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RADIATION RESEARCH 186, 000–000 (2016)
0033-7587/16 $15.00
_2016 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR14476.1
Base Release and Modification in Solid-Phase DNA
Exposed to Low-Energy Electrons
Surakarn Choofong,1 Pierre Cloutier, Le´on Sanche and J. Richard Wagner2
Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences,
Universite´ de Sherbrooke, Sherbrooke, Quebec, Canada
Choofong, S., Cloutier, P., Sanche, L. and Wagner, J. R.
Base Release and Modification in Solid-Phase DNA Exposed
to Low-Energy Electrons. Radiat. Res. 186, 000–000 (2016).
Ionization generates a large number of secondary lowenergy
electrons (LEEs) with a most probable energy of
approximately 10 eV, which can break DNA bonds by
dissociative electron attachment (DEA) and lead to DNA
damage. In this study, we investigated radiation damage to
dry DNA induced by X rays (1.5 keV) alone on a glass
substrate or X rays combined with extra LEEs (average
energy of 5.8 eV) emitted from a tantalum (Ta) substrate
under an atmosphere of N2 and standard ambient
conditions of temperature and pressure. The targets
included calf-thymus DNA and double-stranded synthetic
oligonucleotides. We developed analytical methods to
measure the release of non-modified DNA bases from
DNA and the formation of several base modifications by
LC-MS/MS with isotopic dilution for precise quantification.
The results show that the yield of non-modified bases
as well as base modifications increase by 20–30% when
DNA is deposited on a Ta substrate compared to that on a
glass substrate. The order of base release (Gua . Ade .
Thy ; Cyt) agrees well with several theoretical studies
indicating that Gua is the most susceptible site toward
sugar-phosphate cleavage. The formation of DNA damage
by LEEs is explained by DEA leading to the release of nonmodified
bases involving the initial cleavage of N1-C10,
C30-O30 or C50-O50 bonds. The yield of base modifications
was lower than the release of non-modified bases. The
main LEE-induced base modifications include 5,6-dihydrothymine
(5,6-dHT), 5,6-dihydrocytosine (5-dHU), 5-hydroxymethyluracil
(5-HmU) and 5-formyluracil (5-ForU). The
formation of base modifications by LEEs can be explained
by DEA and cleavage of the C-H bond of the methyl group
of Thy (giving 5-HmU and 5-ForU) and by secondary
reactions of H atoms and hydride anions that are
generated by primary LEE reactions followed by subsequent
reaction with Cyt and Thy (giving 5,6-dHU and 5,6-
dHT). _ 2016 by Radiation Research Society
INTRODUCTION
Ionizing radiation, such as vacuum ultraviolet (UV), X
rays and gamma rays, induces a wide variety of DNA
damage, including single-strand breaks (SSB), doublestrand
breaks (DSB), modifications of purine and pyrimidine
bases, sugar damage, apurinic/apyrimidic sites, base
release, DNA-DNA and DNA-protein crosslinks (1–7). The
effects of ionizing radiation arise from either the indirect
effect, which involves the generation of reactive species
such as hydroxyl radicals by the radiolysis of water, or the
direct effect, which involves the absorption of radiation
energy directly by the target molecule leading to its
excitation or ionization. In the initial energy-depositing
events, ionization produces a large number of secondary
low-energy electrons (LEEs), which have a most probable
energy of less than 10 eV (8). The later species can cause
both indirect and direct mediated damage to DNA. LEEs
with energy below 15 eV can temporarily attach to specific
orbitals localized on DNA and lead to the formation of
molecular transient negative ions (TNIs) (9). These
metastable molecular anions can dissociate into an anion
and a neutral radical (e– ‏ AB ! AB*– ! A ‏ B–) (9) on a
short time scale by a process known as dissociative electron
attachment (DEA).
There are approximately 3 3 104 electrons generated per
MeV of deposited energy with a kinetic energy below 30 eV
(10). Previously, LEEs were shown to induce fragmentation
of the DNA backbone by the formation of SSB or DSB after
irradiation of supercoiled plasmid molecules under ultrahigh
vacuum followed by gel electrophoresis as a sensitive
method for the detection of conformational changes (11–
17). The type of damage was identified in greater detail by
chemical analysis of LEE-induced products remaining
within the irradiated DNA films. Zheng et al. (11, 18, 19)
reported two main reactions: the release of non-modified
bases and the formation of strand breaks using short
Editor’s note. The online version of this article (DOI: 10.1667/
RR14476.1) contains supplementary information that is available to
all authorized users.
1 Scholar in training.
2 Address for correspondence: Department of Nuclear Medicine
and Radiobiology, Faculty of Medicine and Health Sciences,
Universite´ de Sherbrooke, Sherbrooke, 3001, 12th Avenue North,
Quebec, Canada J1H 5N4; email: richard.wagner@usherbrooke.ca.
0
oligonucleotides as models of LEE-induced DNA damage.
