic cells because chromatin is tightly packed and the amount of unbound water is relatively small. To study the direct effect, it is necessary to remove the water responsible for the indirect effect © XXXX American Chemical Society

and to use cryogenic temperatures, which prevent diffusion of intermediate radical species derived from water ionizations. Under these conditions electron parametric resonance (EPR) spectroscopy can be used to characterize radical intermediates.1,3 From the model discussed above one would expect that the strand break site should be independent of the base at that site, and the probability of observing base release should be independent of base type. Studies of oligodeoxynucleotides of known sequence have shown that these assumptions are wrong.4,5 Thus the yield of DNA lesions exceeds the yield of trappable radicals (those radicals that have escaped combination reactions). First it is important to describe the experiments that were performed to arrive at these conclusions. The experiments were performed in variably hydrated plasmid DNA.6 The level of hydration, Γ, was varied from 2.5 to 22.5 mols of water per mole of nucleotide. It was determined by EPR measurements that the yield of 2′-deoxyribose radicals at 4 K was 33 ± 5 nmol/J at Γ = 2.5 and increased to 79 ± 12 nmol/J at Γ = 22.5.7 Next it is important to subject samples to variable radiation doses. About 90% of the radicals are trapped by the bases in DNA, and these radicals have a high cross section for Received: August 24, 2013 Revised: October 11, 2013

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dx.doi.org/10.1021/jp4084844 | J. Phys. Chem. A XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry A

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Combination reactions that go forward to damage are ones that bring together two one-electron oxidations (2oe-ox). This then is the primary interest of the present work. The formation of products such as 8-OxoGua require two one-electron oxidations. The first oxidation is by direct ionization of a base (or by hole transfer to a base) and the second oxidation is by electron transfer to a base radical cation (Base•+) or oxidation by oxygen. One can trace the steps to form these stable end products and some of the pathways have recently appeared in the literature.5 However, it is not altogether clear if some of the steps proposed are energetically favorable. Therefore, new calculations are presented that address the likelihood of the various steps in these reaction pathways actually occurring. The intermediate radicals in a 2oe-ox pathway may or may not be trappable. An example of the latter is a spur reaction (that is, the initial two radicals B(−H)• and B•+ react with a cluster of ionizations). To the extent that these occur at 4 K, there will be a shortfall between 4 K free radical yields and product yields. At 4 K, about half of the radicals initially formed are trapped.16 Upon warming to room temperature, radicals are mobilized and enter into combination reactions, reducing the radical concentration by about 10-fold.15 As a result, there is a large difference between the yield of product observed after dissolving the DNA in water and the yield of radicals in DNA films just prior to dissolution. Because this difference is larger than the shortfall attributed to spur reactions, it means that 2oe-ox reactions occur upon annealing from 4 K to room temperature. At low dose, therefore, there are two distinct pathways for 2oe-ox reactions, those within spurs and those via diffusion of excess electrons and holes. At high dose, defined as >6 kGy, combination reactions occur as a new radiation track intersects a pre-existing radiation track. Holes created by the new track, oxidize free radical trapped from the pre-existing track. Thus, 2oe-ox reactions are generated by multiple track chemistry. In contrast to singletrack chemistry where the concentration of product increases linearly with dose, multiple-track chemistry gives a quadratic response to dose. It is easy, however, to miss deviations from linearity when detection of product requires doses >6 kGy.

destruction. Therefore, the EPR signal dose saturates (in DNA at about 5 kGy). On the other hand 2′-deoxyribose-centered radicals, making up the other 10%, have a low cross section for destruction. Therefore, the concentration of 2′-deoxyribose radicals increases linearly to unusually high doses (∼500 kGy).8,9 It is also important to note that in the solid-state, single-track chemistry accounts for effectively all of the trapped radicals at doses below 5 kGy. Above 10 kGy, a mixture of single-track and multiple-track chemistry occurs.10 In the discussion that follows it will be necessary to consider the contributions of multiple-track chemistry for radiation doses above 10 kGy. The chemical yield of strand breaks was measured in the same plasmid DNA films used for the radical yield measurements. Gel electrophoresis was used to measure the loss of the supercoiled form. Because multiple single strand breaks in one plasmid are scored as only one strand break, it is important to also measure free base release. The yield of single strand breaks was 124 ± 4 nmol/J at Γ = 2.5 and increased to 182 ± 21 nmol/J at Γ = 22.5 whereas the yield of free base release was 134 ± 4 nmol/J at Γ = 2.5 and increased to 189 ± 21 nmol/J at Γ = 22.5.7 The yield of 2′-deoxyribose radical was 33 ± 5 nmol/J at Γ = 2.5 and increased to 79 ± 12 nmol/J at Γ = 22.5.11 Thus the yield of strand breaks exceeds the yield of 2′deoxyribose trapped radicals. The difference between the two yields (124−33 = 91 ± 4 nmol/J) at Γ = 2.5 and 182−79 = 103 ± 21 nmol/J)) at Γ = 22.5 is termed herein the shortfall.5 Next one needs to know what end products are actually observed. As shown here, this depends on the base sequence, and the level of hydration. 1. In d(GCACGCGTGC)2 irradiated films at Γ = 2.5, the five major products are 5,6-diHydroThy, 5,6-diHydroUra, 8-oxoGua, 8-oxoAde, and free base release. The relative yields are in the order 5,6-diHydroUra ∼8oxoGua ∼ fbr > 5,6-diHydroThy > 8-oxoAde.12 It is important to note that this oligomer has all pyrimidines adjacent to a Gua. If a hole is initially formed on Thy, transfer of that hole to a nearby Gua competes with reactions that yield a damaged Thy. 2. In d(CTCTCGAGAG)2 films at Γ = 2.5, the major products include those above plus 5-OHCyt, 5-OHUra, and 5-HOMeUra. The order of yields are 5,6diHydroUra > 5-OHCyt ∼8-oxoGua > 5-OHMeUra > 8-oxoAde > 5-OHUra.13 This oligomer has a pyrimidine stretch and a purine stretch and only one pyrimidine that is adjacent to a Gua. 3. In d(CTCTCGAGAG)2 films at Γ = 15 compared to Γ = 2.5, the yields decrease for 5,6-di-HydroUra, 5,6diHydroThy, and maybe 5-OHMeUra; the yields increase for 8-oxoGua, 8-oxoAde, and free base release, whereas the yields are unchanged for 5-OHCyt and maybe 5-OHUra.14

