RADIATION RESEARCH 186, 360–366 (2016)0033-7587/16 $15.00
Ó2016 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR14385.1

Irradiation at Different Fetal Stages Results in Different Translocation
Frequencies in Adult Mouse Thyroid Cells
K. Hamasaki,a R. D. Landes,b,c A. Noda,a N. Nakamuraa and Y. Kodamaa
Departments of a Molecular Biosciences and b Statistics, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku,
Hiroshima 732-0815, Japan; and c Department of Biostatistics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

implications of these results in interpreting cancer risks
after fetal irradiation are also discussed. Ó 2016 by Radiation

Hamasaki, K., Landes, R. D., Noda, A., Nakamura, N. andKodama, Y. Irradiation at Different Fetal Stages Results in
Different Translocation Frequencies in Adult Mouse Thyroid
Cells. Radiat. Res. 186, 360–366 (2016).

Research Society

While it is generally believed that fetuses are at high riskof developing cancers, including leukemia, after low dosesof radiation, it has been reported that atomic bomb
survivors exposed in utero did not show a dose response
for translocations in blood T lymphocytes when they were
examined at approximately 40 years of age. Subsequentmouse studies confirmed that animals irradiated during thefetal stage did not show evidence of radiation effects inlymphocytes and bone marrow cells when they were
examined after reaching adulthood. However, in a studyof rat mammary epithelial cells, radiation effects wereclearly observed after fetal irradiation. These results
indicate that the fate of chromosome aberrations induced
in a fetus could vary among different tissues. Here we
report on translocation frequencies in mouse thyroid cells,
which were irradiated at different stages of fetal development. Cytogenetic examination was conducted using fluorescence in situ hybridization (FISH) painting ofchromosomes 1 and 3. Adult mice, 2 Gy X-ray irradiatedat 15.5-day-old fetuses (E15.5), showed a higher translocation frequency (30/1,155 or 25.3 3 103) than nonirradiatedadult controls (0/1,007 or 0.1 3 103), and was near thatexperienced by irradiated mothers and non-pregnant adultfemales (43/1,244 or 33.7 3 103). These results are
consistent with those seen in rat mammary cells. However,when fetuses were irradiated at an earlier stage ofdevelopment (E6.5) before thyroid organogenesis, theresulting observed translocation frequency was much lower(3/502 or 5.8 3 103) than that in E15.5 mice. These results
suggest that after fetal irradiation, tissue stem cells record
radiation effects primarily when the exposure occurs incells that have been integrated into tissue. Embryonic stem
cells that have been damaged prior to integration into theniche may undergo negative selection due to apoptosis,
mitotic death or stem cell-niche cell interactions. The

INTRODUCTION

Embryos or fetuses are considered to be highly sensitive
to radiation and can undergo developmental anomalies ordeath, and the threshold dose for malformation to occur inhuman fetuses is estimated to be around 0.1 Gy (1). Studiesof radiation risks leading to cancer after fetal irradiation
originated in the Oxford Survey of Childhood Cancers(OSCC), which was initiated in the 1950s. Specifically, the
OSCC study was a case-control study, and the results
indicated that women whose children later died fromleukemia or cancer before the age of ten exhibited a slightly
higher frequency of having received diagnostic X-rays (e.g.,;15%) during pregnancy compared to mothers whosechildren had no such diseases (e.g., ;10%) (2, 3). The Xray doses to fetuses were later estimated to be approximately 10 mGy (4–7). Therefore, the relative risk ofdeveloping childhood leukemia or cancer was estimated as
1.5 at 10 mGy. In other words, the relative risk at 1 Gy
could increase to as high as ;50 if the increased risks werecaused by X-ray exposures and a linear dose response isassumed (4, 7). The estimated relative risk is more than one
order of magnitude higher than that observed for solidcancers seen in atomic bomb (A-bomb) survivors who were
exposed as adults (8), although such a large relative risk is
not unprecedented. Specifically, the relative risk forleukemia, which appeared within a few years after A-bomb
exposure among those who were at the age of approximatley 10 years, was estimated to be over 50 at 1 Gy (9).
However, as for solid cancers, the magnitude of theincreased relative risk seems substantially smaller than thatof leukemia (10, 11).
After the OSCC study, a number of similar case-control
studies were conducted at various locations around theworld, and the results were consistent while the observed
relative risks were small (e.g., 1.13–1.70) (5, 7). Therefore,

