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Mutation Research 716 (2011) 20– 26

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

jou rna l h omepa g e: www.elsev ier .com/ locate /molmutCo mm uni t y ad d ress : www.elsev ier .com/ locate /mutres

hromosomal aberrations in the bone marrow cells of mice induced byccelerated 12C6+ ions

iaofei Maa,b,c,d,e, Hong Zhanga,c,d,∗, Zhenhua Wanga,c,d, Xianhua Mina,c,d, Yang Liua,c,d,henhua Wua,c,d, Chao Suna,c,d,e, Bitao Hub

Department of Heavy Ion Radiation Biology and Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, PR ChinaThe School of Nuclear Science and Technology, Lanzhou University, Lanzhou 730000, PR ChinaKey Laboratory of Heavy Ion Radiation Medicine of Chinese Academy of Sciences, Lanzhou 730000, PR ChinaKey Laboratory of Heavy Ion Radiation Medicine of Gansu Province, Lanzhou 730000, PR ChinaGraduate University of Chinese Academy of Sciences, Beijing 100049, PR China

r t i c l e i n f o

rticle history:eceived 5 February 2011eceived in revised form 4 July 2011ccepted 28 July 2011vailable online 5 August 2011

eywords:

a b s t r a c t

The whole bodies of 6-week-old male Kun-Ming mice were exposed to different doses of 12C6+ ions orX-rays. Chromosomal aberrations of the bone marrow (gaps, terminal deletions and breaks, fragments,inter-chromosomal fusions and sister-chromatid union) were scored in metaphase 9 h after exposure,corresponding to cells exposed in the G2-phase of the first mitosis cycle. Dose–response relationships forthe frequency of chromosomal aberrations were plotted both by linear and linear-quadratic equations.The data showed that there was a dose-related increase in the frequency of chromosomal aberrations in

2C6+ ionhromosomal aberrationone marrow

all treated groups compared to controls. Linear-quadratic equations were a good fit for both radiationtypes. The compound theory of dual radiation action was applied to decipher the bigger curvature (D2)of the dose–response curves of X-rays compared to those of 12C6+ ions. Different distributions of thefive types of aberrations and different degrees of homogeneity were found between 12C6+ ion and X-rayirradiation and the possible underlying mechanism for these phenomena were analyzed according to thedifferences in the spatial energy deposition of both types of radiation.

. Introduction

The major adverse effects of exposure to ionizing irradiationre cellular lethality and mutation, which are mainly attributedo chromosomal rearrangements arising from energy depositionn the genetic material. A high frequency of chromosomal aber-ations and increased cancer incidence has been detected by theordic Study Group [1–4]. Bone marrow (BM) is one of the organsost sensitive to radiation injury. In medical practice, BM may be

xposed to radiation for diagnosis or radiotherapy of malignancies,esulting in an increased potential for radiation-induced leukemia,rincipally acute and chronic myelogenous leukemia, after an aver-ge latent period of 7 years [1], much less than for the developmentf most solid tumors [2–4]. BM karyotypic abnormalities have also

een detected after autologous BM transplantation for leukemia5,6] and lymphoma [6] following total body irradiation.

∗ Corresponding author at: Department of Heavy Ion Radiation Biology andedicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou

30000, PR China. Tel.: +96 931 4969344; fax: +86 931 8272100.E-mail address: zhangh@impac.ac.cn (H. Zhang).

027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2011.07.016

© 2011 Elsevier B.V. All rights reserved.