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THEORETICAL METHODS To discuss the model calculations, it is necessary to first view the reaction paths. Irradiation of a base causes an electron to be removed, creating a base cation radical B•+, which subsequently deprotonates to give a neutral base radical B(−H)•. The question then is can this product undergo a second oxidation by a base cation radical B•+, B(−H)• + B•+ → B(−H)+ + B. This reaction is thermodynamically possible if the energy to remove an electron from B(−H)• is less than the energy gain from adding an electron to the B•+. This amounts to comparing the ionization potential (IP) of B(−H)• to the electron affinity (EA) of B•+. Thus, if the IP + EA < 0, the reaction is thermodynamically allowed. For electron affinity: EA = Eparent − Efinal (EA > 0 means electron addition lowers the energy). For ionization potential: IP = Efinal − Eparent (IP > 0 means electron removal raises the energy). Adiabatic EA/IP: both parent and final energies calculated at the respective optimized geometries. Vertical EA/ IP: final energies after electron addition/removal calculated from the parent. The procedure is to optimize parent and f inal molecular geometries within the dielectric medium (and vacuum with ε = 1) with DFT methods using the B3LYP functional and the 6-31G(d,p) basis set, test for actual energy minimum with a frequency

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MODELS Any model that explains the above free radical yields must show how the reaction pathways move forward to produce damage whereas most of the free radicals are lost due to combination reactions. For a good discussion of the types of combination reactions seen in DNA, see Bernhard et al.15 Although combination reactions that return an excess electron to a hole are the dominant pathway, these reactions lead back to the parent; i.e., they are not damaging (this assumes that homolytic bond cleavage due to the resultant excited state is negligible). B



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calculation, which also gives an estimate of the zero point energy (ZPE), and calculate the energy at the optimized geometry using B3LYP and the higher basis set 6-311G(2df,3p). With parent and final energy values, calculate AIP and AEA. Calculations were performed with the Gaussian 03 suite of programs.17 The starting structures were normal taken from normal B-DNA models. It is necessary to comment on the choice of basis sets used for these calculations. Though it is true that one often gets slightly different answers by using different basis sets, the interest here is in relative energies. For example, for the calculations presented here it is necessary to compare the ionization potential (IP) of B(−H)• to the electron affinity (EA) of B•+. Although the differences in energies were substantial, the calculations seemed to be independent of the basis sets used. There were, however, some cases where the energy differences were small, and these are discussed later on. Calculations of the vertical ionization potentials of the 2′deoxyribose or base deprotonated radical cations were routine. However, there were problems with optimizing some of the deprotonated nucleoside radical cations that adopted nonplanar geometries. In some cases where cross-links formed whereas others showed C−C and C−O bond elongations as seen previously in thymine radicals.18 For these cases there are uncertainties in the calculated adiabatic ionization energies, so just the vertical ionization potential calculations are reported. It is interesting to note theoretical calculations on one electron oxidation of sugar radicals by Sevilla and co-workers that compare favorably with the results presented herein.19