Editor’s note. The online version of this article (DOI: 10.1667/
RR14385.1) contains supplementary information that is available to
all authorized users.
1 Address for correspondence: Department of Molecular Biosciences, Radiation Effects Research Foundation, 5-2 Hijiyama park,
Minami-ku, Hiroshima 732-0815, Japan; email hamasaki@rerf.or.jp.
360

FETAL IRRADIATION AND TRANSLOCATION FREQUENCY

it appeared to be generally accepted that fetuses exposed todiagnostic X-rays would have higher risks of developingchildhood leukemia and solid cancer. However, while
epidemiological case-control studies can indicate theexistence of an association between fetal radiation exposureand subsequent risks for childhood leukemia and solidcancer, they do not prove a causal association. In fact,
subsequent animal studies have consistently demonstrated
no indication of such extremely high cancer risks after fetal
irradiation (1, 12, 13). The challenge to investigate thisdiscrepancy is ongoing.
Epidemiological studies of A-bomb survivors have notprovided definitive evidence to support the extremely highrisks of childhood cancers (including leukemia) after
exposures in utero (14, 15–17). To explain the apparent
disparate results, various possibilities have been considered,
which include selective loss of radiosensitive individuals
from the survivor cohort due to exposure to relatively large
doses of radiation (18). However, it is now understood thatthe cohort of A-bomb survivors exposed in utero is notlarge enough to negate or support the OSCC data (14);therefore, the results are not incompatible with the OSCC
results (4).
In another published study, translocation frequencies were
examined in blood lymphocytes from about 300 A-bomb
survivors who had been exposed in utero. The translocation
frequencies were examined when the survivors were approximately 40 years old. Contrary to expectations, the translocation frequencies did not increase with an increase in radiationdose (19). Because the mothers showed a clear pattern of doserelated increases in translocation frequencies, the possibility of
dosimetry errors to the fetuses was formally excluded.
Subsequent mouse studies confirmed the survivor data; i.e.,
when mice were irradiated as 15.5-day fetuses (E15.5) andexamined when they reached adulthood (20 weeks old),translocation frequencies were quite low, as was observed in
A-bomb survivors exposed in utero (20). It was thought that
this lack of radiation effect might be unique to hematolymphoid cells, and subsequent experiments focused on mammary
gland epithelial cells in rats. In that study, it was reported thatthe irradiated rat fetuses did show radiation effect in breast
tissues when examined as adults, and the translocation
frequency was nearly the same as that in the mothers as well
as females irradiated as adults (21). These studies may wellindicate that the persistence of radiation-induced chromosome
aberrations after fetal irradiation depends on the tissues being
examined. Additionally, with respect to the settlement of tissue
stem cells into the niche resulting in a functional tissue organ,
the behavior of hematopoietic stem cells (HSCs) during fetal
development is believed to differ from that of nonhematopoietic cells; thus, radiation effect differences among tissues
may also be related to irradiation timing relative to the tissue
developmental stage.
Because of the known susceptibility of the thyroid toradiation-induced carcinogenesis (22) and well-establishedculture methods for primary mouse thyroid cells (23), we

