The influence of DNA concentration in the experiment of DNA damage induced by 7 Li ions and γ rays

doi:10.4236/health.2010.28143

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Vol.2, No.8, 962-967 (2010)

doi:10.4236/health.2010.28143

HEALTH

The influence of DNA concentration in the experiment of

DNA damage induced by 7Li ions and  rays

Fuquan Kong1, Xiao Wang1, Meinan Ni1, Li Sui1, Kui Zhao1,2,3*

1China Institute of Atomic Energy, Beijing, China

2School of Science, Hebei University of Technology, Tianjin, China

3Key Laboratory of Beam Technology and Modification of Ministry of Education, Beijing Normal University, Beijing, China;

*Corresponding Author: kuiz@ciae.ac.cn

Received 4 December 2009; revised 19 January 2010; accepted 23 January 2010.

ABSTRACT

To evaluate the influence of the DNA concentra-

tion in the aqueous solution on DNA radiation

damage, the plasmid DNA in the presence or

absence of Mannitol (scavenger of free radical

OH-) was irradiated by 7Li ions and γ rays at

various DNA concentrations. Gel electrophore-

sis analysis revealed that the DNA damage of

single and double strand breaks induced by

irradiation was dependent on DNA concentra-

tion and became more severe at lower DNA

concentration in the radiation experiment when

all others parameters are the same. In the con-

dition of -ray irradiation, most of double strand

breaks (DSB) damage was neutralized and less

associated with DNA concentration in the pre-

sence of mannitol. However, under 7Li irradia-

tion, the DSB damage could not be cleared by

mannitol but was gradually aggravated with de-

creasing DNA concentrations. Our study sheds

light on the underlying mechanisms in the DNA

radiation damage process. And there are poten-

tial significances for the human space flight,

cancer therapy by heavy ions as well as the ra-

diation security assessment.

Keywords: Heavy Ions; Plasmid DNA; DNA

Concentration;  Rays; Strand Break

1. INTRODUCTION

DNA is considered to be the most important bio-

macromolecule and target responsible for most of the

biological effects in the cells. Many kinds of damage can

be induced by radiation, e.g., base damage, single strand

breaks (SSB), double strand breaks (DSB) and crosslink

of DNA and protein [1]. DNA double strand breaks are

considered to be the most important initial damage initi-

ating serious biological consequences post-radiation. In

particular, unrejoined DSBs may result in cell mutation

or cell death. The DNA damage could be affected by

radiation dose, quality of radiation, dose rate, etc. [2]. In

contrast with the low Linear Energy Transfer (LET) ra-

diations, the heavy-ion irradiation with higher LET and

Bragg peak of dose distribution can produce more com-

plicated track structure and stimulates biological havoc

in organisms, tissues, cells, and DNA [3,4].

In previous experiments, it has been noticed that the

DNA strand-break damage is related to DNA concentra-

tion. Milligan et al. have measured the yield of single

strand breaks G(SSB) induced by γ rays ranging from 0

Gy to 100 Gy and assayed the DNA samples by agarose

gel electrophoresis in the presence or absence of scav-

engers at various DNA concentrations. The result

showed that the G(SSB) had small fluctuation over a

wide range of DNA concentration in the absence of

scavenger. In the presence of DMSO, G(SSB) was pro-

portional to the DNA concentration, rising as the con-

centration increasing. At higher DNA concentration, the

G(SSB) approached a plateau value[5,6]. The similar

result was obtained by SHAO et al. [7] in 2000. How-

ever, these researches were focused on DNA damage

induction at low-LET radiation. So in order to evaluate

the influence of the DNA concentration in the aqueous

solution on DNA radiation damage induced by low-LET

and high-LET radiation, in this work, the high-LET

heavy ions of 7Li was first employed in the plasmid

DNA damage in addition to the low-LET γ rays with

various DNA concentrations in the presence or absence

of free radical scavenger (Mannitol).The distribution of

DNA conformations and the number of DSB per DNA

were statistically analyzed. It is found that the degree of

DNA radiation damage has an obvious relationship with

the DNA concentration, whereas this effect could be

partially attenuated by scavenger at low-LET radiation.