Scheme 1. Two One-Electron Oxidations of Thymine

DNA, a significant fraction of the Thy(Me−H)• radical must be lost upon warming, suggesting the following reaction. b. The next step (reaction 3 in Scheme 1) involves the oeox of the Thy(Me−H)• radical, resulting in the carbocation Thy(Me−H)+. For this to occur it is necessary to transfer an electron from the Thy(Me−H)• radical to a base radical cation. Calculations below are presented to see if this reaction is energetically favorable. c. Upon dissolution in an oxygenated solution, the carbocation will give 5-OHMeUra via reaction 4 in Scheme 1. d. Although yields of 5-HOMeUra were significant in d(CTCTCGAGAG) 2 , 1 3 they were not in d(CGCGAATTCGCG)2.12 This is consistent with the competition the thymine radical cation Thy•+ faces between hole transfer to Gua and deprotonation at >C5CH3. In d(CTCTCGAGAG)2 the thymines are not near a guanine. This then favors deprotonation of thymine at >C5-CH3 and thus a higher yield of 5-OHMeUra, as shown in Scheme 1. So then is it energetically favorable for a base•+ radical cation to oxidize the Thy(Me−H)• radical as shown in reaction 3 of Scheme 1? To assess the probability that an electron will transfer from a dehydrogenation product to a nucleotide cation, it is necessary to compare the respective ionization potentials (IPs) and electron affinities (EAs). The reason is that the starting point of a transfer presumably is the combination of a relaxed XdR cation and a dehydrogenation radical. Thus the electron transfer will be initiated only if AEA of the XdR cation is greater than the AIP of the −Hx radical structure. In Table 1, one sees the adiabatic ionization potential of the Thy(Me−H)• radical in vacuum (ε = 1) is 7.14 eV whereas the adiabatic electron affinity of the guanine cation radical •Gua(+) is 7.49 eV. Given that an electron will transfer from A to B if AEAB > AIPA, this is clearly the case for the Thy(Me−H)• radical. Examining the table seems to indicate this will be the case for any of the base cations and this is also true regardless of the dielectric effect. The main effect of dielectric surroundings arises from the charge on a molecule and the dielectric “reaction field” it generates from the media (Born equation24). The Born equation has the form

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TWO SEQUENTIAL ONE-ELECTRON OXIDATIONS (OE-OX) The goal is to describe an overview of the primary reaction pathways initiated in DNA by the direct effect of ionizing radiation. The indirect effect is excluded by studying DNA in the solid state. EPR is used for identifying radical species, measuring radical concentrations, and for monitoring radical reactions. Irradiating the DNA at 4 K traps radical species that are closely related to the radicals that are initially formed. Annealing the samples to higher temperatures mobilizes these trapped species and activates radical reactions. The final products, DNA lesions, are not radicals and cannot be detected by EPR. However, one can use methodologies such as gel electrophoresis, HPLC, and GC/MS to determine the yields of many of these major lesions. How these products correlate with the radical precursors is part of the model described below, which can be used to explain the observed sequence dependent base damage. 1. Thymine (Scheme 1). a. Oe-ox of thymine gives the thymine radical cation Thy•+ (reaction 1 in Scheme 1), which has been detected at 4 K in poly(AT) and poly(A):poly(T) but not in DNA.16,20 If Gua is adjacent to Thy, hole transfer to form the thymine radical cation to Gua will dominate. Absence of a nearby guanine results in an increased likelihood that irreversible deprotonation from the >C5-CH3 will occur, producing the thymine allyl radical, Thy(Me−H)• (reaction 2 in Scheme 1). Evidence for this step comes from studies of thymidine in glasses, in poly d(AT) and in DNA.21,22 The Thy(Me−H)• radical is stable and persists in 1-MeThy crystals on warming to 200 C.23 Its distinctive five-line EPR signature is easy to detect. Because it has not been detected at room temperature in

⎛1 ⎞ ΔE ∼ Q 2⎜ − 1⎟ ⎝ε ⎠ C



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Table 1. Base Cation AEA’s (eV) vs ε and 3-α Radical AIP’s (eV) vs ε base cation AEA’s (eV) vs ε ε 1 2.5 5 10 20 40 80

•

G(+) 7.49 6.29 5.90 5.70 5.61 5.56 5.53

•

A(+)

7.94 6.71 6.31 6.10 6.00 5.95 5.92

3-α radical AIP’s (eV) vs ε •

C(+) 8.43 7.15 6.71 6.50 6.39 6.34 6.31

•

T(+)

