RADIATION RESEARCH 178, 600–603 (2012)0033-7587/12 $15.00
Ó2012 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR3074.1

Translocations in Spleen Cells from Adult Mice Irradiated as Fetuses are
Infrequent, but Often Clonal in Nature
Mimako Nakano,1 Yoshiaki Kodama, Kazuo Ohtaki and Nori Nakamura
Department of Genetics, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku, Hiroshima 732-0815, Japan

clear dose response results from survivors who were
exposed to radiation at various ages (7). Because cytogenetic damage is one of the best characterized biomarkers ofradiation exposures and represents one of the mostimportant classes of DNA damage that may contribute to
the development of malignancy, further investigations wereconducted in mice. The mouse results not only confirmed
the lack of a dose response after irradiation as fetuses, butalso disclosed that neonates (3–4 days old) had the samecharacteristics (8). In this study, spleen cells were examinedwith a 2-color FISH method by painting chromosomes 1
and 3 when the animals reached 20 weeks of age. Further,
irradiation of mice at various ages (but with all cytogenetic
examinations made at a fixed age of 20 weeks) showed thatthe translocation frequency was low when mice were
irradiated at 1–2 weeks after birth, but thereafter it became
progressively higher with increasing age at the time ofirradiation and reached a plateau level when they were
irradiated at 6 weeks of age or older. (It is noted that humanneonates may not show the same characteristics because
humans appear to be born relatively more mature thanmouse neonates.) Genetic makeup with regard to theknockout conditions of the p53 gene (either heterozygous
or homozygous) did not affect these results. Interestingly,
although the frequencies were generally low, thosetranslocations that were occasionally detected within thesame individuals often looked identical (i.e., they appeared
to be clonal descendants). This seems to be a uniquecharacteristic associated with fetal irradiation, if it isconfirmed; this is because clonal translocations are usuallyassociated with high aberration frequencies in A-bomb
survivors (9) or those who were exposed to radiation during
the Chernobyl nuclear plant accident (10).
The aims of the present study are twofold. First, to apply
the mFISH method to the same spleen cell samples thatwere examined previously to understand how oftentranslocations observed in the same animals are clonallyrelated. Second, to confirm that the low translocation yield
is not caused by a technical bias due to the usage of a partial
genome painting method (FISH painting of chromosomes 1
and 3 only). Although the probability would be low, if thelymphocyte pool consisted of a small number of hematopoietic stem cells (e.g., 10–20), detecting translocations

Nakano, M., Kodama, Y., Ohtaki, K. and Nakamura, N.
Translocations in Spleen Cells from Adult Mice Irradiated as
Fetuses are Infrequent, but Often Clonal in Nature. Radiat.
Res. 178, 600–603 (2012).

We previously reported that mouse fetuses or neonates
exposed to 2 Gy of X rays showed an unexpectedly low
incidence of chromosome damage in lymphocytes, bonemarrow, and spleen cells when the mice were subsequently
examined at 20 weeks of age. However, cells bearing
translocations were occasionally observed that, on the basisof 2-color whole chromosome painting appeared to be clonal
descendants. Unfortunately, this approach typically did not
permit unequivocal confirmation of their clonality. To
overcome this problem, multi-color FISH (mFISH) was
employed, which assigns all 21 individual chromosome typesof the mouse a unique color. After mFISH analyses of thesame cell samples studied previously, it was confirmed thatspleen cells of 20-week-old mice irradiated either as 15.5-day
fetuses or as 3- to 4-day-old neonates showed translocation
frequencies close to zero. Translocations previously suspected
as being clonal in nature were confirmed as such by mFISH,
which also revealed the presence of an additional clone notpreviously detected or suspected. Since no evidence of
clonality was observed in the irradiated mother, we concluded that in both fetuses and neonates, there exists a small
fraction of stem cells that are distinct from the bulk of thestem cell compartment in terms of their ability to acquire andtransmit radiation-induced chromosome damage throughclonal expansion. Ó 2012 by Radiation Research Society

INTRODUCTION

It has long been thought that fetuses are sensitive toradiation in terms of increased risks for childhood leukemia
and solid cancers (1–5). Therefore, it was surprising to findthat fetuses exposed to atomic-bomb radiation did not show
a dose-related increase of translocation frequencies in blood
lymphocytes when they were examined at around 40 yearsof age (6). This observation was in sharp contrast to the
1 Address for correspondence: Department of Genetics, Radiation
Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku,
Hiroshima 732-0815, Japan: e-mail: ykodama@rerf.or.jp.