The formation of strand breaks involved direct cleavage of
the phosphodiester bond leaving a non-modified phosphate
group at one terminus and a modified as yet unidentified
residue on the other terminus. Based on these results, we
have proposed that LEEs are initially captured by DNA
bases, giving a transient negative ion (TNI), which is then
transferred to an antibonding orbital of either the N1-C1
bond, leading to dissociation of this bond and prompt base
release, or the sugar-phosphate moiety, leading to cleavage
of the C-O bond at either the C50 or C30 positions of the
deoxyribose ring. These reactions are extensively supported
by published theoretical investigations (20–22). In similar
published studies using pure sources of LEEs, several base
modifications have been identified that were induced by the
reaction of LEEs with TTT, including 5,6-dihydrothymine
(5,6-dHT), 5-hydroxymethyluracil (5-HmU) and 5-formyluracil
(5-ForU) (23, 24).The role of LEE-induced DNA
damage during the exposure of cells to ionizing radiation
remains difficult to evaluate. Because LEEs are very
reactive and have a short interaction range (;10 nm),
electron beams can only be formed under vacuum
conditions. Thus, irradiation of solid DNA films with such
beams greatly restricts the chemical reactions of primary
reactive species. However, it is essential to probe the initial
chemical steps that are common under both extreme and
ambient conditions to extrapolate the mechanisms of LEEinduced
DNA damage to those occurring in cells. With this
approach in mind, we have devised a novel system to
demonstrate the reactivity of LEEs under standard ambient
conditions of temperature and pressure (SATP) (26). This
system also allows for the introduction of primary reactants,
such as H2O and O2, which can change the course of
reaction of the initial radical and ionic species believed to be
generated by LEE reactions. In this system, liquid DNA
samples are freeze dried as a thin film onto either glass or
tantalum (Ta) substrates. After exposure to X rays (1.5 keV)
under SATP, the formation of DNA damage on the glass
substrate can be attributed solely to the interaction of X-ray
photons, whereas damage on the Ta substrate can be
attributed to both the direct interaction of X rays and the Xray-
induced emission of LEEs from the Ta substrate. The
emitted electron distribution exhibits a peak at 1.4 eV and
an average energy of 5.85 eV. For comparison, we
previously reported the enhancement of SSB, DSB, and
DNA-DNA crosslinks induced by LEEs and 1.5 keV X rays
when supercoiled plasmid DNA was deposited on a Ta
substrate under vacuum conditions (27). Afterwards, the
supercoiled plasmids were irradiated in air (28) and under a
nitrogen atmosphere at SATP (25, 26). More detailed
studies with supercoiled plasmid DNA have been performed
by introducing specific primary reactants during irradiation.
Interestingly, the addition of H2O, O2 and N2O into this
system greatly enhanced the formation of SSB and DSB,
particularly for DNA irradiated on a Ta surface with the
emission of LEEs (27–32). For the current study, we
continued similar investigations using linear double-stranded
DNA from calf thymus and oligonucleotides, as a target
under SATP conditions. Instead of analyzing conformational
modifications to DNA as in previous investigations,
we used liquid chromatography (LC) and tandem mass
spectrometry (MS/MS) to identify specific products remaining
on the surface of the irradiated glass and Ta
substrates; these include base release and base modifications.
MATERIALS AND METHODS
Labeled 20-deoxyadenosine (15N5), 20-deoxyguanosine (15N5), cytosine
(15N3), thymine (15N2) and 8-oxo-7,8-dihydro-20-deoxyguanosine
(15N5) were purchased from Cambridge Isotope Laboratories, Inc.
(Tewksbury, MA). Labeled adenine (15N5) and guanine (15N5) were
prepared from the corresponding 20-deoxynucleoside by hydrolysis in
1% of formic acid at 378C for 24 h followed by purification and
quantification of the nucleobase on reversed-phase HPLC. Labeled
5,6-dihydro-20-deoxythymidine (the nucleoside of 5,6-dHT) and 5,6-
dihydro-20-deoxyuridine (the nucleoside of 5,6-dHU) were prepared
by radiolysis, following a previously published procedure (33).
Briefly, 10 mM (15N2)-thymidine and (15N3)-20-deoxycytidine in 10
ml deaerated (i.e., bubbled with N2 for 30 min) aqueous solution were
exposed to gamma radiation (8,000 Gy, dose rate 10.7 Gy/min) in the
presence of cysteine (0.5 mM). Quantification of these compounds
was determined by HPLC-UV using an authentic nonlabeled standard
of the nucleoside of 5,6-dHT assuming that 5,6-dHT and 5,6-dHU
have the same UV absorption at 220 nm. For 5-hydroxymethy-20-
deoxyuridine and 5-formyl-20-deoxyuridine (the nucleosides of 5-
HmU and 5-ForU), the synthesis of isotopic internal standards has
been reported elsewhere (34). A number of experiments were
performed with linear double-stranded calf thymus DNA (CT-DNA;
Sigma Canada/Gentec International, Markham, Canada). CT-DNA
was prepared by extraction and precipitation for the removal of
proteins and other impurities to obtain a stock solution of purified CTDNA
in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The CT-DNA
concentration was determined by measuring the ratio of UV
absorption at 260. The ratio of absorption at 260–280 nm was 1.95
indicating that the amount of protein remaining in DNA was less than
10%. Oligonucleotides (ODNs) were purchased from Alpha DNA
(Montreal, Canada) and purified before use by HPLC-UV. To produce
double-stranded ODN-AB, ODN A [50-TGA CTG CAT ACG CAT
GTA GAC GAT GTG CAT-30 (30-mer)] was mixed with an equal
amount of ODN B [50-ACT GAC GTA TGC GTA CAT CTG CTA
CAC GTA-30 (30-mer)] and hybridized in a PCR thermal cycler.
Similarly, double-stranded DNA was produced from self-complementary
ODN-DDD [CGC GAA TTC GCG; Dickerson-Drew dodecamer
(12-mer)].
Irradiation of DNA
Thin films of DNA were prepared by depositing a solution of 12 ll
of DNA containing 600 ng of DNA, which was diluted 200–500-fold
from concentrated stock DNA solution with Nanopuree water. DNA
was deposited on either a glass or Ta substrate followed by
lyophilization according to the method of Alizadeh et al. (35). Film
thickness was approximately 10 nm, which corresponds to 5
monolayers of DNA assuming a density of DNA of 1.7 g/cm3 (36).
The surface diameter of the films was 6.0 6 0.2 mm. Before
depositing the DNA solution, the glass and Ta substrates were
carefully washed with ethanol and H2O, and dried over N2 gas. The
samples were then frozen at_608C and lyophilized into a solid film by
opening a vacuum chamber under low pressure (5 mTorr). The
instrument used to irradiate lyophilized DNA samples under SATP is
0 CHOOFONG ET AL.
described in detail elsewhere (32, 35, 37). Briefly, an X-ray source in a
stainless steel chamber is evacuated down to 5 mTorr by a mechanical
pump. A nitrogen plasma discharge with an 8 mA current is applied
between a cold-cathode and an aluminum foil target to generate Ka X
rays with energy of 1.5 keV. The X rays traverse a flight tube that is
continuously flushed with helium gas followed by a thin envelope of
Mylare and impinge on the target in a small chamber filled with N2.