ε

Thy(Me−H)•

5mCyt(Me−H)•

8.61 7.24 6.75 6.50 6.38 6.31 6.28

1 2.5 5 10 20 40 80

7.14 5.72 5.22 4.97 4.83 4.77 4.73

6.75 5.44 4.98 4.75 4.64 4.58 4.55

and yields virtually the same effect for all ions with the same charge magnitude within a given dielectric medium. Also note that most of the effect appears for ε < 5, as is evident in Table 1. Because DNA contains a small amount of 5-methylcytosine, the last column of Table 1 lists the ionization potential for the 5-methylcytosine radical (labeled as 5mCyt(Me−H)•). One sees that the 5mCyt(Me−H)• radical will transfer an electron to all of the base radical cations (Base•+), and again this is true regardless of the dielectric effect. 2. Cytosine (Scheme 2). a. Oe-ox of Cyt gives the cytosine radical cation Cyt•+ (reaction 1 in Scheme 2). If Gua is adjacent to Cyt, hole transfer to Gua will dominate. As the intrastrand distance between Cyt and Gua increases, the rate of hole transfer will decrease and other reactions will become competitive. b. The cytosine radical cation Cyt•+ may deprotonate at C1′ of the 2′-deoxyribose to give the neutral dCyt(C1′−H)• radical, as shown in reaction 3 of Scheme 2. The next step (reaction 4) involves the Oe-ox of dCyt(C1′−H)• radical, resulting in the carbocation dCyt(C1′−H)+. For this to occur it is necessary to transfer an electron from the dCyt(C1′−H)• radical to a base radical cation. Below calculations are presented to see if this reaction is energetically favorable. If a fraction of the dCyt(C1′−H)• radical persists at room temperature, oxidation by O2 will occur following dissolution and subsequent hydration will lead to the same end products (as shown in reaction 5 of Scheme 2). c. Reaction 6 leads from dCyt(C1′−H)• to deoxyribonolactone. i. Attack of C1′ carbocation by water yields free base, as shown in reaction 6 of Scheme 2. ii. This pathway is used to explain why the yield of free Cyt is nearly 2× larger than any other base in AT rich DNA but not in CG rich DNA.5 Hole transfer to Gua competes with deprotonation at C1′, such that reaction 4 proceeds as long as Cyt is not adjacent to Gua. d. Alternatively, Cyt•+ may reversibly deprotonate at N4 to give the iminyl radical Cyt(N4−H)•, as shown in reaction 2 of Scheme 2. The Cyt(N4−H)• radical has been observed in crystalline cytosine at 10 K25 but has not been observed in solid-state DNA. Calculations below show that a second oe-ox of the iminyl radical (reaction 7 in Scheme 2) is unfavorable in that none of the base cations (base•+) will accept an electron from Cyt(N4−H)•. Reaction 4 in Scheme 2 involves the Oe-ox of the dCyt(C1′− H)• radical, resulting in the carbocation Cyt(C1′−H)+. For this to occur, it is necessary to transfer an electron from the

Scheme 2. Two One-Electron Oxidations of Cytosine

dCyt(C1′−H)• radical to a base radical cation. For this to be energetically favorable, it is necessary that an electron will transfer from A to B if AEAB > AIPA. As a first step, calculations were performed on just the 2′-deoxyribose moiety. In Table 2, one sees the adiabatic ionization potential of the dRib(C1′−H)• radical in vacuum (ε = 1) is 5.92 eV whereas the adiabatic electron affinity of the guanine cation radical •Gua(+) is 7.49 eV. Thus AEAB > AIPA for the dRib(C1′−H)• radical. Examining Table 2 it seems as if this will be the case for any of the base cations, and is also true regardless of the dielectric effect. Because reactions 3 and 4 in Scheme 2 actually involve the cytosine nucleoside, it is necessary to perform these calculations on the bases with the 2′-deoxyribose moiety included. Problems were encountered in optimizing some of the XdR− Hx cation structures for the adiabatic ionization energies (particularly for deprotonations at −H2′). These dehydrogenation radicals exhibited normal structural geometries only for calculations of vertical ionization energies. The results are presented in Table 3. To assess the probability of electron transfer from dehydrogenation radicals to a nucleoside cation, it is assumed that electron transfer will be feasible only if the vertical electron D



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Table 2. Adiabatic EA’s of the Base Cations and IP’s of 2′-Deoxyribose Structures (eV) base cation AEAs (eV) vs ε ε

•

1 2.5 5 10 20 40 80

G(+) 7.49 6.29 5.90 5.70 5.61 5.56 5.53

•

A(+)

dRib(−Hx′) AIPs (eV) vs ε

•

•

C(+)

7.94 6.71 6.31 6.10 6.00 5.95 5.92

8.43 7.15 6.71 6.50 6.39 6.34 6.31

T(+)