This work will redound to understanding of ionizing

F. Q. Kong et al. / HEALTH 2 (2010) 963-968

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radiation damage to the organisms and provides basic

experimental data for the human space flight, cancer

therapy by heavy ions as well as the radiation security

assessment.

2. MATERIALS AND METHODS

2.1. DNA Sample

The purified DNA sample, double-stranded pUC19

plasmid (2686-bp long) was purchased from TaKaRa

Biotechnology Co., Ltd., (Dalian, China) at a concentra-

tion of 500 ng/μl in TE buffer. The original forms of

DNA sample are a mixture of supercoiled (SC) form

(~90%) and open circular (OC) form. Mannitol was sup-

plied by Beijing 306 Hospital at a concentration of 1.1

M and was diluted to 600 mM for experiment.

2.2.  Rays and 7Li Ions Irradiation

DNA samples in a series of concentrations were irradi-

ated by γ rays of 60Co source located in China Institute

of Atomic Energy (CIAE) at the dose of 482 Gy (dose

rate = 10 Gy/min) at room temperature.

Appling 37.3 MeV 7Li ions (LET = 70.2 keV/μm, in

aqueous solution) generated by HI-13 heavy ion tandem

accelerator of CIAE, the DNA samples in various concen-

trations were irradiated at the dose of 500 Gy (48.4 Gy/

min) at room temperature. The range of 7Li ions in water

is about 307 μm. For all irradiations, a 20 μl aliquot of

DNA solution was dropped onto the middle of a 5 μm-

thick Mylar film and sealed with another Mylar film. An

Au-Si surface barrier semiconductor detector was de-

ployed to monitor the number of incident ions, then radia-

tion dose can be determined by formula (1). All presented

values of energy, LET and dose are at the entrance surface

of the solution. After irradiation, the samples were pre-

served in the micro centrifuge tube at –20˚C prior to be

used. The irradiated dose is defined as follows:

Dose(Gy) = 1.6 × 10-9 × LET(keV/μm) × F(ions/cm2) ×

1/ρ (g/cm3) (1)

where F is the fluence of ions and ρ is the density of the

medium.

2.3. Agarose Gel Electrophoresis

The control and irradiated DNA samples were loaded on

1% agarose gel and electrophoresed for 90 min at 4V/cm.

After electrophoresis, the gel was stained with ethidium

bromide and visualized on a UV-transilluminator. The

DNA bands were photographed and analyzed by Al-

phaImager Imaging System (Alpha Innotech Corpora-

tion, San Leandro, CA) to quantitate the fraction of the

three kinds of DNA conformation (i.e., supercoiled, open

circular and linear form of the DNA molecules).

3. RESULTS

3.1 The Result of γ Rays Irradiation

DNA samples were irradiated by γ rays at a series of

concentration from 10 to 100 ng/μl by the dose of 482

Gy. In Figure 1, lanes 1, 8 are unirradiated control sam-

ples. From lane 2 to lane 7, the DNA concentrations are

100、50、40、30、20 and 10 ng/μl, respectively ( without

mannitol) and samples in lane 9 to lane 14 are arranged

in the same order (in the presence of 600 mM mannitol).

It is shown that at lower DNA concentration, the SC

form DNA disappears and the fraction of linear (L) form

DNA increases. Meanwhile, the fraction of OC form

DNA reduces with the decreasing concentration in the

absence of mannitol in DNA solution. Moreover, the

short linear DNA fragments appears when the concentra-

tion is lower than 50 ng/μl, until all of DNA molecules

are broken into very short fragments at the concentration

of 10 ng/μl. This tendency indicates that DNA is dam-

aged more seriously at lower DNA concentration. In

Figure 1(b), 600 mM mannitol was added in the DNA

solution. Comparing to Figure 1(a), the linear form dis-

appears, on the contrast, most of the original SC form

molecules are preserved and its fraction dropped by de-

creasing DNA concentration. Nevertheless, the SC mo-

lecules tends to transform into OC conformation under

DNA concentration of 30 ng/μl, indicating that the SSB

damage could not be eliminated by scavenger at lower

DNA concentration. Generally, it is confirmed that DNA

damage can be neutralied by mannitol to a great extent

but is still depends on DNA concentration.