361

chose thyroid epithelial cells as the second nonlymphoidtissue to examine the radiation effects in those exposed as
fetuses, that is, to determine if the frequency was low as inlymphoid cells or high as in mammary epithelial cells. We
also considered the timing of in utero irradiation relative toorganogenesis in both thyroid epithelial cells and spleen
lymphocytes.
MATERIALS AND METHODS
Mice and Irradiation
Pathogen-free B6C3F1 mice (8 weeks old, both male and female)
were purchased from the Experimental Animal Co. (Hiroshima,
Japan). Approximately two weeks after their arrival, mice were mated,and pregnant females at day 6.5 (E6.5 fetuses) or 15.5 (E15.5 fetuses)
after mating were whole-body X-ray irradiated with 2 Gy. This study
used a Shimadzu HF-320 machine (Kyoto, Japan) operating at 220
kVp, 8 mA, with a 0.5 mm aluminum and 0.3 mm copper filter at adose rate of 1.0 Gy/min, and a Faxitront CP-160 machine (Tucson,
AZ) operating at 160 kVp, 6.3 mA with a 0.5 mm aluminum and 0.21
mm copper filter at a dose rate of 0.77 Gy/min. Nonpregnant adultfemale mice (9–15 weeks old) were also irradiated to evaluate
translocation frequencies after adult exposures. We examined 96 mice
after fetal irradiations (74 mice at E15.5 and 22 mice at E6.5), 17
mothers, 61 irradiated adult females (nonpregnant) and 36 nonirradiated adult females (control group) (Table 1). All mice were kept at an
animal facility in the Radiation Effects Research Foundation (RERF;
Hiroshima, Japan) and housed in sterile cages placed in a microisolator and fed a sterile regular diet ad libitum. The study plan wasapproved by the Experimental Animal Care Committee at RERF.
Preparation of Primary Cultures of Thyroid Epithelial Cells
When the animals irradiated at day E6.5 or E15.5 reached 8 to12
weeks old, mice were euthanized with carbon dioxide and the thyroid
lobes were removed (timeline shown in Fig. 1). Primary cultures were
established with the method described in Jeker et al. (23) with slight
modifications. Briefly, minced lobes from several animals of the samegroup (exposed or control) were mixed and digested with type 1A
collagenase (125 U/ml, Sigma-Aldricht, St. Louis, MO) and Dispase I(500 PU/ml; Sanko Junyaku, Japan) dissolved in 1 ml of Eagle’s
minimal essential medium (EMEM) (Wako Pure Chemical Industries,
Osaka, Japan) for 15 min in a 378C incubator while the tubes were
gently rotated. Moderately dispersed lobes were then plated into 24-wellculture dishes (one lobe equivalent was plated into 2 wells). Culture
media consisted of Ham’s F12 media (Sigma-Aldrich) supplemented
with 10% fetal bovine serum (FBS) (HyClonee Laboratories, Logan,
UT), 5 lg/ml of human transferrin (Sigma-Aldrich), 10 lg/ml of bovine
insulin (Sigma-Aldrich), 4 ng/ml of hydrocortisone (Wako), 10 ng/ml ofsomatostatin (Sigma-Aldrich), 2 ng/ml of Gly-His-Lys acetate salt
(Sigma-Aldrich) and 0.2 mIU/ml of thyrotropic hormone from bovine
pituitaries (Sigma-Aldrich). In addition, 50 lg/ml of gentamicin
antibiotic (Sigma-Aldrich) was added. After culturing for one day,dishes were washed with phosphate buffered saline (PBS) and the
media was replaced with fresh media. Thyroid epithelial cells were
cultured for about 65 h at 378C in a 5% CO2 incubator before they were
harvested for chromosome tests.
Spleen T Lymphocytes
Spleens were also removed from some animals, and spleen cells
were cultured as described previously (20). Briefly, spleen cells weresuspended in RPMI 1640 media supplemented with 20% FBS andphytohemagglutinin (PHA) (0.18 mg/ml; Remel Europe Ltd./Thermo
Scientific, Basingstoke, UK). Cells were then cultured for 26 h.

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HAMASAKI ET AL.