600

CLONAL TRANSLOCATIONS IN MICE IRRADIATED AS FETUSES

involving only a few painted chromosomes could haveoverlooked translocations that occurred between two
unpainted chromosomes.
MATERIALS AND METHODS
Animals and Cells
Details have been described previously (8). Briefly, pregnant
B6C3F1 mice at 15.5-day p.c. or 3- to 4-day-old neonates were
exposed to 2 Gy of X rays. When the offspring or neonates reachedapproximately 20 weeks of age, their spleen cells were isolated andcultured for metaphase preparations. Spleen cell samples from 8
animals were examined in the present study. These included samples
from 4 and 3 mice irradiated as fetuses and neonates, respectively, andfrom one mother. Samples that showed low or high translocation
frequencies in the previous study with FISH painting of chromosomes
1 and 3 were selected from the fetal or neonatal irradiation group. The
mouse ID numbers shown in the present study correspond to thoseused in the previous study (8). Fixed cells were used (some of whichwere previously examined) which had been kept at 208C in Carnoy’s
fixative to prepare new metaphases. Metaphase slides were also usedthat had been examined previously, and that had been found to containpossibly identical translocations (see below).
Multi-Color FISH (mFISH)
Metaphase chromosomes were hybridized with a set of chromosome probes (mFISH probes) according to the manufacturer’s
instructions (Cambio, Cambride, UK). Spectrum gold, spectrum red,
spectrum far red, spectrum green, and Cy5.5 were used as thefluorochromes. The resulting mFISH figures were analyzed with a
Genus/Chromofluor System (Applied Imaging International, Newcastle upon Tyne, UK). Five images were sequentially acquired
through fluorochrome-specific optical filters, and pseudo-colors wereassigned to each image based on gray scale intensity values using
custom-designed software. One hundred metaphases were analyzed
for each animal with the exception of the N2-6 individual, for which
73 metaphases were scored.
Examination of Metaphases Previously Suspected to Contain Clonal
Translocations
In the previous study, only chromosomes 1 and 3 were painted.
Thus, to identify clonal translocations, it was necessary to conduct
additional FISH painting on the same metaphases to identify the
unpainted cooperating chromosomes that had been involved in thetranslocation events. However, because there were no clues regarding
the interacting chromosomes, this was a difficult task. Therefore, inthe present study, mFISH data was first analyzed from newly prepared
metaphases from spleen cells from the same mice to obtain clues about
the interacting chromosomes. Subsequently, probes for the most likely
candidate chromosomes were selected and used on the metaphasesthat were previously screened and known to contain possible clonaltranslocations. Every translocation detected in the previous study wasphotographed with color film (ISO 800, Fuji, Tokyo, Japan), and themicroscope coordinates were recorded. Chromosome slides were thenput into a 100% ethanol solution for 30 min to remove the cover slips,
washed twice in a 100% ethanol solution for 2 min to remove themounting medium, and air dried. Slides were then soaked in 70%
formamide/23SSC at 728C for 2 min to denature the DNA. They werethen cooled quickly with successive rinses; twice with 23SSC at 48C,and dehydrated in an ethanol series (70, 85 and 100%), and air-dried.
Metaphase slides suspected to contain clonal translocations were thenrehybridized with a new set of chromosome probes. Translocations
were judged as identical (i.e., clonal) if the two chromosomes involved

601

in each translocation were the same, and the estimated chromosome
break points were reasonably similar.
Calculation of the Genome-Equivalent Translocation Frequency
The previously used 2-color FISH method detected translocations
involving chromosome 1 or chromosome 3, and the genomeequivalent frequency of translocations (FG) was estimated with theequation of Lucas et al. (11). That is:
FG ¼ 

Frg
2:05 3 fg ð1  fg Þ þ fr ð1  fr Þ  fr fg 

where Frg represents the observed translocation frequency, and fg and fr
are the fractions of the genome painted with green (chromosome 1)
and red (chromosome 3), respectively. Since chromosomes 1 and 3
comprise 7% and 6% of the genome, respectively (12), four times theobserved frequency is equal to the genome-equivalent frequency, orFG ¼ Frg/0.24.