The fractions of three kinds of DNA forms versus dif-

ferent DNA concentrations without or with 600 mM

mannitol are shown in Figure 2 and Figure 3 respec-

tively. It is revealed in Figure 2 that the fraction of OC

form dramatically rises from 0% to 75% and the fraction

of linear form decreases from 100% to 25% with the

increasing DNA concentrations in the absence of man-

nitol. In the presence of 600 mM mannitol (Figure 3),

Figure 1. Gel electrophoresis image of DNA after irradiated by

γ rays at various concentrations.

F. Q. Kong et al. / HEALTH 2 (2010) 963-968

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Figure 2. Distribution of DNA OC form (■) and L form (●)

irradiated by γ rays at various DNA concentrations without

mannitol.

Figure 3. Distribution of DNA OC form (■) and SC form (▲)

irradiated by γ rays at various DNA concentrations with 600

mM mannitol.

linear form of DNA molecule disappears, which means

there is no DSB damage when OH˙is eliminated. The SC

form is maintained at the concentration above 30 ng/μl.

The conformational change from SC to OC only occurs

at lower DNA concentrations (20 ng/μl and 10 ng/μl).

Even though, there is still 60% of original SC form at the

concentration of 10 ng/μl. Therefore, it can be postulated

that almost all DNA damage by γ rays irradiation is

caused by diffusion of free radical OH˙ and associated

with DNA concentration, which is consistent with re-

lated reports [8-11].

3.2. The Result of 7Li Ions Irradiation

3.2.1. The Result of DNA Damage in the Absence

of Mannitol

Figure 4 shows the gel electrophoresis image and form

distribution of DNA irradiated by 7Li ions at different

concentrations in the absence of mannitol. In Figure 4(a),

lane 1 is the control sample and DNA concentrations are

100, 50, 30, 20 and 10 ng/μl from lane 2 to lane 6, re-

spectively. It is shown in Figure 4(a) that the OC form

decreases but the linear form increases with the decreas-

ing DNA concentrations, which is similar with the result

irradiated by γ rays. Different from the disappearing of

SC form irradiated by γ rays, the SC form decreases ir-

radiated by 7Li ions with the decreasing DNA concentra-

tion. The fraction of SC is less than 15% and the fraction

of linear form is only 10% at the DNA concentration of

100 ng/μl. The maximum fraction of linear form is up to

55% at the DNA concentration of 10 ng/μl in the ab-

sence of mannitol(shown in Figure 4(b)).

3.2.2. The Result of DNA Damage in the Presence

of 600 mm Mannitol

Figure 5 shows the gel electrophoresis image and form

distribution of DNA irradiated by 7Li ions at different

concentrations in the presence of 600 mM mannitol. It is

shown in Figure 5(a) that, since DNA are protected by

(a)

(b)

Figure 4. (a) Gel electrophoresis image after irradiated by 7Li

ions at various DNA concentrations without mannitol; (b) Dis-

tribution of DNA OC form (■), L form (●) and SC form(▲)

after irradiated by 7Li ions at various DNA concentrations

without mannitol.

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mannitol compared to the Figure 4(a), the influence of

DNA concentration is still exit. However, different from

the γ-ray result, the linear form still exists in the pres-

ence of 600 mM mannitol, indicating that part of DSB

damage comes from the direct ionizing energy deposi-

tion onto the DNA molecules and does not disappear

because of any radical scavenger. The data in Figure 5(b)

shows that the severity of DNA damage is attenuated

since the most of OH˙radicals are eliminated by manni-

tol. Hence the maximum fraction of undamaged SC form

reaches about 70% at higher DNA concentrations. How-

ever the DNA damage is still aggravated with decreasing

DNA concentration from the decreasing of SC form and

increasing of linear form.

3.3. The DSBs/DNA Induced by  Rays and

7Li Ions

Based on the data presented in above, the number of

DSB per single DNA molecule (DSBs/DNA) is calcu-

lated by Eq.(2) [12].

DSBs/DNA = f(LI)/[1 – f(LI)] (2)

where f(LI) is the fraction of linear form of DNA.