The humidity in the irradiation chamber is near zero, as monitored by
a hygrometer. In the irradiation chamber, 72 lyophilized DNA films
are arranged for X-ray exposures at varying times. During the
irradiation cycle, three samples for both glass and Ta substrates served
as controls; these were prepared and stored under exactly the same
conditions as irradiated samples except that they were not exposed to
X rays. The remaining samples were exposed in triplicate to X rays on
a glass and Ta substrate for 1.5 and 3 h. To electrically grounded
samples irradiated on Ta, a small drop of silver was placed at the
corner of the Ta substrate. A Gafchromice dosimetry media type HDV2
film (Ashlande, Bridgewater, NJ) was used as a dosimeter. The
density of Gafchromic films was measured using an HPt ScanJet
4400C instrument (HP Inc., Palo Alto, CA) with an output resolution
of 300 dpi in true color as analyzed with ImageJ software (National
Institutes of Health, Bethesda, MD) (38). The density of film was
plotted versus the irradiation time (min) to give a slope of 0.0393
min_1, which was used to convert the irradiation time to photon
fluence (photon/cm2) (32). The calibration factor was reduced by 0.41
due to the addition of a thin Mylar envelope (6 lM) on top of the
Gafchromic film during irradiation to prevent photon scattering from
soft X rays.
Preparation of Samples for Analysis
DNA was recovered from the substrates and processed for either
base release or base modifications. For base release, 2 3 15 ll of TE
buffer was used to redissolve the dried drop of DNA on top of the
glass or Ta substrate. The contents of four dried drops of DNA were
combined to give a total of 2,400 ng of DNA. The sample of DNA
was diluted with TE buffer to 120 ll. Stable isotopic standards of the
four DNA nucleobases were added with the final concentration of 0.05
(Cyt), 0.05 (Gua), 0.04 (Thy) and 0.02 lM (Ade). DNA was
precipitated by the addition of glycogen 0.1 mg_ml_1 (20 ll), H2O (220
ll) and 0.3 M acetate solution at pH 5 (40 ll) followed by brief
vortexing and the addition of isopropanol (1 ml). To maximize
precipitation of DNA, the sample was stored at_208C for 1 h and then
centrifuged at 10,000g for 30 min at 48C. The supernatant was
withdrawn and dried by evaporation on a Savante SpeedVace
system. The natural DNA nucleobases, as well as the isotopic
standards, were extracted twice from the dried residue with 300 ll of
acetonitrile followed by sonification, centrifugation and evaporation of
the acetonitrile fraction. The samples were kept at _208C until they
were dissolved in 100 ll of 0.01% acetic acid immediately before
analysis by LC-MS/MS.
In the case of base damage analysis, 12.5 ll of a solution containing
P1 nuclease enzyme (1 unit_ll_1) in acetate buffer (50 mM, pH 5.0)
was added to the dried drop of DNA and incubated for 30 min before
initial recovery and subsequent washing with additional 12 ll. This
step was incorporated into the analyses because it gave more
consistent recovery of DNA, likely due to the sticking or binding of
DNA molecules onto the substrate surface. As for base release, four
dried spots of DNA were combined to obtain a total of 2,400 ng (50
ll). The appropriate isotope standards of 5,6-dHT (0.2 lM), 5,6-dHU
(0.5 lM), 5-HmU (0.2 lM), 5-ForU (0.2 lM) and 8-oxoG (0.005 lM)
were added to DNA sample before P1 digestion. The sample was dried
by evaporation on a SpeedVac system and redissolved in 35 ll H2O.
After adjusting to pH 7.5 by the addition of 5 ll of 50mM ammonium
bicarbonate, DNA was further digested with 2.5 ll of snake venom
phosphodiesterase (0.002 unit/ll) and 2.5 ll of alkaline phosphatase
(1 unit/ll) followed by incubation for 1 h at 378C. Then, 5 ll of 120
mM acetic acid was added to lower the pH to 5 before analysis by LCMS/
MS.
Quantification of the Yields by LC-MS/MS
The products from DNA damage were separated by LC (Nexera X2
system; Shimadzu, Kyoto, Japan) equipped with binary solvent
delivery pumps (LC-30AD), an autosampler (SIL-30AC), a column
oven (CTO-20AC) and a UV/Vis detector (SPD-20A) operating at 220
and 260 nm absorbance. Two types of reversed-phase columns were
used. For the separation of base release products (Cyt, Thy, Gua and
Ade), we used an HSS T3 with 1.8 lm particle size; 100 3 2.1 mm
(i.d.) protected by a VanGuarde precolumn (Waterst Corp., Milford,
MA), whereas for the separation of base modification products (8-
oxoG, 5-HmU, 5-ForU, 5,6-dHU and 5,6-dHT), we used an
Acclaime Polar Advantage II C18 with 5 lm particle size, 120 A°
25032.1 mm (i.d.) (Thermo Fisher Scientifice Inc., Waltham, MA)
protected by a precolumn of the same material. The temperature of the
column was maintained at 308C. Different gradients were used for the
separation of base release and base modification products. For base
release products, the mobile phase was a gradient prepared from
0.01% (v/v) acetic acid (pH 3.5) (solvent A) and 50:50 methanol:
acetonitrile (solvent B). Mobile phase composition was increased to
10% solvent B within 5 min and raised to 70% in 1 min and held for 4
min. Subsequent reconstitution of the starting conditions in 1 min and
re-equilibration with 0% solvent B for 4 min resulted in a total
analysis time of 15 min at a total flow rate of 0.5 ml/min. The
separation was followed by a wash cycle with solvent B and reequilibrated
period of 1 min before the next injection. The injection
volume of samples was 30 ll. For base modifications, the gradient
started at 90% solvent A [5 mM ammonium formate (pH 5.0)] and
10% solvent B (90% acetonitrile) and increased linearly to 30%
solvent A and 70% solvent B at a total flow rate of 0.16 ml/min. The
volume of injection was 30 ll. For both base release and base
modifications, the compounds were quantified by tandem MS. The
system consisted of an API 3000 tandem mass spectrometer system
(MS/MS) operating with a turbo-ion spray source (AB-Sciex,
Concord, Canada). Nitrogen was the nebulizing gas collision gas.