Experiments with heavy ions have repeatedly demonstratedthat heavy ions are considerably more effective than low linearenergy transfer (LET) radiation in causing biological damage at alllevels of biological organization. A heavy ion track is character-ized by the co-ordinates of ionization and excitation of electronsand this is mainly attributed to the considerable density of energydisposition along the track [7]. Another significant difference isthat when heavy ions enter the target medium, a large amount ofenergy is dissipated mainly by secondary electrons (�-ray), whichare non-uniformly ionized around the track core and could travelbetween several and many micrometers, producing a latent track;a damage zone surrounding the track core [8]. The spatial energydisposition within the latent track is rather non-uniform. Com-pared to low LET photons such as X-rays and gamma (�)-rays,exposure to heavy ions results in complex and irreparable clus-tered DNA damage, most of which leads to cell death, but is lesseffective in producing oxidative DNA damage though free radi-cals [9–11]. The growing investment in heavy-ion radiotherapy isfuelled by its success in localizing most of the radiation dose at a

tumor site. Furthermore, heavy-ion radiotherapy exploits the highrelative biological effectiveness (RBE) in the bragg-peak region,thereby having the potential advantage of providing a higher localeffect in tumors. Local effects or damage to adjacent organs and

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2.6. Statistical analysis

X. Ma et al. / Mutation

ealthy tissue is thereby kept to a minimum. Among the vari-us types of accelerated charged particles, the clinical advantagesf 12C6+ ions in treating various types of tumors compared to-rays or �-rays has been confirmed by dedicated centers for

reatment with charged particles in Japan, Germany and China12–15].

There are abundant reports concerning the in vitro damageffects of heavy ions on chromosomes in haemopoietic systemselated to deep space missions [16–26], but information on then vivo induction of chromosomal damage in BM cells is scant, par-icularly related to medical practice. The ratio of complex/simplexchanges (defined as chromosome exchanges involving at leasthree breaks in two or more chromosomes) was proposed as a fin-erprint of exposure to heavy ions [27–31]. Supporting evidenceas mainly collected in in vitro cultured lymphocytes using mul-

icolor fluorescence in situ hybridization (m FISH) for detection ofxchanges and multicolor banding in situ hybridization (m BAND)or inter-/intrachromosomal exchanges. Using these techniques,ande et al. [32,33] detected a significantly elevated frequency ofomplex chromosome translocations and inter-/intrachromosomalxchanges in a dose-dependant manner in peripheral lympho-ytes collected from healthy plutonium workers occupationallyxposed to heavy ions many years ago, demonstrating the long-erm stability and transmissibility of these complex translocations32]. The frequency of complex translocations was highly corre-ated with plutonium dose to the bone marrow [32]. Howevero statistically significant increase in the yield of complex-typexchanges or inter-/intrachromosomal translocations has beenetected in lymphocytes collected from astronauts after missions,uggesting the that use of complex-type exchanges as a biomarkerf radiation quality after low-dose exposure in mixed radiationelds such as the environment of low-Earth orbit is inappropri-te [16,21,26]. Apart from complex translocations, an increasedrequency of dicentrics and simple translocations has frequentlyeen detected in lymphocytes collected from astronauts at various

ntervals after short- or long-term space flight compared to pre-ight baseline measurements [17,23,24]. In contrast to the datan lymphocytes, information on chromosomal damage in bonearrow induced by heavy ions in vivo is limited and incomplete.

rooks et al. [34,35] observed an increase in the frequencies ofhromosomal aberrations and micronuclei as linear functions ofose in rat BM cells collected 1–8 h post-exposure to 56Fe ions.arked decreases of the mitotic index as a function of dose were

lso observed [34]. The technique of mFISH has been used onhromosomes in BM cells of mice by Rithidech et al. [36] to eval-ate in vivo cytogenetic effects of 56Fe ions. In contrast to theesults from in vitro human peripheral lymphocytes, their in vivoata did not detect any significant differences in the frequencyf complex chromosomal exchanges in BM cells collected fromice exposed to 56Fe ions or � rays [36]. Presently there are no

eports concerning the in vivo cytogenetic effects of 12C ions onells of the BM. With the increase in the number of 12C ion ther-py centers around the world and improvements in the survivalate of cancer patients, information concerning chromosomal dam-ge induced in BM cells by 12C ions is of particular importanceecause the damage is directly connected with radiation-induced

eukemia.To investigate the effect of radiation quality on the yield and

omplexity of aberrations, the whole body of mice was exposedo either X-rays or 270 MeV/u 12C6+ ions in the plateau region,he latter resembling the exposure conditions of healthy tissueuring 12C6+ radiotherapy. To account for particle-induced mitoticatastrophe which may cause underestimation of the frequency of

berrations, chromosomal damage was measured 9 h after irradia-ion in BM cells, corresponding to cells exposed in the S/G2 phasef the first mitosis cycle.