TABLE 1
Summary Results of Mice 2 Gy X-Ray Irradiated as Fetuses in Thyroid StudyAge at 2 Gy irradiation
Number of cultures
Number of mice
Number of cells
Number of stable events
Stable frequency (95%CI)
Number of unstable events
Unstable frequency (95% CI)

Fetuses (E6.5)

Fetuses (E15.5)

Adultsa (9–15 week)

No irradiation (controls)

6
22
502
3b
5.8 (1.5, 14.7)
00.3 (0, 4.5)

11
74
1,155
30
25.3 (17.3, 35.5)4
3.4 (1.1, 7.9)

18
78
1,244
43
33.7 (24.5, 44.9)
33
25.9 (18.0, 35.8)

6
36
1,007
0
0.1 (0, 2.3)00.1 (0, 2.3)

Note. Stable and unstable aberration frequencies estimated by the Bayesian model are expressed per 1,000 cells.
aThese include mothers of the irradiated fetuses.
b
These include one clonal event, which was found in six cells.
Cell Harvest and Slide Preparation
Colcemid (100 ng/ml; Serva Electrophoresis, Heidelberg, Germany)
was added during the last 18 h of the thyroid cell cultures and the last
2 h of the spleen cell cultures. Cells were then harvested after trypsin
treatment, treated with a hypotonic solution of potassium chloride, andfixed with 3:1 methanol/acetic acid. Chromosome slides were madeusing a standard air-drying method (20).
Fluorescence In Situ Hybridization (FISH)
FISH painting of metaphase chromosomes was performed aspreviously described (20). Briefly, metaphase chromosomes weredenatured and hybridized for 18 h with whole DNA probes (Applied
Spectral Imaging Inc., Carlsbad, CA) for mouse chromosomes 1 and 3
labeled with FITC and Rhodamine, respectively. Slides were thenwashed and counterstained with DAPI in an anti-fade/glycerol solution
and stored at 208C until use. In most cases, 100–200 metaphase cells
were scored with FISH per culture, which consisted of thyroid cells
from about six mice. Aberrant chromosomes bearing color changes
were detected in the arms of FISH-painted chromosomes (i.e.,translocations, insertions and dicentrics). Chromosome aberrations werecounted, as we have described previously (20); translocations anddicentrics were counted as single events while insertions were counted
as two translocations. When cells bearing apparently identicaltranslocations in terms of breakpoint locations in FISH-labeled andnonlabeled chromosomes were observed, we considered these to beclonal cells and the aberrations were counted as a single event.
Immunostaining of Cultured Thyroid Cells
To confirm that the cells in culture were primarily derived fromthyroid epithelial cells, thyroglobulin protein levels were examined withimmunostaining. Briefly, cells cultured in slide chambers (8 chambers
per slide) were fixed and incubated with polyclonal rabbit anti-human
thyroglobulin antibody (Dako Inc., Carpinteria, CA), and subsequently
with Alexa Fluort 488-labeled goat anti-rabbit IgG (HþL) (Molecular
Probest, Grand Island, NY) as the second antibody. More than 90% of
cultured cells showed the same levels of thyroglobulin expression as
observed in FRTL-5 cells (a thyroid cell line derived from Fisher rats)
used for positive control even on day 10 after the initiation of thecultures (unpublished results). Therefore, the cells harvested after 65 hof culture and used for chromosome analyses were considered to be
primarily thyroid epithelial cells.