−H1′

−H2′

−H3′

−H4′

−H5′

8.61 7.24 6.75 6.50 6.38 6.31 6.28

5.92 4.64 4.21 4.00 3.90 3.84 3.82

6.08 4.71 4.23 3.97 3.84 3.78 3.75

6.23 4.88 4.41 4.16 4.04 3.98 3.95

5.24 4.01 3.61 3.40 3.30 3.26 3.24

6.31 4.99 4.53 4.31 4.19 4.13 4.08

Table 3. Vertical EA’s and IP’s of Nucleoside Structures (eV) VEAs (eV) XdR

VEA of XdR•+

AdR CdR GdR TdR UdR

7.26 7.59 6.88 7.59 7.97

VIPs (eV) of the dehydrogenation products −H1•

−H2•

−H3•

−H4•

−H5Me•

7.47 8.96 8.16

8.07 8.75 9.75

6.81

affinity of the XdR cation is greater than the vertical ionization potential of the −Hx radical structure. In Table 3 one sees that all −H1′• radicals have VIP’s < VEA of the CdR radical cation. All −H2′• radicals have VIP’s > VEA’s of all XdR cations except that the VIP of the −H2′• from CdR < VEA of the UdR radical cation. All −H3′• radicals have VIP’s < VEA’s of all XdR cations except for GdR. All −H4′• radicals have VIP’s < VEAs of pyrdR cations. −H4′• radicals from CdR and GdR have VIP’s < VEA’s of AdR cations, and all −H4′• radicals have VIP’s > VEA of GdR cations. All −H5′• radicals have VIPs > VEAs of purdR cations, whereas the −H5′• VIPs of TdR exceed VEA’s of all XdR cations except for UdR. These results predict that it is thermodynamically feasible that all −H1′• radicals eventually will yield an electron to any XdR cation. However, the conclusion is mixed for the other −Hx• radicals. In particular, the −H2′• structure appears unlikely to initiate an electron transfer to any of the XdR cations for normal DNA. Reactions 3 and 4 in Scheme 2 are of interest because they represents a transfer of oxidative damage from a base to the backbone. Oxidative damage is generally viewed as flowing from the DNA backbone to the bases, not the other way around.26 If electron loss from cytosine leads to oxidative damage of its 2′-deoxyribose, then that damage is base-specific. Such a possibility is of interest because there are DNA sequences where base sequence appears to influence the probability of 2′-deoxyribose damage.27 There is an alternative pathway for the cytosine radical cation, Cyt•+ depicted in Scheme 2 involving reaction 2, deprotonation at N4 to give the iminyl radical Cyt(N4−H)• followed by a second oxidation of this iminyl radical. The calculated vertical ionization potential of the iminyl radical Cyt(N4−H)• is 8.96 eV so a second oe-ox is unfavorable for all of the base radical cations (Base•+). 3. Guanine (Scheme 3). a. Oe-ox of Gua gives the guanine cation radical Gua•+, shown in reaction 1 of Scheme 3). b. Deprotonation of Gua•+ gives the Gua(N1−H)• radical (reaction 2 in Scheme 3). At 4 K deprotonation is faster than hole transfer by hopping but it is not faster than tunneling to Gua within one or two base pairs on the

−H1′•

−H2′•

−H3′•

−H4′•

−H5′•

6.26 5.71 6.42 6.28 6.43

8.16 7.86 8.22 8.35 8.47

7.15 7.13 7.25 7.11 7.22

7.33 7.20 7.20 7.34 7.53

7.42 7.55 7.34 7.71 7.82

Scheme 3. Two One-Electron Oxidations of Guanine

same strand. The Gua(N1−H)• radical is trapped in DNA constituents containing Gua irradiated at 10 K.28 This radical is unstable at higher temperatures, decaying in the 120−230 K range.29 At room temperature no radicals assignable to the one-electron oxidation of Gua have been observed. Therefore, a nonradical product is formed that yields 8-oxoGua as shown in the next step. c. Oe-ox of Gua(N1−H)• radical (reaction 3 in Scheme 3) would give a carbocation, Gua(N1−H)+, that upon dissolution should give 8-oxoGua (reaction 4). To examine the energetics of reaction 3 in Scheme 3, the adiabatic ionization potential of the Gua(N1−H)• radical has been computed to be 7.64 eV (Table 4). To oxidize the Gua(N1−H)• radical, it is necessary to find an XdR cation with an AEA greater than this AIP. Only CdR and TdR barely meet this requirement. Table 4 shows that this reaction might be favorable for the Pyr•+ but not for the Pur•+. On the other hand, the vertical ionization energy of the Gua(N1−H)• radical is 8.16 eV. One can see from Table 3 that this reaction is not favorable for any Base•+. E



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Table 4. Adiabatic EA’s and IP’s of Nucleoside Structures (eV) AEAs (eV) XdR

AEA of XdR•+

AdR CdR GdR TdR UdR

7.56 7.71 7.14 7.84 8.18

AIPs (eV) of the dehydrogenation products −H1•

−H2•

−H3•

−Hx•

−H5Me•

7.84 8.69 7.64

7.63 7.32 7.34

6.40

−H1′•

−H2′•

−H3′•

−H4′•

−H5′•

5.45 5.05 5.26 5.29 5.44

5.55 5.76 6.31 5.85 5.98

4.95 6.14 6.41 6.44 6.51

6.10 5.55 5.77 5.83 5.98

4.82 6.69 6.65 6.85 6.91

again this reaction is not feasible at least at the level of theory calculated here. Because deprotonation of the adenine cation at N6 does not yield satisfactory results, it is necessary to look at other sites for deprotonation. Though it is common for the adenine cation to deprotonate at N6, in deoxyadenosine the cation deprotonates at C1′.32 However, one cannot create 8-oxoAde from a 2′deoxyribose radical. The most direct way to create 8-oxoAde would be by OH− addition to C8 of the adenine cation radical. So then the question becomes, can any of the base cation radicals (XdR•+) oxidize this adenine OH− adduct? Calculations show that this is very likely to occur. The calculated AIP of this OH− adduct is only 6.56 eV. Therefore, looking at the AIP’s of base cation radicals (XdR•+) in Table 4, one sees that all of the can oxidize the adenine OH− adduct.