3.3.1. The DSBs/DNA Induced by  Rays

In Figure 6, the curve shows the DSBs/DNA at different

DNA concentrations in the absence of mannitol except

the concentration of 10 ng/ul since the fraction of linear

form is 100% at this concentration. It is indicated that

the yield of DSBs per DNA averagely increases as the

decreasing DNA concentration, especially at the very

lower DNA concentration. In the presence of 600 mM

mannitol, the f(LI) is zero (shown in Figure 1 and Fig-

ure 3) so the DSBs/DNA can not be calculated from the

formula (2) which means that there is no DSB in DNA

samples after irradiated by γ rays. It is indicated that the

major damage from γ rays irradiation is the free radical

and most free radicals can be neutralized by mannitol.

So one DNA was attacked by more free radicals at very

lower DNA concentration without mannitol.

3.3.2. The DSBs/DNA Induced by 7Li Ions

In Figure 7, it is shown that the DSBs/DNA increase

when the DNA concentrations decrease either with or

without scavenger. Since the yield of DSB decreases in

evidence when DNA samples are added 600 mM man-

nitol compared with the yield of DSB without mannitol.

But the DSB damage still considerable in the presence of

mannitol, which implies that these DSB sites are resulted

from the local multiple damage induced by direct ioniz-

ing of 7Li ions, thus cannot be prevented completely by

scavenger [13] and is dependent on DNA concentration.

4. DISCUSSIONS

After irradiated by γ rays, the DNA concentration influ-

ences the experimental result when all others parameters

are the same. It would probably be result of interaction

between DNA and free radical OH˙. The plentiful yield

of OH˙is generated by the radiolysis of water when the

DNA aqueous solution is irradiated by γ rays. During

irradiation, the concentration of free radical in water

could be assumed roughly steady and may be higher

than the concentration of DNA molecules. At lower

(a)

(b)

Figure 5. (a) Gel electrophoresis image after irradiated by 7Li

ions at various DNA concentrations with 600 mM mannitol; (b)

Distribution of DNA OC form (■), L form (●) and SC form(▲)

after irradiated by 7Li ions at various DNA concentrations with

600mM mannitol.

Figure 6. The DSBs/DNA at various DNA concentration irra-

diated by γ rays.

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Figure 7. The yield of DSBs/DNA at various DNA concentra-

tion irradiated by 7Li ions. ■ and ▲ show the DSBs/DNA

without mannitol and with 600 mM Mannitol, respectively.

DNA concentration, more number of free radicals react-

ing with one DNA molecule, i.e. a single DNA molecule

is under attack statistically by more free radicals and

DNA is damaged seriously as well as the change of DNA

forms is obvious. On the other hand, the gross amount of

free radicals is much lower than that of DNA molecules

especially at higher DNA concentration in the presence

of mannitol, which result that the number of free radicals

reacting with DNA is reduced. Therefore the effect of

DNA concentration associated with strand break can be

neglected. Thus the DNA form is less affected by DNA

concentration when the DNA concentrations larger than

20ng/μl as shown in Figure 3. At the same time, the lin-

ear DNA fragments vanished, only the small variety of

OC form occurs at the lowest DNA concentration. It is

indicated that the influence of free radical is main reason

of DNA damage. Once cleared by scavenger, the number

of residual free radicals is not enough to produce double

strand breaks. And this implies that DNA damage largely

come from indirect effects during DNA damage induced

by γ rays.

Comparing Figure 2 and Figure 4(b), it is found that

the change of DNA forms after irradiating by γ rays is

more obvious than that irradiated by 7Li ions when the

DNA concentration decreases in the absence of mannitol.

In the presence of 600mM mannitol, there are no L form

but only SC form and OC form existence after irradiated

γ rays (shown in Figure 3). However, there are still

some linear molecules left despite the existence of man-

nitol after irradiated by 7Li ions and their fraction in-

creases with the decrease of DNA concentrations (shown

in Figure 5). These parts of DNA damage should be the

result of direct ionizing energy deposition of 7Li ions and

thus cannot be eliminated by scavenger. Under γ rays

irradiation, the DNA, γ photon and the free radicals in-

duced by photons distribute uniformly in water so that

the probability of interaction between DNA and the free

radicals is relatively high, hence DNA damage is severe.