rch 716 (2011) 20– 26 21

2. Materials and methods

2.1. Animals

Male outbred Kun-Ming mice (6 weeks old) provided by Lanzhou University(Lanzhou, China) were used under identical breeding conditions. To measure chro-mosomal aberrations, a total of 60 mice were randomly divided into groups of 6animals. The doses for both X-rays and 12C6+ ion irradiation were 0.5 Gy, 1 Gy, 2 Gy,4 Gy or 6 Gy. A control group, consisting of 6 mice that received no irradiation, wasalso included. For cell cycle detection, a total of 48 mice were randomly divided intogroups of 4 animals. The doses for both X-rays and 12C6+ ion irradiation were 0.5 Gyor 6 Gy. A control group consisting of 4 mice that received no irradiation was alsoincluded.

2.2. Irradiation procedure

Each mouse was positioned in a chamber which was attached to the irradia-tion equipment at the Heavy Ion Research Facility in Lanzhou (HIRFL, Institute ofModern Physics, Chinese Academy of Sciences, Lanzhou, China). The whole body ofeach mouse was irradiated with 12C6+ ion beams at energy of 270 MeV/u and LETof 10 keV/�m, with a dose rate of 0.3 Gy/min. The doses of the beams were deter-mined using an air ionization chamber. The 12C6+ ion dose of 0.5 Gy corresponded toa fluence of 3.0 × 107 particles/cm2. Animals irradiated with X-rays were similarlygiven whole body irradiation by an X-ray therapy machine (Elekta BMEI (Beijing)Medical Equipment Co. Ltd, China) at a source-to-surface distance (SSD) of 100 cm,with a dose rate of 2 Gy/min.

2.3. Preparation of BM cells and chromosomes

BM cells were obtained according to the technique of Yosida and Amano [37].Briefly, femurs were removed immediately after animal sacrifice and BM was flushedout with physiological saline using a syringe. The BM cell suspension was cen-trifuged at 1000 × g for 5 min. The supernatant was discarded, and the pellet wasresuspended in 5 mL hypotonic solution of pre-warmed 0.075 M KCl for 20 min.The suspension was then centrifuged at 1000 × g for 8 min. The supernatant wasagain discarded, the pellet was resuspended in 5 mL of a fixative solution (aceticacid/methanol, 1:3, v/v), centrifuged (1000 × g for 8 min) and the supernatant wasdiscarded again, the cells were resuspended in two additional changes of the fixa-tive mixture. After the final centrifugation, cells were thoroughly mixed and weredropped from a height of 10 cm onto precleared cold glass slides and air dried. Glassslides were coded according to the number of the animal. Air-dried preparationswere aged for a week before Giemsa staining.

2.4. Meta-phase chromosome analysis

The coded slides were examined under 100× magnification using a bright-fieldmicroscope (Nikon, Japan). Nearly three hundred well-spread metaphases wereanalyzed per group for abnormalities. Chromosome aberrations observed in BMmetaphase were scored as follows: (1) gaps (chromosome and chromatid types),identified as an achromatic site along the length of a chromatid (or chromosome)that was less than the width of the chromatid (or chromosome); (2) fragments (chro-mosome and chromatid types), identified as unrejoined acentric fragments derivedfrom a chromosome or chromatid severance; (3) terminal deletions, identified as achromatid that was less than the length of the other sister-chromatid, and breaks,identified as large segment loss in one chromatid; (4) inter-chromosomal fusion,identified when the terminal of two chromatids of two different chromosomes fusedtogether; and (5) sister-chromatid union, identified when two chromatids of thesame chromosome were fused together. Fig. 1 shows representative photomicro-graphs of the five abnormal types. The mitotic index was also scored. Five hundredcells from each animal were scored to determine the mitotic index.