So then is there an alternative pathway to produce 8-oxoGua in thin films of DNA? One possibility would involve hydration of Gua•+ at C8 to form •GOH. Although this reaction is observed in aqueous solution, it has also been observed in a single crystal of guanine:HCl:H2O at 10 K.30 So then the question becomes, can any of the base cation radicals (XdR•+) oxidize •GOH? Calculations show that this is very likely to occur. The calculated AIP of •GOH is only 5.76 eV. Therefore, looking at the AEA’s of base cation radicals (XdR•+) in Table 4, one sees that all of the base cations can oxidize •GOH. 4. Adenine (Scheme 4). a. Oe-ox of Ade gives the adenine radical cation Ade•+ (shown in reaction 1 of Scheme 4). Hole transfer from

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Scheme 4. Two One-Electron Oxidations of Adenine

CONCLUSIONS One-electron oxidation of thymine produces the radical cation Thy•+. Hole transfer from Thy•+ will dominate if a Gua lies close by. Absence of a nearby Gua results in an increased likelihood that irreversible deprotonation from the C5-methyl will occur, giving the stable thymine allyl radical, Thy(Me−H)•. Because this Thy(Me−H)• radical has not been observed in DNA, a significant fraction must either gain or lose an electron. Electron addition to Thy(Me−H)• with subsequent protonation reverses the damage, re-forming thymine. Electron loss from Thy(Me−H)• results in a nonradical carbocation. This product reacts in water to give the observed 5OHMeUra end product. The crucial step considered here is the transfer of the electron from the Thy(Me−H)• radical. Calculations presented here show that any base cation can oxidize a Thy(Me−H)• radical regardless of the dielectric effect (the same is true for 5MeCyt). Thus to produce the observed 5-OHMeUra end product, two one-electron oxidations are required. The same situation is present in studies of cytosine. Deprotonation of the radical cation Cyt•+ must compete with hole transfer to a nearby base. There are two sites that cytosine can deprotonate from to give the neutral dCyt(C1′−H)• radical or the iminyl radical Cyt(N4−H)•. Calculations presented here show that a second oxidation of the iminyl radical by a base cation is unfavorable. On the other hand, calculations show that one-electron oxidation of any dRib(C1′−H)• by any of the DNA base cations is favorable and results in a carbocation. Dissolution of the sample would then lead to release of free cytosine and deoxyribonolactone. As pointed out by Sharma et al.,5 there is an interesting explanation of the 2× increase in free Cyt release in AT rich DNA compared to CG rich DNA. Hole transfer to Gua competes with deprotonation at C1′, such that reaction 4 in Scheme 2 proceeds as long as Cyt is not adjacent to Gua. Guanine is considered to be the most probable site in DNA for one-electron oxidation. The guanine cation deprotonates to form the neutral Gua(N1−H)• radical and has been observed

the Ade•+ to a nearby guanine is favored as a consequence of guanine’s lower oxidation potential. However, if guanine is not within a few base pairs, other reactions may compete with hole transfer to guanine. b. One reactions involves deprotonation from N6 to give the Ade(N6−H)• radical (reaction 2 in Scheme 4), which has been observed in single crystals of adenosine at 10 K.31 c. Oe-ox of Ade(N6−H)• yields the carbocation Ade(N6− H)+, shown in reaction 3, Scheme 4. d. Here too, dry DNA gives 8-oxoAde. The yield is 5−10× smaller than 8-oxoGua.12 To examine the energetics of reaction 3 in Scheme 4, the adiabatic ionization potential of the Ade(N6−H)• radical has been computed to be 7.84 eV (Table 4). None of the XdR•+ have AEAs this large, so it is not feasible for any nucleoside cation radicals of oxidize the Ade(N6−H)• radical. The vertical ionization energy of the Ade(N6−H)• radical is 8.33 eV. This is higher than any of the VEA’s of the XdR•+ listed in Table 3, so F



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were performed by William Nelson on three 64-bit quad core computers. Special thanks to Duke Windsor (GSU) for help in keeping these machines up and running while this work was being completed. Thanks to Yuriy Razskazovskiy (ETSU) for many helpful discussions.