RESULTS

Translocation Frequencies Measured with mFISH

Table 1 summarizes the translocation frequencies obtained with the mFISH method along with the frequencies
previously obtained with the 2-color FISH method. The
results are expressed as genome-equivalent translocation
frequencies so that direct comparisons can be made. It isevident that the paired results are in reasonable agreement;
specifically, the frequency in the mother was 25% with themFISH method and 20% with the 2-color FISH method.
Also, the mean translocation frequencies of mice irradiatedas fetuses or neonates were nearly the same with either
method (about 4%), which was clearly lower than that of themother. (The frequency of 4% itself does not specifically
represent a particular event or parameter, however, because
this is a mean of arbitrarily selected samples of animals as
mentioned in the Materials and Methods.)
Clonal Translocations

In the previous study that used the 2-color FISH method,
only one clonal translocation event could be confirmed,
although several candidate clones were recognized (8).
Thus, in the present study, attempts were made to detect
additional translocations because mFISH is the method best
suited for this purpose (i.e., the two chromosomes involvedin a translocation are generally identifiable). The results areincluded in Table 1. For example, mFISH analysis detected
5 translocations in spleen cells of the F2-1 mouse, and 4 ofthe 5 were identical, t(6;6), whereas, no translocations weredetected previously in this spleen cell sample. Likewise, in
the F2-7 mouse, the mFISH method detected 9 translocations of which 5 were judged as clonal. Specifically, one
cell had t(1;12), and 4 cells had t(3;4), and both of themwere detected in the previous study, i.e., 5 cells had t(1;12)
and 2 cells had t(3;4). In the N2-7 mouse, cells bearingt(1;4) or t(1;13) were found in both the previous and present
slides repeatedly and were considered as clonally derived. It

602

NAKANO ET AL.

TABLE 1Results of mFISH and 2-Color FISH Analyses of Spleen
T-Lymphocytes of Mice Irradiated as Fetuses or Neonates

Mouse IDb

mFISH
Translocation frequency
clone informationc

Mother
M2-1

25/100 (25%)
No clones detected
Irradiated as fetusese
F2-1
5/100 (5%)
4 3 t(6qþ;6q)F2-2
0/100 (0%)
F2-7
9/100 (9.0%)
1 3 t(1q;12qþ)
4 3 t(3q;4qþ)F2-8
1/100 (1%)
(Mean %)
(3.8%)
Irradiated as neonatesN2-2
1/100 (1%)
N2-6
N2-7
(Mean %)

4/73 (5%)
No clones detected
5/100 (5%)
1 3 t(1q;4qþ)
2 3 t(1qþ;13q)(3.7%)

2-color FISHa
Translocation frequencyd
clone information
38/800 (20%)
No clones detected
0/800 (0%)
0/800 (0%)
16/800 (8.4%)
5 3 t(1q;12qþ)
2 3 t(3q;4qþ)18/800 (9.5%)
18 3 t(1q;19qþ)(4.5%)
2/800 (1%)
No clones detected
3/800 (1.6%)
No clones detected
14/800 (7.4%)
2 3 t(1q;4qþ)
8 3 t(1qþ;13q)(3.4%)a

The 2-color FISH data shown here are new data except for theclonal translocation t(1q;19q þ) found in F2-8 individual which wasreported previously (8).
bThe animal IDs correspond to those in the previous report (8).
c
For example, 4 3 t(6q þ;6q) stands for 4 cells had the samet(6q þ;6q).
dPercentages in the parentheses indicate genome-equivalentfrequencies of translocations so that direct comparisons can be madebetween the mFISH and 2-color FISH data. The frequencies in thiscolumn are reproduced from the previous report (8).
eOffspring F2-1, F2-2, F2-7 and F2-8 were born to the mother M2-1.

should be noted that although many translocations that werepreviously suspected to be clonal could be identified asclones in the present mFISH analysis, there are still
metaphases in the previously examined slides in which it
was difficult to determine the unpainted interacting
chromosomes because re-hybridization with the new FISH
probes did not work sufficiently well [i.e., t(1;12?) in 3 cells
and t(3;4?) in 5 cells of the F2-7 mouse].
As a considerable fraction of the translocations wereconfirmed as being clonally related, the translocation
frequencies of the clone bearing animals was consequently
reduced substantially because clonal translocations should
be counted as single events. For example, when adjusted forclonality, the translocation frequency of the F2-1 mouse withthe mFISH method (i.e., the observed translocation frequency FG ¼ 5%) becomes 2% (two events among 97 cells). In
contrast, in the mother mouse, the translocation frequency
was much higher and no clonal translocations were detectedamong 100 cells scored with the mFISH method.