The products together with the corresponding isotopic standards were
detected by multiple reaction monitoring (MRM) of preselected
molecular and fragment ions using the positive ionization mode.
Parameters for LC-MS/MS measurement, including MRM transitions,
retention times and collision energies are summarized in Table 1, and
calibration curves showing a linear response at different mixtures of
natural and isotopic label are shown in Supplementary Fig. S1 (http://
dx.doi.org/10.1667/RR14476.1.S1).
TABLE 1
LC-MS/MS Parameters for the Analysis of DNA
Damage
Products
MRM transition (Da)
Retention
time (min)
Collision
energy (eV)
Natural
standard
Isotopic
standard
Free base release
Cyt 112.2/95.1 115.1/97.0 0.6 30
Thy 127.0/110.1 131.2/114.0 2.7 25
Ade 136.1/111.1 141.0/123.0 1.9 30
Gua 152.0/135.0 137.1/125.1 1.7 30
Modified base
5,6-dHT 245.1/117.1 247.0/117.1 12.2 15
5,6-dHU 230.6/117.1 233.0/117.1 13.1 20
5-HmU 259.1/125.0 261.1/127.0 9.4 15
5-ForU 257.0/141.0 259.0/143.0 11.3 15
8-oxoG 284.2/168.1 289.2/173.1 11.9 15
LEE-INDUCED DNA DAMAGE 0
Calculation of the Yield of Damage
The amount of product in pmol was estimated from the ratio of
integrated peak areas obtained for the natural and isotopic standards
with the same retention time multiplied by the concentration of
isotopic standard added to the sample during sample preparation. The
yields of damaged DNA were determined by the slope of a linear
regression of the total moles of product (pmol) versus photon fluence
(photons_cm_2) and then converted to nmol/J by Eq. (1). The yield of
products induced by LEEs was taken as the difference in the yield of
products between that produced on a glass substrate compared to that
on a Ta substrate. Equation 1 was used to convert units from nmol per
1014 photons/cm2 to nmol/J, where A is the yield in nmol, r is an
average radius of a drop 0.3 cm; the X-ray energy is 1,486 eV.
nmol
J
¼
A nmol
1014 photons
_
pr2
1 cm2 _
1 photon
1486 eV
_
1 eV
1:6310__19 J
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RESULTS
Double-stranded CT-DNA was exposed to soft X rays as
a thin dry film on either a glass or Ta substrate under a N2
atmosphere and 0% relative humidity. The glass substrate
mimics the direct effects of X rays, while the Ta substrate
enhances the effects of LEEs, generated by the interaction
of X rays with DNA, by producing an additional
distribution of low-energy photoelectrons emanating from
the Ta surface.
We examined the release of non-modified DNA nucleobases
(Cyt, Thy, Ade and Gua) and the formation of base
modifications including: 7,8-dihydroguanine (8-oxoG); 5-
hydroxymethyluracil (5-HmU); 5-formyluracil (5-ForU);
5,6-dihydrothymine (5,6-dHT); and 5,6-dihydrouridine
(5,6-dHU). A total of seven products were measured in
our analyses (Fig. 1). The release of four DNA bases was
measured by LC-MS/MS analysis of nucleobases in the
supernatant of irradiated samples after precipitation of DNA
(Fig. 2). The formation of base damage was measured in
parallel by initial enzymatic hydrolysis of DNA to its
component nucleosides followed by analysis of this mixture
by LC-MS/MS (Fig. 3). In both cases, the appropriate
isotopic standards were added before sample workup to take
into account the loss of products during subsequent steps in
the preparation of samples for analysis and variation in the
detection efficiency by mass spectrometry. Thereby, the
yield of product was precisely determined by comparison of
FIG. 1. The products measured in our analyses.
FIG. 2. Analysis of base release by LC-MS/MS performed by multiple-reaction monitoring in the positive
mode (Table 1). The natural products (solid line) and isotope standards (dashed line) are shown for the analyses
of cytosine (Cyt), thymine (Thy), adenine (Ade) and guanine (Gua).
0 CHOOFONG ET AL.
the ion signals for the natural and isotopic compound during
the same chromatographic run.
To determine the radiolytic yields of DNA damage, the
amount of base release and base modifications was
measured as a function of photon fluence (Fig. 4). For
these analyses, thin DNA films were irradiated and pooled
(4 3 0.6 lg ¼ 2.4 lg) in triplicate at 0, 1.5 and 3 h. When
extended to longer irradiation times (0–6 h), the formation
of products remained linear with dose. As shown in Fig. 4,
the results using three time points fit well to a linear
function (R2 . 0.85). Thus, we assume that all products
were formed by a single photon or electron event.
Comparisons of yields from samples that were simply
deposited on the Ta substrate without connection to an
electronic ground showed that grounding the substrate
increased the yield of products by approximately 10%. The
increase of damage observed in grounded compared to nongrounded
samples can be explained by the recombination of
ejected electrons and electron holes of Ta in the nongrounded
case. When the sample is grounded, however, the
initial holes instantaneously combine, since Ta is a metal
and a good conductor. Thus, the current studies were
performed with electrically grounded substrates. An overall
assessment of the results indicates that damage to DNA was
20–30% greater when irradiated on the Ta substrate
compared to the glass substrate (Table 2). These results
are in general agreement with previously published studies
using the same system, but with alternative targets (29, 32,
35, 37). While the yield difference between the two
substrates can be attributed only to LEEs, the amount of
damage on the glass substrate arises from both direct
ionization of DNA and the reaction of LEEs generated in
step with ionization. It has been estimated that 0.2% of the
incident soft X-ray photons interact with a 10 nm thick
FIG. 3. Analysis of base modifications by LC-MS/MS performed by multiple-reaction monitoring in the
positive mode (Table 1). The natural products (solid line) and isotope standards (dashed line) are shown for the
analyses of 8-oxo-7,8-dihydroguanine (8-oxoG), 5-hydroxymethyl-20-deoxyuridine (5-HmU), 5-formyl-20-
deoxyuridine (5-ForU), 5,6-dihydro-20-deoxythymidine (5,6-dHT) and 5,6-dihydro-20-deoxyuridine (5,6-dHU).