2.5. Cell cycle analysis

The cell cycle distributions of mouse BM cells were measured at 9, 40 and 72 hfollowing irradiation. The procedure for cell cycle analysis was performed accord-ing to the routine standard method. Briefly, BM cells were collected and pelleted bycentrifugation. The pellets were suspended in 5 mL ice-cold 70% ethanol and incu-bated overnight. The fixed cells were centrifuged and the pellets were washed oncewith PBS. After resuspension in 0.5 mL PBS, the cells were incubated with PI solution(0.1% Triton X-100, 10 �g/mL PI, and 100 �g/mL DNase-free RNase A) for 30 min atroom temperature in the dark. The stained cells were immediately measured on aFACScan flow cytometer (Becton Dickinson) in combination with the BD Modfit LTanalysis package.

All data are expressed as mean ± standard deviation (S.D.). Statistical differ-ences between controls and treated groups were determined by Student’s t-test.Differences were considered significant at p < 0.01 or p < 0.05.

22 X. Ma et al. / Mutation Research 716 (2011) 20– 26

Fig. 1. Photographs of metaphase chromosome of BM and primary spermatocyte cells collected at 9 h after whole-body exposure to 12C6+ ions. Panels A–E: metaphasec rimar( entric

3

3a

dmttfite

hromosome of bone marrow cells; panel F: diakinesis–metaphase chromosome of pt), breaks (b), sister-chromatid union (SCU), intra-chromosome fusions (intra-f), ac

. Results and discussion

.1. Comparison of the aberration yields in metaphase BM cellsfter exposure to high energy 12C6+ions and X-rays

In order to avoid underestimating the frequency of aberrationserived from mitotic catastrophe induced by particle irradiation,itotic chromosomes were collected at 9 h after irradiation. At this

ime point, the majority of the analyzed metaphases corresponded

o cells exposed in the inter-phase (mainly G2-phase) stage ofrst mitosis. Only structural aberrations induced by the differentreatments were enumerated in the present study, with specialmphasis on gaps, terminal deletions, fragments and fusions. All

y spermatocytes. Arrows indicate chromatid gaps (g), chromatid terminal deletions chromosome (acen chr), muti-breaks (muti-b) and double minutes (DM).

these types of structural abnormalities and their frequencies forboth control and treated groups are presented in Table 1. Numeri-cal chromosome abnormalities such as polyploidy and aneuploidywere not evaluated. Chromosome aberrations, whatever the type,showed a significant increase in a dose-dependent manner in micetreated with either 12C6+ ion or X-rays. Terminal deletions andbreaks represented the highest percentage of abnormalities (about70.38% of the total aberrations at the highest dose 6 Gy for 12C6+ ionand about 74.19% for X-rays) which were also manifest in meiosis

bivalents in primary spermatocytes (Fig. 1F), while chromosomalfusions (Fig. 1 A and B) were the least frequent lesions, represent-ing only 2.2% at 6 Gy dose for 12C6+ ion and 0.0% at all dose groupsfor X-rays. A different distribution of terminal deletions and breaks

X. Ma et al. / Mutation Research 716 (2011) 20– 26 23

Tab

le

1Th

e

dis

trib

uti

on

of

chro

mos

omal

aber

rati

ons

ind

uce

d

by12

C6+

ion

s

or

X-r

ays

in

BM

cell

s

of

mic

e.