In the F2-8 mouse, all of the 18 translocations detected
previously were t(1;19), but none of them was detected in
the present mFISH study. This is probably attributable to achance observation of analyzing 100 cells with mFISH
compared with 800 cells with 2-color FISH (the expected
number of translocations is 2.3 with the estimated prediction
interval of 0.645.6 to 5.6 in 100 cells if the observed
frequency 18/800 is regarded as the true frequency) (13).
DISCUSSION

Previously, a 2-color FISH method was used to examine
19 adult mice that were exposed to 2 Gy of X rays as fetuses
or neonates. The majority of the animals (9 out of 12 mice
irradiated as fetuses, and 6 out of 7 mice irradiated asneonates) had only 0–4 translocations which involved thepainted chromosomes 1 or 3 among 800 cells examined, ora genome-equivalent frequency (FG) of less than 2%. This
result is in sharp contrast to that of the mother (FG of 20%
and 25%) (8). In contrast, a minor fraction of the animals (3out of 12 mice irradiated as fetuses, and 1 out of 7 irradiatedas neonates) had 10 or more translocations among 800 cells(FG of þ5%), and the translocations were often identical as
confirmed with the present mFISH study (Table 1). Thus,
the frequency of cells with a translocation (without
correcting for the clonal aberrations) appears to show either
close to zero or larger than 5% in FG, and the latter often
contains clonal translocations. The results indicate thatwhen the stem cell pool of 15.5-day fetuses started torecover from radiation damage after an exposure to 2 Gy, asmall number of stem cells could record radiation damage
as persisting translocations and subsequently clonallyexpanded. The results are in line with the observations that
10–11-day-old fetuses already carry a large number ofhematopoietic stem cells that may contribute to at least 10%of adult hematopoiesis (14) whereas fetal stem pool still
does not seem to be a simple homogeneous cell population.
This observation is in sharp contrast to the findings
among the A-bomb survivors exposed after birth, namely,
clonal translocations were found preferentially among thosesurvivors with high frequencies of translocations (i.e., those
who received large doses of radiation) (9). This isreasonable because in adults, a large number of hematopoietic stem cells were already present at the time ofradiation exposure, and exposure to larger doses affected alarger fraction of the stem cell populations chromosomally.
It also facilitated clonal expansions of the surviving (andaffected) stem cells through the recovery processes taking
place after the extensive cell death caused by the exposure.
The present results shed light on a previous finding that
one A-bomb survivor exposed in utero was found to bearclonal aberrations; i.e., 6 translocations and 3 inversions
were identical out of 48 aberrant cells when 144 cells were
examined with the G-banding method (15). The mother’s
estimated uterine dose was 1.5 Gy with the DS02 dosimetry

CLONAL TRANSLOCATIONS IN MICE IRRADIATED AS FETUSES

system. These A-bomb results stood out and appeared
unusual, but are now supported by the present study.
The reasons why irradiated fetuses cannot record
radiation damage may be related to the specific nature ofhematopoitic tissues. Our recent studies indicate that both
rat mammary epithelial cells (Nakano et al., unpublisheddata) and mouse thyroid cells (Hamasaki and Kodama et al.,unpublished data) irradiated as fetuses do record radiation
damage when examined as adults. It is well known that
hematopoitic stem cells move during fetal life and mostenter the bone marrow niche several weeks after birth inmice (16). Additionally, their gene expression levels change
before and after settlement in the bone marrow niche. For
example, the expression level of the ATM gene is reportedly
considerably lower before entering the bone marrow niche(17). This observation indicates that hematopoietic stem
cells might be incapable of triggering the ATM-dependent
repair of so-called dirty double-strand breaks of DNA
induced by irradiation. This hypothesis might be testable bycreating animals with forced expression of the ATM gene(and/or additional related genes) in fetuses.
In summary, ordinary cytogenetic tests of blood T cells
are not applicable to retrospective biodosimetry for those
who were exposed to radiation as fetuses and possibly as
neonates. However, because it seems likely that othertissues with the exception of hematolymphoid cells may
record radiation damage after fetal irradiation, a possibility
remains to use cells that are available with the least invasive
method, such as skin cells.
ACKNOWLEDGMENTS
We thank Dr. Richard Wakeford for his valuable comments to the initial
draft, Dr. Leon Kapp for his careful reading of the manuscript andcomments, Mr. Munechika Misumi for his statistical help, Ms. M. Miura,
K. Muramoto, T. Matsumoto and Mr. S. Kaneoka for their excellent
technical assistance, and Ms. M. Utaka for manuscript preparation. The
Radiation Effects Research Foundation (RERF), Hiroshima and Nagasaki,
Japan, is a private, non-profit foundation funded by the Japanese Ministryof Health, Labour and Welfare (MHLW) and the U.S. Department ofEnergy (DOE), the latter in part through DOE award DE-HS0000031 to
the National Academy of Sciences. This publication was supported by
RERF Research Protocol RP 8–93. The views of the authors do notnecessarily reflect those of the two governments.
Received: May 22, 2012; accepted: July 17, 2012; published online:
November 8, 2012