LEE-INDUCED DNA DAMAGE 0
DNA film and that approximately half of the energy of these
photons is absorbed by the film (32, 39). On the other hand,
the interaction of X rays with Ta generates LEEs (average
energy ¼ 5.8 eV) with an efficiency of approximately
0.047% (35). Assuming that all of the secondary electrons
emitted by the Ta substrate are absorbed by the DNA film
(35), one can calculate that there is fivefold greater energy
absorbed by the interaction of X rays with DNA compared
to the energy delivered to DNA by secondary electrons
generated by the interaction of X rays with Ta. In addition,
it should be noted that 95% of photo-emitted electrons from
the Ta surface have energies below 30 eV (26).
The total yield of base release (Cyt, Thy, Ade and Gua)
was 60.4 and 83.6 nmol/J after irradiation of CT-DNA on
glass and Ta substrate, respectively (Table 2). Similar
results were obtained with two double-stranded oligonucleotides
with defined sequences [ODN-AB (30-mer) and
ODN-DDD (12-mer)]. The contribution of LEE for base
release from DNA target molecules followed the order of
Gua . Ade . Thy ; Cyt. The difference between glass
and Ta substrates for CT-DNA indicated a slightly higher
release of Gua (8.3) and Ade (8.0) compared to that of Thy
(3.6) and Cyt (3.3). In DNA oligomers, however, LEEinduced
release of Gua was at least twofold greater than that
of Ade [5.5 compared to 2.1 nmol/J for ODN-AB (30-mer)
and 9.2 compared to 3.9 nmol/J for ODN-DDD (12-mer)
(Table 2)]. The total yield of base modifications within CTDNA,
including 5,6-dHU, 5,6-dHT, 5-HmU, 5-ForU and 8-
oxoG, was 38.6 and 43.4 nmol/J after irradiation of DNA on
a glass and Ta substrate, respectively (Table 3). Again,
similar results were obtained for the oligonucleotides. The
major products of direct irradiation on glass as well as on Ta
substrates were 5,6-dHU and 5-Meth*. It should be noted
that 5,6-dHU arises from the deamination of the corresponding
5,6-dihydrocytosine products in aqueous solution,
and thus, it is the ultimate reduction product of Cyt in DNA.
Interestingly, the yield of 5,6-dHT was approximately
fivefold less than that of 5,6-dHU. The formation of 5-HmU
and 5-ForU from the oxidation of Thy in DNA was
combined (5-Meth*) because they arise from the same
intermediate and the ratio of each product largely depends
on subtle variations in sample preparation (see Discussion).
In contrast to base modifications, 8-oxoG was a minor
product representing ,6% of the total products.The total
yield of base release compares fairly well with previously
reported yields of base release of 73 and 67 nmole/J using
dry salmon sperm DNA and M. luteus DNA, respectively,
in the solid state, under similar conditions of hydration and
FIG. 4. Graphs representing the total formation of products in DNA
films deposited on glass and tantalum (Ta) substrates as a function of
photon fluences. Panel A: Base release of guanine. Panel B: Base
modification giving 5,6-dihydro-20-deoxyuridine (5,6-dHU).
TABLE 2
Yields of Base Release Products from DNA
Type of ODN Products
nmol/J
X rays X rays ‏ LEEs LEEs
CT-DNA Cyt 6.8 6 1.5 10.1 6 0.7 3.3
Thy 3.5 6 0.5 7.1 6 0.9 3.6
Ade 16.5 6 2.5 24.5 6 0.1 8.0
Gua 33.6 6 4.8 42.0 6 5.6 8.3
Total 60.4 6 9.4 83.6 6 7.3 23.2
ODN-AB Cyt 9.2 6 0.2 11.4 6 0.6 2.2
Thy 3.1 6 0.4 4.8 6 0.5 1.6
Ade 33.2 6 0.5 35.3 6 6.3 2.1
Gua 34.1 6 4.5 39.6 6 1.2 5.5
Total 79.6 6 0.0 91.0 6 0.0 11.5
ODN-DDD Cyt 5.7 6 0.7 6.8 6 1.7 1.1
Thy 2.0 6 0.3 3.2 6 0.5 1.3
Ade 12.4 6 2.5 16.3 6 2.5 3.9
Gua 17.4 6 5.2 26.5 6 1.6 9.2
Total 37.4 6 8.7 52.8 6 6.3 15.5
Notes. G values (nmol/J) for CT-DNA (dsDNA), ODN-AB (30-
mer, dsDNA) and ODN-DDD (12-mer, dsDNA) from X rays (glass
substrate), X rays plus LEEs (Ta substrate) and LEEs only (glass
minus Ta substrates) for free bases release. Average of three
independent experiments (n ¼ 3) with standard deviation. LEEs ¼ X
rays – X rays ‏ LEEs. Statistical significance: P , 0.05.
0 CHOOFONG ET AL.
irradiation (40, 41). The lower base release values in our
study compared to other published studies may be attributed
in part to the delayed release of additional DNA bases after
incubation of DNA after irradiation (40). In the latter study,
the authors reported an increase of 25% in Cyt and 40% in
Thy but no significant increases in either of the purine bases
after further incubation of DNA for 5 days at 228C. Delayed
base release exemplified by hydroxyl radical-induced DNA
damage arises from the formation of initial radicals on the
sugar moiety followed by subsequent cleavage of the Nglycosidic
bond through a series of relatively slow reactions
(hours to days) (42). Assuming that a similar conversion of
sugar radicals leading to release of the nucleobase occurs in
our samples, one can expect an increase in Cyt and Thy
yields after irradiation incubation of DNA. Although we
attempted to examine delayed base release, the results were
inconsistent because of interference with the spontaneous
release of DNA bases. Thus, we measured only immediate,
prompt release of non-modified DNA bases, noting that this
likely represents the majority of base release.