Under 7Li ions radiation, the situation is different due to

the presence of track structure of high-LET 7Li ions. A

lot of secondary electrons and free radicals induced by

7Li ions aggregate near the tracks and even form com-

plex track structure [14]. The concentrations of secon-

dary electrons and free radicals reduce exponentially

along the radial distance of track. Thus the probability of

interaction between DNA and ions reduces exponentially

along the radial distance of track in the same tendency

and only DNA close enough to the track structure can be

damaged. So DNA damage induced by 7Li ions is allevi-

ated than that of γ rays as the decreasing DNA concen-

trations. At higher-LET radiation, the interaction be-

tween DNA and heavy ions is a combined effect of di-

rect ionizing and indirect free radical, [15,16] which is

different from that of the low LET irradiation. When the

DNA concentration increases, there are more DNA

molecules around the track of ions, the probability of

interaction between a DNA molecule and an ion track

decreases accordingly. So the yield of strand breaks de-

creases as the increasing DNA concentration. Based on

the same reason, in the presence of scavenger, even

though most of free radicals have been eliminated，the

SSBs and DSBs induced by direct ionizing still exist and

decrease at higher DNA concentration. It is shown that

DNA is damaged more severe by 7Li ions on the contrast

to γ ray irradiation as shown in Figure 3 and Figure 5,

further verifying that DNA damages induced by heavy

ions are the common results of direct and indirect inter-

action. So the DSBs/DNA still increases with decreasing

DNA concentrations regardless of the presence of scav-

enger, because of direct interaction of 7Li ions (shown in

Figure 7). But the influence in the conditions of without

mannitol and with 600 mM mannitol is different. In Fig-

ure 7, when the DNA samples without mannitol, any

decrease in DNA concentration makes the yield of DSBs

increase apparently within the range of concentration of

this experiment. However the yield of DSBs changes

less at the higher DNA concentration when DNA with

600 mM mannitol. It is indicates that DNA concentration

influence deeper in the course of DNA damage induced

by indirect free radical than by direct ionizing.

5. CONCLUSIONS

In this paper, it is found that the DNA concentration is

an important parameter in the course of DNA damage

induced by 7Li ions and γ rays. Different DNA concen-

tration may be result in dissimilar results, which are ne-

glected in previous experiment and theory. These effects

might be caused by associated factors including the free

radical, the track structure of heavy ions, the probability

of interactions as well as aggregation of DNA. This

F. Q. Kong et al. / HEALTH 2 (2010) 963-968

967

[7] Shao, C.L., Yu, Z.L. and Masahiro, S. (2000) Reaction

rate coefficients of hydroxyl radical-induced DNA single

and α-type double-strand breaks. Radiation and Envi-

ronmental Biophysics, 39(2), 121-124.

finding there are potential significances for the human

health especially for the space flight, cancer therapy by

heavy ions as well as the radiation security assessment

and would be a challenge to the theoretic research and

strongly calls for further experiments. [8] Yang, M.J, Kong, F.Q., Zhan, Y., et al. (2007) A study of

protective action of different scavengers on DNA damage

induced by γ ray. Nuclear Techniques, 30, 255- 258.

6. ACKNOWLEGEMENTS [9] Aslam Siddiqi, M. and Bothe, E. (1987) Single- and dou-

ble- strand break formation in DNA irradiation in aque-

ous sloution: Dependence on dose and OH radical scav-

enger concentration. Radiation Research, 112(3), 449-

463.

The authors thank the crew of HI-13 Tandem accelerator for their

cooperation during DNA irradiation process. The authors thank pro-

fessor Yizhong Zhuo and professor Zhongwen Wang for their valuable

criticism.

[10] Hutchinson, F. (1985) Chemical changes induced in

DNA by ionizing radiation. Progress in Nucleic Acid Re-

search and Molecular Biology, 32, 115-154.

[11] Zhou, L.J., Fang, Y.Z., Zhang, Y.P., et al. (1993) The

effect of γ-ray irradiation on plasmid pBR 322 DNA.

Journal of Radiation Research and Radiation Processing,

11(1), 28-30

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