Gro

up

s

No.

of

cell

scor

edD

istr

ibu

tion

of

aber

rati

ons

(abe

rrat

ion

s/ce

ll)

Tota

l no.

ofab

erra

tion

s/ce

lls

(%)

Gap

s

(%)

Term

inal

del

etio

ns

and

brea

ks

(%)

Inte

r-ch

rom

osom

efu

sion

s

(%)

Frag

men

ts

and

DM

(%)

Sist

er-c

hro

mat

idu

nio

n

(%)

Con

trol

0

Gy

380

0.00

2

±

0.00

(0.0

)

0.06

9

±

0.03

0.00

0

±

0.00

(0)

0.02

7

±

0.09

(0.)

0.00

0

±

0.00

(0)

0.09

7

±

0.09

(1.1

)12

C6+

ion

0.5

Gy

362

0.00

7

±

0.01

(0.9

)

0.13

2

±

0.03

(3.1

)

0.00

5

±

0.00

(1.7

)

0.04

4

±

0.02

(4.6

)

0.00

0

±

0.00

(0.5

) 0.

191

±

0.15

(2.7

)1

Gy

362

0.02

0

±

0.01

(0.8

)

0.23

4

±

0.09

(3.9

)

0.00

9

±

0.01

(1.9

)

0.04

7

±

0.03

(5.1

)

0.00

0

±

0.00

(0.8

) 0.

301

±

0.18

* (10

.4)

2

Gy

370

0.03

9

±

0.01

(3.1

)

0.70

2

±

0.23

(18.

3)

0.03

8

±

0.01

(7.1

)

0.26

8

±

0.02

(14.

1)

0.00

1

±

0.01

(3.7

) 1.

029

±

0.28

**(3

1.3)

4

Gy

310

0.08

9

±

0.03

(6.7

)

1.95

0

±

0.91

(24.

1)

0.07

5

±

0.02

(15.

1)

0.50

6

±

0.04

(33.

6)

0.00

3

± 0.

01(5

.1)

2.61

2

±

0.38

**(6

0.4)

6

Gy

270

0.23

1

±

0.06

(8.1

)

2.58

7

±

0.98

(27.

9)

0.09

3

±

0.03

(16.

3)

0.77

1

±

0.02

(37.

2)

0.00

5 ±

0.02

(7.0

)

3.67

6

±

0.45

**(7

5.6)

X-r

ay

0.5

Gy

341

0.00

9

±

0.00

(0.3

)

0.09

5

±

0.02

(1.8

)

0.00

±

0.00

(0)

0.02

8

±

0.01

(1.2

)

0.00

0 ±

0.00

(0)

0.12

3

±

0.11

(1.8

)1

Gy

312

0.00

6

±

0.01

(0.7

)

0.13

5

±

0.07

(4.1

)

0.00

±

0.00

(0)

0.06

4

±

0.01

(3.4

)

0.00

0 ±

0.00

(0)

0.19

1

±

0.11

* (6.

2)2

Gy

331

0.04

2

±

0.01

(4.1

)

0.20

6

±

0.01

(12.

1)

0.00

0

±

0.00

(0)

0.14

1

±

0.03

(14.

0)

0.00

1

±

0.00

(0)

0.39

1

±

0.19

(27.

1)**

4

Gy

291

0.04

0

±

0.03

(6.1

)

0.90

3

±

0.32

(25.

3)

0.00

0

±

0.00

(0)

0.26

7

±

0.13

(34.

6)

0.00

0

±

0.00

(0)

1.31

1

±

0.28

(58.

0)**

6

Gy

284

0.15

0

±

0.0(

5.9)

1.92

3

±

0.97

(32.

1)

0.00

±

0.00

(0)

0.58

2

±

0.26

(39.

1)

0.00

0

±

0.00

(0)

2.64

4

±

0.30

(71.

1)**

(%)

rep

rese

nts

the

per

cen

tage

s

of

cell

wit

h

the

ind

icat

ed

typ

e

of

aber

rati

ons.

Dat

a

rep

rese

nt

mea

n

±

S.D

. Th

e

sign

ifica

nce

of

dif

fere

nce

s

betw

een

rad

iate

d

grou

ps

and

con

trol

s

was

det

erm

ined

by

Stu

den

t’s

t-te

st.