DISCUSSION
Base release is a well-studied phenomenon in radiationinduced
DNA damage. It is believed to arise from ionization
of the sugar moiety leading to an electron hole that
subsequently induces cleavage of the N-glycosidic bond and
release of the non-modified DNA bases [Fig. 5, pathway A
(1)]. Interestingly, the yield of organic radicals detected on
the deoxyribose moiety in DNA at 48K is several-fold less
than the yield of base release, referred by Bernhard and
colleagues as the shortfall (41, 43–45). This has been
interpreted by implicating redox reactions between carboncentered
radicals of the deoxyribose with reactive DNA
base radicals or in some cases with guanine, which has the
lowest oxidation potential of the four normal bases. Similar
reactions may occur in our irradiation system. In addition,
there are alternative pathways that may explain base release
by the reaction of LEEs with DNA involving DEA. These
reactions most likely involve initial cleavage of the Nglycosidic
bond, the C50-O50 bond or the C30-O30 bond as
shown in Fig. 5, pathway B (19, 20). Each of these reactions
leads to a neutral radical and a ground state anion. Nglycosidic
bond cleavage most likely gives a nucleobase
anion together with a neutral carbon-centered radical at C10
of the deoxyribose moiety. In contrast, both C-O bond
cleavage reactions likely give a neutral carbon-centered
radical at either C30 or C50 of the deoxyribose moiety
together with a corresponding phosphate anion because of
the high electronegativity of the phosphate group. Although
the fate of C30 and C50 centered radicals arising from the CO
bond cleavage in DNA is not known, it is possible that
they also lead to base release through subsequent radical
and ion reactions. In theoretical studies, the above reactions
have been proposed on thermodynamic arguments, showing
a relatively low barrier particularly toward the C30-O30
cleavage compared to the other reactions with very lowenergy
electrons (,2 eV) (46–48). The resulting carboncentered
radicals likely do not survive in the solid state at
room temperature and subsequently undergo redox reactions
into stable products. The release of Thy and other
DNA bases from short oligonucleotides using pure sources
TABLE 3
Yields of Base Modification Products from DNA
Type of ODN Products
nmol/J
X rays X rays ‏ LEEs LEEs
CT-DNA 8-oxoG 0.5 6 0.0 0.8 6 0.1 0.3
5-Methb 13.1 6 2.1 14.6 6 2.6 1.4
5,6-dHT 4.4 6 0.7 4.5 6 1.0 0.1
5,6-dHU 20.6 6 2.3 23.5 6 2.1 3.0
Total 38.6 6 5.1 43.4 6 0.7 4.8
ODN-AB 8-oxoG 0.2 6 0.0 0.2 6 0.0 0.0
5-Methb 7.6 6 0.9 10.2 6 3.5 2.6a
5,6-dHT 4.1 6 0.9 5.4 6 0.9 1.4a
5,6-dHU 8.1 6 1.2 11.3 6 1.0 3.2
Total 20.0 6 3.0 27.1 6 1.4 7.1
ODN-DDD 8-oxoG 0.1 6 0.0 0.2 6 0.0 0.0
5-Methb 1.4 6 0.1 2.4 6 0.1 1.1
5,6-dHT 1.5 6 0.1 1.6 6 0.1 0.1
5,6-dHU 7.7 6 0.3 9.7 6 0.5 2.0
Total 10.7 6 0.1 13.9 6 0.2 3.2
Notes. G values (nmol/J) for CT-DNA (dsDNA), ODN-AB (30-
mer, dsDNA) and ODN-DDD (12-mer, dsDNA) from X rays (glass
substrate), X rays plus LEEs (Ta substrate) and LEEs only (glass
minus Ta substrates) for base modification products. Average of three
independent experiments (n ¼ 3) with standard deviation. LEEs ¼ X
rays – X-rays ‏ LEEs. Statistical significance: P , 0.05; aP ¼ 0.05–
0.10; b5-Meth ¼ 5-HmU plus 5-ForU.
FIG. 5. Proposed pathways of base release from DNA by initial
ionization (panel A) and by reaction with LEEs (panel B). Note that
deprotonation of the 2-deoxyribose radical cation arising from
ionization may occur from several sites resulting in neutral carboncentered
radicals, which in turn transform into modified 2-deoxyribose
moieties with liberation of non-modified nucleobases. In the case of
LEE reactions, the 2-deoxyribose radicals are different from those
arising from ionization, which probably gives carbon-centered radicals
at C10 (N-C cleavage), C30 (C30-O) cleavage and C50 (C50-O). The
latter radicals may then transform to give base release directly via C10-
centered radicals or indirectly through C30- and C50-centered radicals.
LEE-INDUCED DNA DAMAGE 0
of LEEs may also be explained by a competition of different
DEA processes (18, 19, 23, 49, 50). Here, we show that
base release is a predominant reaction of DNA exposed to
soft X rays and that it is enhanced by 14–40% when soft X
rays are combined with LEEs generated at the DNA-Ta
interface. Using the same system, we previously reported
that Thy was the major LEE-induced product of thymidine
(286 nmol/J) (37). The high yield of base release with
thymidine as a target may be attributed to highly efficient
base release from monomers and short oligonucleotides
observed using LEE-generating systems (18, 19, 23, 49–
51). In comparison, the total yield of LEE-induced release
of non-modified bases from DNA varied between 11.5–23.2
nmol/J indicating that base release is greatly suppressed in
DNA compared to short oligonucleotides. The more
efficient base release from short oligonucleotides and
monomeric targets can be attributed to the lack of
competitive channels for the decay of initial transient
anions and the more efficient base release from the termini
of small molecular weight targets (19, 49).