in DNA constituents at 10 K. There are, however, no radicals assignable to one-electron oxidation of Gua at room temperature. Therefore, it is necessary to identify a scheme in which a nonradical precursor that yields 8-oxoGua upon dissolution. One explanation could involve one-electron oxidation of the Gua(N1− H)• by a base cation to give the carbocation, Gua(N1−H)+, that upon dissolution should give 8-oxoGua. It was shown above that the cations of CdR and TdR (Pyr•+) can oxidize Gua(N1−H)•. Also it may be possible to consider a competitive reaction to deprotonation of the guanine cation, that is hydration of the guanine cation to produce •GOH. Calculations show that this C8OH adduct can oxidize Gua(N1−H)•. One-electron oxidation of adenine gives the radical cation Ade•+, which deprotonates to form Ade(N6−H)•. A second oxidation yields the carbocation Ade(N6−H)+. Dissolution of the carbocation yields 8-oxoAde. Calculations have shown that none of the nucleoside cations will oxidize the Ade(N6−H)• radical. As with the situation in guanine, it is necessary to examine other species that may oxidize Ade(N6−H)•, such as the •AOH adduct. This study has shown that there are energetically favorable pathways for formation of the major products observed in irradiated pyrimidines. Things are not as straightforward for the purines. Because the redox properties of the radical intermediates have not been measured, it is necessary to use computational theory to calculate redox properties. One can say, for example, that at the level of theory used herein Pyr•+ radical cations can oxidize Gua(N1−H)• but not Ade(N1−H)•. There are, however, purine C8 OH adducts that will oxidize the purine deprotonated cation radicals. The schemes presented here show which two one-electron oxidation pathways are energetically feasible. No attempts have been made to show that the reaction pathways are kinetically feasible. There is, however, good evidence for the first three steps in each pathway involving one-electron oxidation of the DNA bases. These cations are good acids and rapidly deprotonate. The sites of deprotonation have been determined by detailed EPR/ENDOR experiments.1,3 At the end of each scheme are the final products that can only be formed by a second one-electron oxidation. The uncertainty in the schemes then lies in the combination of an XdR cation and a dehydrogenation radical. At this point we know which combinations are energetically feasible, but not if these reactions are kinetically feasible.

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REFERENCES

(1) Bernhard, W. A.; Close, D. M. DNA Damage Dictates the Biological Consequences of Ionizing Irradiation: The Chemical Pathways. In Charged Particle and Photon Interactions with Matter; Mozumder, A.;, Hatano, Y., Eds.; Marcel Dekker: New York, 2003; pp 431−470. (2) Bernhard, W. A. Radical Reaction Pathways Initiated by Direct Energy Deposition in DNA by Ionizing Radiation. In Radical and Radical Reactivity in Nucleic Acid Chemistry; Greenberg, M., Ed.; John Wiley and Sons: Hoboken, NJ, 2010; pp 41−68. (3) Close, D. M. Radical ions and their reactions in DNA constituents: ESR/ENDOR studies of radiation damage in the solid state. Radiat. Res. 1993, 135, 1−15. (4) Sharma, K. K.; Razskazovskiy, Y.; Purkayastha, S.; Bernhard, W. A. Mechanisms of strand break formation in DNA due to the direct effect of ionizing radiation: the dependency of free base release on the length of alternating CG oligodeoxynucleotides. J. Phys. Chem. B 2009, 113, 8183−8191. (5) Sharma, K. K.; Tyagi, R.; Purkayastha, S.; Bernhard, W. A. Oneelectron oxidation of DNA by ionizing radiation: competition between base-to-base hole-transfer and hole-trapping. J. Phys. Chem. B 2010, 114, 7672−7680. (6) Strand breaks can be detected with unusually high sensitivity using plasmid DNA as a target. Assays rely on the differential electrophoric mobility of conformational isomers that are supercoiled (break free), relaxed (contain at least one single strand break) or linearized (contain a double strand break). For example, 250 ng of a 10 kb plasmid employed for measuring single strand breaks with a detection sensitivity of ∼4 × 10−14 mol. More details can be found in Purkayastha, S.; et al. J. Phys. Chem. B 2005, 16967−16973. (7) Sharma, K. K.; Milligan, J. R.; Bernhard, W. A. Multiplicity of DNA single-strand breaks produced in pUC18 exposed to the direct effects of ionizing radiation. Radiat. Res. 2008, 170, 156−62. (8) Wang, W.; Becker, D.; Sevilla, M. D. The influence of hydration on the absolute yields of primary ionic free radicals in g-Irradiated DNA at 77 K. Radiat. Res. 1993, 135, 146−154. (9) Purkayastha, S.; Bernhard, W. A. What is the initial chemical precursor of DNA strand breaks generated by direct-type effects? J. Phys. Chem. B 2004, 108, 18377−18382. (10) Mozumder, A.; Magee, J. L. Theory of radiation chemistry, VII. Structure and reactions in low LET tracks. J. Chem. Phys. 1966, 45, 3332−3341. (11) Purkayastha, S.; Milligan, J. R.; Bernhard, W. A. An investigation into the mechanisms of DNA strand breakage by direct ionization of variably hydrated plasmid DNA. J. Phys. Chem. B 2006, 110, 26286− 26291. (12) Swarts, S. G.; Gilbert, D. C.; Sharma, K. K.; Razskazovskiy, Y.; Purkayastha, S.; Naumenko, K. A.; Bernhard, W. A. Mechanisms of direct radiation damage in DNA, based on a study of the yields of base damage, deoxyribose damage, and trapped radicals in d(GCACGCGTGC)2. Radiat. Res. 2007, 168, 367−381. (13) Sharma, K. K.; Bernhard, W. A. Direct damage to the backbone of DNA oligomers is influenced by the OH moiety at strand ends, by the type of base, and by context. J. Phys. Chem. B 2009, 113, 12839− 12843. (14) Sharma, K. K.; Swarts, S. G.; Bernhard, W. A. Mechanisms of direct radiation damage to DNA: the effect of base sequence on base end products. J. Phys. Chem. B 2011, 115, 4843−4855. (15) Bernhard, W. A.; Mroczka, N.; Barnes, J. Combination is the dominant free radical process initiated in DNA by ionizing radiation: An overview based on solid-state EPR studies. Int. J. Radiat. Biol. 1994, 66, 491−497.