*p

<

0.05

vs. c

ontr

ol.

**p

<

0.01

vs. c

ontr

ol.

Fig. 2. Dose–response curves for the total number of aberrations per cell scored inBM cells collected at 9 h after exposure to 12C6+ ions or X-rays. At each data point, thevertical lines represent the standard error of the mean (data from six mice in eachgroup). For each type of radiation, dotted lines represent the dose–response curvesfitted by linear-quadratic equations: y = 0.011D2 + 0.571D − 0.072 (R2 = 0.988) for12C6+ ions; and y = 0.064D2 + 0.04D + 0.084 (R2 = 0.999) for X-rays. Similarly, for each

type of radiation, the solid line lines represent the dose–response curves fitted by lin-ear equations: y = 0.637D − 0.115 (R2 = 0.8628) for 12C6+ ions; and y = 0.426D − 0.166(R2 = 0.945) for X-rays.

was also found between exposure to 12C6+ ion and X-rays: in X-rayexposure, the aberrations were dominated by chromatid breaks,whereas in 12C6+ ion exposure, the distribution was dominated bychromatid terminal deletions – complete disintegrations of termi-nal parts of chromosomes – which was regarded as a manifestationof the more local energy density in a particle track [38], so theaberration spectrum in BM cells was found to depend on irradi-ation quality but not on dose. The formation of chromatid fusionscould be interpreted to mean that in the case of 12C6+ ion exposurethe ionization density was high enough that it simultaneously pro-duced two double strand breaks (DSB) in two sections of chromatinthat were occasionally interlaced. The resulting ruptured sticky endof each individual chromatin fragment could then randomly jointogether by non-homologous end joining (NHEJ) resulting in a fusedstructure when the chromatin condensed in meta-phase. It doesnot appear likely from these results that long-distance recombina-tion occurs between damage at separate sites within the nucleus,since the observation of Jakob et al. [39] suggests that the mobilityof damaged foci is rather limited. The dislocation of the damagedfoci covered a distance of less 0.5 �m [34]. This type of aberrationcould trigger breakage–fusion–bridge cycles in subsequent mito-sis cycles [40], which create chromosomal instability that has thepotential to affect many chromosomes within a cell, as proposedin 1941 by McClintock [41,42], and associated with tumor cell pro-gression [43]. This suggests that heavy ions probably possess morepotential than X-rays for carcinogenesis. These five types of aber-rations were not induced in G1 cells, since chromosome damageinduced in G1 cells would give rise to chromosome-type aberrations[44]. However, as presented in Fig. 1, all of the observed aberra-tions in the present study were chromatid-aberrations, so theseabnormal structures were probably formed in S/G2 phase when

DNA duplication had finished.

Both linear and linear-quadratic equations were used to testthe fit of the data of total number of aberrations per cell (Fig. 2).In general, only the linear equation was a good fit for 12C6+ ion

2 Research 716 (2011) 20– 26

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4 X. Ma et al. / Mutation

xposure, while the linear-quadratic equation was a good fit bothor X-rays and 12C6+ ion exposure, thus the theory of compoundual radiation action (CTDRA) was the best fit model based on theresent data [45]. Based on the linear terms of the equation forose response curves, 12C6+ ions were 1.5 times more effectivehan X-rays in inducing chromosomal damage, while based on theuadratic-terms, the curvature (D2) was less significant for 12C6+

on irradiation than for X-ray irradiation. This may be interpretedccording to the CTDRA that in the case of 12C6+ ion exposure, twohromosome parts were damaged simultaneously by the same par-icle which then interacted to produce the aberrations; while in thease of X-ray exposure, two chromosome parts were damaged indi-idually by two separate photons which then interacted to producehe aberration. So as dose increases, ionization event proximity isnhanced and hence the quadratic-term predominates [46].