As shown in Table 2, LEE-induced base release was
largest for Gua with 48% of the total base release products,
followed by Ade (26%), Thy (13%) and Cyt (14%) taking
the average values for three DNA targets. This is consistent
with the more favorable capture of LEEs and corresponding
increases of damage in DNA sequences with an increasing
number of G bases (52–54). Guanine appears to have the
greatest ability to capture electrons because of its high
dipole moment and energies of TNI states. On the basis of
LEE (,2 eV) transmission yields, Ray et al. concluded that
electron capture is proportional to the number of G bases in
short DNA oligomers, and that the electron does not stay on
the bases per se, but rather is transferred to the sugarphosphate
backbone. Transfer of the electron to the
phosphodiester bond is supported by theoretical studies
indicating a low-energy barrier for transfer of the electron
from a transient anion of Gua within a nucleotide
monophosphate unit to an antibonding r*-orbital of the
C30-O30 bond, leading to favorable cleavage of this bond.
Indeed, transient anions of DNA bases have been directly
observed with presolvated electrons in aqueous solution
with fs laser photolysis, of which Gua anions were most
likely to undergo dissociation (55). A recently published
theoretical study predicted that the energy barrier for
dissociation would be elevated for a TNI state containing
Cyt and Thy nucleotides, because they undergo protonation,
whereas the effect is less overbearing for Ade and absent for
Gua radical anions, which do not undergo protonation (54).
The C30-O30 bond appears to give the lowest barrier to
dissociation although other sites, which include C10-N1 (Nglycosidic
bond) and C50-O50, may also be considered to
reach an energetically feasible state. Our results, showing a
preference for the release of Gua, thereby support numerous
studies that have identified Gua as a weak link toward LEEinduced
phosphodiester bond cleavage.
In this work, we measured the yields of five base
modifications in DNA as a function of dose: 5-hydroxymethyluracil
(5-HmU); 5-formyluracil (5-ForU); 5,6-
dihydrouridine (5,6-dHU); 5,6-dihydrothymine (5,6-dHT),
5,6-dihydrouracil (5,6-dHT); and 8-oxoguanine (8-oxoG)
(Table 3). The major product was 5,6-dHU. The formation
of 5,6-dHU can be explained by the addition of electrons
onto Cyt of DNA followed by protonation of the Cyt anion
radical to give an intermediate 5,6-dihydro-6-cytosinoyl
radical (Fig. 6, pathway A). The latter radical likely
undergoes reduction and protonation to 5,6-dihydrocytosine.
Because loss of the 5,6-double bond of Cyt renders the
exocyclic amino group highly susceptible to deamination,
5,6-dihydrocytosine will quantitatively convert into 5,6-
dHU in aqueous solution. A similar pathway explains the
formation of 5,6-dHT from the reaction of electrons with
Thy in DNA (not shown). The formation of 5,6-dHU and
5,6-dHT may involve both thermalized and kinetically hot
electrons. Initial transient negative ions arising from the
attachment of LEEs may degenerate into more stable Cyt
and Thy anion radicals, thereby contributing to the yield of
5,6-dihydropyrimidines. In the case of LEEs, it is
reasonable to propose an alternative pathway to give 5,6-
dHU and 5,6-dHT, which involves the addition of H atoms
(H_) or hydride anions (H_) (Fig. 6, pathway B). For
example, a dominant feature of LEE interactions with DNA
bases is the release of either H atoms (H_) or hydride anions
(H_) from target molecules. In the gas phase, H_ are
generated when targets are bombarded with LEEs of ,3
eV, whereas H_ fragments are only observed at higher
energies (.5.5 eV) (56–58). In the condensed state, the
desorption of H_ and to a lesser extent diatomic fragments
of DNA bases only become apparent as a broad resonance
peak above 8 eV (59, 60). In the current system, a range of
LEEs are emitted from the Ta substrate between 0–30 eV
FIG. 6. Proposed pathways for base modifications involving the
formation of 5,6-dihydrocytosine in DNA by initial ionization (panel
A) and by reaction with LEEs (panel B). Both thermalized and
kinetically hot electrons arising from ionization can react with
pyrimidines (Cyt and Thy) to give the corresponding pyrimidine
radical anion. In contrast, the formation of 5,6-dHU by LEEs likely
involves the additional reaction of hydrogen atoms (H
_
) or hydride
anion (H_) that are generated by initial reactions of LEE with other
components of DNA involving C-H bond cleavage. The reaction is
shown only for Cyt in which the initial product (5,6-dihydrocytosine)
is susceptible to deamination in aqueous solution leading quantitatively
to the final product: 5,6-dHU.
0 CHOOFONG ET AL.
with an average energy of 5.85 eV. Thus, both H_ and H_
may be generated by initial reactions of LEEs with DNA at
the DNA-Ta interface. The reaction of H_ with Cyt and Thy
gives the same intermediates that are formed by the
deprotonation of the corresponding Cyt and Thy radical
anions, while the addition of an H_ leads directly to anionic
species that undergo protonation to give the final products
(Fig. 6, pathway B).
The next prominent type of base modifications, following
5,6-dihydropyrimidines, was 5-hydroxymethylyracil (5-
HmU) and 5-formyluracil (5-ForU) (Fig. 7). Because the
ratio of 5-HmU and 5-ForU varied between samples, and
because they arise from the same intermediate radical, 5-
(uracilyl)methyl, we combined these two products, referring
to them as methyl oxidation products of Thy (5-Meth*;
Table 3). The ratio of 5-HmU and 5-ForU in samples likely
varies because they may be exposed to different amounts of
oxygen after their removal from the irradiation compartment
and dissolution in aqueous aerated solution. The formation
of 5-ForU absolutely requires oxygen to convert intermediate
5-(uracilyl)methyl radicals into peroxyl radicals and
hydroperoxides, which subsequently undergo dehydration
into 5-ForU. In contrast, 5-HmU is likely formed in the film
by the oxidation of 5-(uracilyl)methyl radicals. Previous
electron paramagnetic resonance studies underline the
relatively long lifetime of 5-(uracilyl)methyl radicals
compared to other neutral radicals of Thy and Cyt in the
solid phase (61). Thus, one can assume that a percentage of
these radicals are either reduced or oxidized in the solid
film, giving Thy methyl anions and Thy methyl cations,
respectively. The anion reverts back to Thy while the cation
gives 5-HmU directly after hydration in aqueous solution.