REFERENCES
1. Stewart A, Webb J, Giles D, Hewitt D. Malignant disease inchildhood and diagnostic irradiation in utero. Lancet 1956; 2:447–8.

603

2. Stewart A, Webb J, Hewitt D. A survey of childhood
malignancies. Br Med J 1958; 1:1495–1508.
3. Bithell JF, Stewart AM. Pre-natal irradiation and childhood
malignancy: a review of British data from the Oxford Survey. Br
J Cancer 1975; 31:271–87.
4. Doll R, Wakeford R. Risk of childhood cancer from fetal
irradiation. Br J Radiol 1997; 70: 130–9.
5. Wakeford R, Little MP. Risk coefficients for childhood cancer
after intrauterine irradiation: a review. Int J Radiat Biol 2003;
79:293–309.
6. Ohtaki K, Kodama Y, Nakano M, Itoh M, Awa AA, Nakamura
N. Human fetuses do not register chromosome damage inflictedby radiation exposure in lymphoid precursor cells except for asmall but significant effect at low doses. Radiat Res 2004;
161:373–9.
7. Kodama Y, Pawel D, Nakamura N, Preston D, Honda T, Itoh M, etal. Stable chromosome aberrations in atomic bomb survivors:
results from 25 years of investigation. Radiat Res 2001; 156:337–
46.
8. Nakano M, Kodama Y, Ohtaki K, Nakashima E, Niwa O,
Toyoshima M, et al. Chromosome aberrations do not persist in thelymphocytes or bone marrow cells of mice irradiated in utero orsoon after birth. Radiat Res 2007; 167:693–702.
9. Nakano M, Kodama Y, Ohtaki K, Itoh M, Awa AA, Cologne J, etal. Estimating the number of hematopoietic or lymphoid stem cells
giving rise to clonal chromosome aberrations in blood T
lymphocytes. Radiat Res 2004; 161:273–81.
10. Salassidis K, Schmid E, Peter RU, Braselman H, Bauchinger M.
Dicentric and translocation analysis for retrospective dose
estimation in humans exposed to ionizing radiation during theChernobyl nuclear power plant accident. Mutat Res 1994; 311:39–
48.
11. Lucas JN, Poggensee M, Straume T. Translocations between two
specific human chromosomes detected by three-color ‘‘chromosome painting’’. Cytogenet Cell Genet 1993; 62:11–2.
12. Disteche CM, Carrano AV, Ashworth LK, Burkhart-Schultz K,
Latt SA. Flow sorting of the mouse Cattanach X chromosome, t(X;7) 1 Ct, in an active or inactive state. Cytogenet Cell Genet
1981; 29:189–97.
13. Krishnamoorthy K, Peng J. Improved close-form prediction
intervals for binomial and Poisson distributions. J Stat Plan Infer
2011; 141:1709–18.
14. Gothert JR, Gustin SE, Hall MA, Green AR, Gottgens B, Izon DJ,
et al. In vivo fate-tracing studies using the Scl stem cell enhancer:
embryonic hematopoietic stem cells significantly contribute toadult hematopoiesis. Blood 2005; 105:2724–32.
15. Ito M, Honda T. Cytogenetic study of in utero exposed individuals,
part 2. J Nagasaki Med Assoc 1986; 61:369–72.
16. Bowie MB, McKnight KD, Kent DG, McCaffrey L, Hoodless PA,
Eaves CJ. Hematopoietic stem cells proliferate until after birth andshow a reversible phase-specific engraftment defect. J Clin Invest
2006; 116:2808–16.
17. Bowie MB, Kent DG, Dykstra B, McKnight KD, McCaffrey L,
Hoodless PA, et al. Identification of a new intrinsically timed
developmental checkpoint that reprograms key hematopoietic stem
cell properties. Proc Natl Acad Sci U S A 2007; 104:5878–82.
Epub 2007 Mar 22.