mice; the median number of animals represented in each culture was 6.
In each culture, two types of aberrations were counted: stable type(translocations, insertions) and unstable type (dicentrics). For eachaberration type, the number of aberrations and the number of cells perculture were counted. The observed aberrations were modeled with
Poisson distributions. The single parameter in each treatment groupspecific Poisson distribution was Vk, where k represents the treatmentgroup aberration frequency (expressed per 1,000 cells). Using a
Bayesian approach to the data analysis, the 8 ks (4 treatment groups 3
2 types of aberrations) were modeled with a gamma distribution
having hyperparameters a and b. Finally, independent uniform (0, 106)
priors were assigned to a and b. For purposes of comparison, ratios ofaberration frequencies and their 95% credible intervals (CIs) arepresented. (Credible interval is a Bayesian term analogous to aconfidence interval.) We analyzed counts of aberrations in spleen T
lymphocytes with a model similar to that used in the thyroid study.
Differences were: 1. Cells from a single mouse rather than pooled cells
from several mice provided the V cells; and 2. Only stable aberrations
were counted, thus, there were 4 ks instead of 8 ks, as in the thyroid
study. In both the thyroid and spleen studies, CIs for ratios notcontaining the null value, 1, were statistically significant at the 0.05
significance level. WinBUGS version 1.4 was used to perform theseanalyses, and sensitivity analyses were performed to evaluate theimpact of the priors on the final results. A more thorough descriptionof the statistical model and WinBUGS code used in this analyses isfound in the Supplementary Information (http://dx.doi.org/10.1667/
RR14385.1.S1).

RESULTS

Because thyroid lobes from several mice (approximately
6) were pooled for primary cultures due to the difficulty of
establishing primary cultures from single mice, the results
including summary statistics and estimated aberration

Statistical Analyses
In the thyroid study, there were four treatment groups: nonirradiatedcontrols; mice irradiated as adults, at E15.5 or E6.5. From each of thefour treatment groups (Table 1), cultures of thyroid cells wereobtained. Each culture contained V cells pooled from between 1 to 9

FIG. 1. Timeline of the embryonic stages at irradiation and at
tissues harvesting for chromosome analysis.

FETAL IRRADIATION AND TRANSLOCATION FREQUENCY

frequencies (per 1,000 cells) are shown with the group ofanimals (i.e., irradiated or nonirradiated and age at
exposure; see Table 1). Specifically, the estimated frequencies of stable-type aberrations (primarily translocations andsome insertions involving chromosome 1 or 3) were
estimated to be very near 0 per 1,000 cells in nonirradiatedadult female mice (i.e., controls).2 In the females irradiatedas adults (including the mothers), the stable-type aberration
frequency of 33.7 per 1,000 cells was 249 times greater[95% CI (13.8, .108)] than controls; and the frequency of
E15.5-irradiated mice (examined at 8 to 12 weeks of age)was 25.3 per 1,000, which was 188 times greater [25.3, 95%
CI (10.3, .108)] than controls. The stable aberration
frequency after 2 Gy irradiation in adult females was 1.3
times higher than in adult mice irradiated as E15.5 fetuses,
but the difference was not statistically significant [CI 95%(0.8, 2.1)]. Therefore, adult mouse thyroid cells that were 2
Gy X-ray irradiated at E15.5 tended to retain radiationinduced stable-type aberrations at levels similar to theirmothers.
Noting that timing of irradiation may be a reason fordifferences seen in radiation effects among tissues, we also
explored timing of irradiation in thyroid development.
Specifically, prior to thyroid organogenesis, E6.5 fetuses
were 2 Gy irradiated using the same methods as for theE15.5 (post-organogenesis) mice. The E6.5 group’s stabletype aberration frequency was 5.8 per 1,000, which wassignificantly higher than that in the control group’s [by 43
times, 95% CI (1.8, .107)], but was significantly lower by
0.23 times, 95% CI (0.06, 0.63) the frequency in E15.5
group. Consequently, it can be concluded that, in contrast to
thyroid cells irradiated at a post-organogenesis stage,
precursor cells for thyroid glands irradiated at a preorganogenesis period were less capable of recording
radiation effects.
Translocation frequencies were also examined in adultspleen cells of mice irradiated during the fetal stage (Table2) or as adults. Compared to their mothers’ frequency of
48.3 per 1,000, the frequency of 3.4 per 1,000 in adults thatwere irradiated in utero at E15.5 was 0.071 times [95% CI(0.047, 0.103)] the mothers’ frequency. Similar results wereobtained from adult mice irradiated in utero at E6.5;
namely, E6.5 group’s frequency of 1.1 per 1,000 was 0.023
times [95% CI (0.009, 0.048)] the mothers’ frequency.
Further, the frequency from E6.5 irradiated mice was 0.32
times [95% CI (0.120, 0.711)] that from E15.5 irradiatedmice. For adult mice having never been irradiated (i.e.,
controls), the observed translocation frequency was 0.0[95% CI (0.0, 0.9) per 1,000 cells]. Of note, clonaltranslocations were found after irradiation in both theE15.5 fetuses as well as the E6.5 fetuses, although the
2 The observed frequency was 0; however, the Bayesian model
estimated the frequency to be slightly above 0. This type of result
also occurred for unstable aberration frequencies in controls and E6.5
mice (see Table 1), and for control aberration frequencies in the
spleen (see Table 2).