AUTHOR INFORMATION

Corresponding Author

*D. M. Close: e-mail, [email protected]; fax, 423-929-6905. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Much of this material covers work done in collaboration with Professor William Bernhard at the University of Rochester and Professor William Nelson at Georgia State University. William Nelson died unexpectedly in December 2010. Professor Bernhard was invited to Georgia State to present the first William Nelson Memorial Lecture in April 2011. The lecture, entitled “Damage Due to Ionizing Radiation: from DNA to Protein and Back Again” introduced much of the work presented here. William Bernhard died in May 2012. An In Memoriam summarizing his work recently appeared in Radiation Research.33 Some of the calculations presented here G



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(16) Spalletta, R. A.; Bernhard, W. A. Free radical yields in A:T polydeoxynucleotides, oligodeoxynucleotides, and monodeoxynucleotides at 4 K. Radiat. Res. 1992, 130, 7−14. (17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant; et al. Gaussian03, Revision E.01; Gaussian, Inc.: Wallington, CT, 2004. (18) Close, D.; Forde, G.; Gorb, L.; Leszczynski, J. Model calculations of radiation-induced damage in thymine derivatives. Struct. Chem. 2003, 14, 451−454. (19) Kumar, A.; Pottiboyina, V.; Sevilla, M. D. One-electron oxidation of neutral sugar radicals of 2′-deoxyguanosine and 2′deoxythymidine: A density functional theory (DFT) study. J. Phys. Chem. B 2012, 116, 9409−9416. (20) Spalletta, R. A.; Bernhard, W. A. Influence of primary structure on initial free radical products trapped in A:T polydeoxynucleotides x-irradiated at 4 K. Radiat. Res. 1993, 133, 143−150. (21) Sevilla, M. D.; Engelhardt, M. D. Mechanisms for radiation damage in DNA constituents and DNA. Faraday Discuss. Chem. Soc. 1978, 63, 255−263. (22) Weiland, B.; Hüttermann, J. Free radicals from x-irradiated ’dry’ and hydrated lyophilized DNA as studied by electron spin resonance spectroscopy: analysis of spectral components between 77 K and room temperature. Int. J. Radiat. Biol. 1998, 74, 341−358. (23) Schmidt, J. An ESR analysis of a heat-stable radical in gammairradiated single crystals of 1-methylthymine. J. Chem. Phys. 1975, 62, 370−375. (24) Born, M. Volume and hydration warmth of ions. Z. Phys. 1920, 1, 45−48. (25) Hole, E. O.; Nelson, W. H.; Sagstuen, E.; Close, D. M. Electron paramagnetic resonance and electron nuclear double resonance studies of x-irradiated crystals of cytosine hydrochloride. Part I. Free radical formation at 10 K after high radiation doses. Radiat. Res. 1998, 149, 109−119. (26) Sevilla, M. D.; Bernhard, W. A., Mechanism of Direct Radiation Damage to DNA. In Radiation Chemistry: From Basics to Applications in Materials and Life Sciences; Spotheim-Maurizot, M., Mostafavi, M., Douki, T., Belloni, J., Eds.; EDP Sciences: Les Ulis Cedex A, France, 2008; pp 191−201. (27) Sharma, K. K.; Purkayastha, S.; Bernhard, W. A. Unaltered free base release from d(CGCGCG)2 produced by the direct effect of ionizing radiation at 4 K and room temperature. Radiat. Res. 2007, 167, 501−507. (28) Hole, E. O.; Nelson, W. H.; Close, D. M.; Sagstuen, E. ESR and ENDOR study of the guanine cation: Secondary product in 5′-dGMP. J. Chem. Phys. 1987, 86, 5218−5219. (29) Wang, W.; Razskazovskii, Y.; Sevilla, M. D. Secondary radical attack on DNA nucleotides: reaction by addition to DNA bases and abstraction from sugars. Int. J. Radiat. Biol. 1997, 71, 387. (30) Close, D. M.; Nelson, W. H.; Sagstuen, E. Radical formation in x-irradiated single crystals of guanine hydrochloride monohydrate. II, ESR and ENDOR in the range 10−77 K. Radiat. Res. 1987, 112, 283− 301. (31) Close, D. M.; Nelson, W. H. ESR and ENDOR study of adenosine single crystals x-irradiated at 10 K. Radiat. Res. 1989, 117, 367−378. (32) Close, D. M.; Nelson, W. H.; Sagstuen, E.; Hole, E. O. ESR and ENDOR study of single crystals of deoxyadenosine monohydrate xirradiated at 10 K. Radiat. Res. 1994, 137, 300−309. (33) Close, D. M.; Sevilla, M. D.; Coleman, N. In Memorium. Radiat. Res. 2012, 175, 101−103.

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DNA Damage by the Direct Effect of Ionizing Radiation: Products

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