The distribution of the number of aberrations per metaphase inhe 12C6+ ion-exposed cell population was rather inhomogeneousven in cells on the same slide. For example, at 0.5 Gy 12C6+ ionrradiation, the number of aberrations in a metaphase could be asew as 1 or occasionally as many as 8; for X-ray exposure at 6 Gy,owever, the number was rather homogeneous, between 1 and 3berrations per metaphase. This difference could be related to theifferential spatial energy disposition properties of the two radia-ion types. Following X-ray exposure, ionizations were randomlyistributed leading to a homogeneous distribution of aberrations

n individual cells. However, following 12C6+ ion irradiation, theonization tracks would be non-uniformly distributed. Thereby thenhomogeneously distributed aberrations in the case of 12C6+ ionxposure were determined by the different numbers of direct parti-le hits per cell nucleus [38,47,48]. In the present study, for BM cellsith a mean geometrical nuclear cross-section of about 25 �m2, the

xposure to a 0.5 Gy, 270 MeV/u 12C6+ ion (3.0 × 107 particles/cm2)esulted in a mean number of 7 direct hits per cell nucleus. Fromoisson statistics it follows that 8% of the nuclei received fewerhan 3 hits, while 9% were traversed four times, 12% were traversedve times, 14% were traversed six times and 40% were hit sevenimes or more. The non-uniform energy deposition inside a par-icle track also contributes to the inhomogeneity. Track structurean be divided into two different areas: (1) a partially melted innerore region with a diameter of 7 pm (at energy of 270 MeV/u) butensely dissipated 60–65% of total energy; (2) an outer zone calledhe penumbra with a diameter of about 150 �m where 35–40%f the total energy is non-uniformly spread over a disproportion-tely larger region and drops precipitously to very low levels [49].hus energy dispositions in cells that lie within the realm of a trackary significantly according to their distance to the center core andontribute to the inhomogeneity.

Since the response to X-ray irradiation was close to linear inhe present study, it was assumed that a comparison of the mono-

ial coefficient to derive the values of RBE was justified. An RBEf 1.5 was derived from the comparison. Even though LET is oftenmplied to specify the quality of a therapy beam, its value does notake into account the stochastic nature of energy deposition. The

easurement of microdosimetric spectra in a simulated site withizes comparable to the diameter of cell and chromatin or evenNA could offer more information on radiation quality. Hultqvistt al. [39] calculated the dose-mean linear energy,¯yD, for simulatedbject diameters in the range 2–100 nm in water irradiated with2C with initial energies of 290 MeV/u. The yD is constant through-ut the plateau, and the beam energy used in the present studyas 270 MeV/u, very closed to the energy they calculated. Thus itas assumed that a direct application of their calculations to the

resent beam was justified. At the plateau, ¯yD , is 28 keV/�m for aimulated object size of 2.3 nm (comparable to the diameter of theNA double helix); 26 keV/�m for a size of 10 nm (comparable to

he diameter of nucleosomes); 20 keV/�m for a size of 30 nm (com-

Fig. 3. Calculated yD-ratios for a C beam with primary energy 270 MeV/u andX-rays beam as a function f simulated diameter (data based on the calculation ofHultigvist et al. and Grindborg et al.). Dotted line represents the RBE of 1.5.

parable to the diameter of a 30 nm chromatin fiber) and 18 keV/�mfor a size of 100 nm (comparable to the diameter of chromonemafiber). The ratios of the yD-values at plateau to those calculated forX-rays [50] are shown in Fig. 3. The ratios were compared with theRBE measured in the present study. It can be seen from Fig. 3 thatthe yD-ratio for a volume of about 10 nm in diameter is approxi-mately equal to the RBE. This (simulated object diameter of 10 nm)is consistent with the calculations of Lillhok et al. [51] and Hultqvistet al. [39], which suggests that the yD-ratio determined at 10 nm tobe most closely related to RBE values in cases of neutron and protonirradiation. This suggests that energy deposited within the range ofdiameter of nuleosomes or chromatin fibers is most responsible forchromosome damage. Thus, there is good reason to believe that theeffectiveness of ionization in inducing chromosomal damage or cellkilling which involves the micrometer range would be connectedwith energy deposition in the nanometer range of site size.