Indeed, the relatively high amount of 5-ForU compared to
5-HmU indicates that a good percentage of 5-(uracilyl)-
methyl radicals accumulate in the dry film of DNA after Xray
exposures and LEEs at 228C under an atmosphere of N2.
However, we cannot rule out the possibility that 5-
(uracilyl)methyl radicals are chemically reduced by the flux
of electrons onto the dry film, restoring Thy to its initial
state. The loss of 5-(uracilyl)methyl radicals due to oneelectron
reduction may lead to an underestimation of this
pathway.The formation of 5-Meth* products may be
explained by either the deprotonation of Thy radical cations
(Fig. 7; pathway A) or the reaction of LEEs with Thy such
that the C-H bond dissociates to give a neutral 5-
(uracilyl)methyl radical and either a H_ or a Thy anion
accompanied with a H_ (Fig. 7, pathway B). There are
detailed published studies of the deprotonation of thymidine
radical cations in the solid state as well as in aqueous
solution (61, 62). While the formation of 5-(uracilyl)methyl
radicals has been reported by LEEs in the gas and
condensed phases, the resonance for C-H bond cleavage
at the methyl group is weak and appears at high energy (10–
11 eV) (63, 64). In previously published studies, our group
demonstrated the ability of LEE reactions with TTT to
produce 5-Meth* products after bombardment with monoenergetic
electrons of initial energy of 10 eV, as well as with
thymidine as a target using the same system as in the current
experiments (23, 37).Interestingly, the yield of 5,6-dHU
from Cyt in DNA was several-fold greater than that of 5,6-
dHT from Thy in DNA. The ratio of these two products
greatly varies depending on the type of DNA and level of
hydration (41, 65). Within double-stranded Cyt-rich oligonucleotides,
the ratio was approximately 10:1 in favor of the
formation 5,6-dHU (66). This effect has been attributed to
the transfer of stable radical anions from Thy to Cyt and the
relatively rapid deprotonation of Cyt radical anions by Hbonding
with Gua in double-stranded DNA (66, 67). In the
current study, the formation of 5,6-dihydropyrimidines was
biased toward the formation of 5,6-dHU after irradiation of
DNA targets on a glass substrate. However, the amount of
5,6-dHT diminished to relatively low levels when only the
contribution of LEEs was taken into consideration. This
result suggests that the bias toward the formation of 5,6-
dHU may be due to a greater tendency of TNI states of Cyt
to decay into a stable radical anion compared to those of
Thy. A possibility is that there are other more probable
pathways of decay of initial Thy TNI states, such as DEA
involving cleavage of the C-H bond of the methyl group and
formation of 5-Meth* products. A difference in the ratio of
5,6-dHU and 5,6-dHT may also reflect differences in the
reactivity of H_ and H– with DNA bases.
The yield of 8-oxoG was minor, representing only 1–2%
of the total yield of base damage under analyses (Table 3).
Although changes in 8-oxoG were observed when DNA
was irradiated on Ta compared to glass, they were relatively
small compared to other base modifications. In general, our
results indicate that 8-oxoG is not a major product of either
direct ionization or the reaction of LEEs in the solid state.
The lack of formation of 8-oxoG suggests that guanine
radical cations are diverted to alternative pathways. For
example, hydration followed by one-electron reduction can
FIG. 7. Proposed pathways for base modifications involving the
formation of methyl oxidation products (5-HmU and 5-FoU) of Thy in
DNA by initial ionization (panel A) and reaction with LEEs (panel B).
The ionization of Thy in DNA followed by deprotonation leads to the
formation of the 5-(uracilyl)methyl radical). Similarly, one can
propose that the same radical arises from the reaction of LEEs by
DEA involving C-H cleavage of the methyl group of Thy together
with the release of hydride anions (H_). The latter radical
subsequently transforms into common oxidation products of Thy: 5-
HmU and 5-FoU.
LEE-INDUCED DNA DAMAGE 0
give 2,6-diamino-4-hydroxy-5-formamidopyrimidine,
which was not measured in the current study.
CONCLUSIONS
DNA damage by LEEs was assessed under standard
ambient temperature and pressure by comparing X-ray
damage induced to dry films of linear double-stranded DNA
deposited on glass and Ta substrates. Two types of DNA
damage remaining in the films after LEE bombardment
were identified by LC-MS/MS: non-modified base release
of Cyt, Thy, Ade and Gua; and modifications of Cyt (5,6-
dHU), Thy (5,6-dHT, 5-HmU, 5-ForU) and Gua (8-oxoG)
detected as modified nucleosides. The results demonstrate
that the yields of DNA damage on Ta substrates due to the
emission of LEEs produces 20–30% more damage compared
to X rays alone. The total release of non-modified
DNA bases is approximately equal to the amount of total
base modifications. This ensemble of results indicates the
formation of common radiation-induced DNA damage
products from both ionization and LEE-induced DEAmediated
processes.
SUPPLEMENTARY INFORMATION
Fig. S1. Calibration curves for base release (panel A) and
base modifications (panel B).
ACKNOWLEDGMENTS
We thank Dr. E. Alizadeh for technical support with sample preparation
as well as radiation processing. We also thank G. Madugundu for his help
with the optimization of LC-MS/MS analysis. This work was supported by
grants from the Natural Science and Engineering Research Council of
Canada (NSERC) and the Canadian Institutes of Health Research (CIHR).
Received: March 30, 2016; accepted: July 25, 2016; published online:
Month 00, 2016
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LEE-INDUCED DNA DAMAGE 0

الهام اسودی 1