3.2. Cell cycle progression

The mitotic index of mouse BM cells was measured at 9 h post-exposure to various doses of 12C6+ ions. The equation that fit thedata best was:

Mitotic cells1000 total cells

= 60 − 9.7D (R2 = 0.8718)

where D is the dose in grays. The slope is three times smaller thanthe value measurement by Brooks et al. [34,35] in rats at 1–8 hpost-exposure to 56Fe ions (1000 MeV/u).

The percentages of mouse BM cells in different phases of the cellcycle collected at different times following 12C6+ ion irradiation areshown in Table 2. The percentages of cells in G2/M phase rose from6.4.1% at control level to 9.9%, while the number of cells in S-phase

decreased from 28.1% at control level to just 3.8% at 9 h after expo-sure to 4 Gy 12C6+ ions. These results suggest that G1-phase andG2-phase cells were being arrested or delayed while S-phase cellswere still cycling, resulting in a significantly higher percentage of

X. Ma et al. / Mutation Research 716 (2011) 20– 26 25

Table 2cell cycle data of BM cells collected at different times after exposure to 12C6+ ions.

0 Gy 0.5 Gy 4 Gy

% G0/G1 % S % G2/M % G0/G1 % S % G2/M % G0/G1 % S % G2/M

9 h 65.5 ± 2.8 28.1 ± 2.2 6.4 ± 2.9 74.2 ± 1.4 18.6 ± 0.1 7.2 ± 1.4 86.2 ± 0.2 3.8 ± 0.1 9.9 ± 0.225 h 65.5 ± 2.8 29.1 ± 2.2 5.3 ± 0.9 / / / 82.8 ± 0.5 8.1 ± 1.2 8.5 ± 0.5

2725

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40 h 66.5 ± 2.7 28.3 ± 2.2 5.2 ± 0.7 64.8 ± 4.1

72 h 65.7 ± 2.3 28.6 ± 2.9 5.5 ± 0.6 70.3 ± 3.4

2/M phase cells between 9 and 40 h post-irradiation. At 40 h fol-owing 0.5 Gy irradiation, a considerable proportion of cells seemedo have started cycling again, as documented by the appearancef S-phase signals, and the increase in the percentage of S-phaseells was directly proportional to the decrease in the number of G1-hase cells, consistent with the results of Rithidech et al. [52] whichuggests that there is an inverse correlation between the numberf G1-phase cells and S-phase cells following particle irradiation.he level of S-phase was also significantly lower at all collectedoints post-exposure to 4 Gy compared to 0.5 Gy. The level of S-hase dramatically increased at 72 h after exposure to 4 Gy 12C6+

ons but remained significantly lower (p < 0.05) relative to controlevel, reflecting a lag phase of recovery after exposure to high doses.

In conclusion, 12C6+ ion irradiation was effective at inducinghromosomal aberrations in the BM cells of mice, even at a lowose of 1 Gy. Our results (metaphase chromosome scoring at 9 hfter irradiation) may provide useful information for assessmentf genetic risks in humans exposed to heavy ions. There is a needor further investigation of the relationship between chromoso-

al aberrations induced by heavy ions and secondary hematologicalignancies, and identification of reasonable measures effective

t reducing chromosomal aberrations caused by heavy ion irradia-ion in the course of radiotherapy. The next step will be to calculatehe relative biological effectiveness (RBE) in inducing secondaryematologic malignancies that would make standard cancer riskstimates for X-rays applicable to heavy ions.

onflict of interest

None.

cknowledgements

The authors acknowledge the accelerator crew at the HIRFL,ational Laboratory of Heavy Ion Accelerator in Lanzhou. This

esearch was supported by the National Basic Research Program ofhina (2010CB834202), the National Natural Science Foundation ofhina (10835011) and the Scientific Technology Research Projectsf Gansu Province (0702NKDA045).

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