363

frequency of clonal events was definitely lower than thatafter adult irradiations (Table 2)2.
Although we did not statistically compare translocation
frequencies between thyroids and spleens within adult mice
exposed to irradiation in utero, the thyroid appears to be
more susceptible to induction of stable-type aberrations by
irradiation; within each of the E6.5 and E15.5 irradiatedmouse groups, the thyroid stable aberration frequency was,respectively, over 5 and 7 times greater than thecorresponding spleen’s frequency (comparing estimatedfrequencies within treatment groups between Tables 1 and2).
Unstable-type aberrations (dicentric chromosomes) werealso detected in the thyroid cell cultures (Table 1). In
contrast to the results for stable-type aberrations, considerable difference was observed in the frequencies between
mice exposed as E15.5 fetuses (3.4 3 103) and adults,
including the mothers (25.9 3 103). Because cells bearing
unstable-type aberrations are subject to negative selection
after cell divisions, these results likely reflect the fact that
adult mice irradiated as fetuses had more thyroid cell
divisions than adult mice irradiated as adults.
DISCUSSION

During the development of most organs in the mouse,tissue stem cells settle into the appropriate niche and begin
to function at E7–E14 (the organogenesis stage), but the
precise timing for migration, settling and subsequentdifferentiation may differ among tissues (24). The thyroid
gland is already observed in E8.5 fetuses and the glandundergoes a split into multiple buds by E15, but with nofollicle formation yet (25). In contrast, the hematolymphoidsystem is exceptional because the majority of HSCs do not
locate into their bone marrow niches prior to birth, but onlyat 3–4 weeks after birth (26).
Given these considerations, past and present results canbe interpreted among other possibilities, as follows. Whenfetal stem cells were irradiated before migrating into their
final niches, most of the aberrant cells could have been lost
and were consequently unable to participate in organogenesis. Examples are lymphoid and bone marrow cells
irradiated at the fetal stage in mice (20), blood lymphocytes
in A-bomb survivors exposed in utero (19) and mousethyroid cells irradiated at E6.5 (in the current study). In
contrast, when fetal stem cells were irradiated after locating
into their niches, aberrant cells were able to persist as they
did in the mothers, and subsequently contribute toorganogenesis as was seen in rat mammary cells (21) andmouse thyroid cells (in the current study using irradiation at
E15.5). Also, Jacquet et al. reported that cells from mouseembryos irradiated at day E8 and examined 7 h later showed
high frequency of chromatid aberration but very low
frequency of exchange-type chromatid aberration (27),which is an indication of deficient DNA rejoining. Thismodel is shown schematically in Fig. 2.

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HAMASAKI ET AL.

FIG. 2. Schematic model of the observed differences intranslocation frequencies in different tissues of adult mice after fetal
irradiation. Panel A: When stem cells are irradiated before
implantation into the niche, damaged cells are negatively selected
and can only poorly contribute to organogenesis. Panel B: In contrast,
such a negative selection does not occur when stem cells are irradiatedafter implantation into the niche.

An important question remains concerning the mechanism that might be involved in the elimination of aberrant
stem cells from organogenesis when they are irradiated prior
to settling into their niches in developing organs. Two
alternative models may be proposed to explain theobservations. In one model, stable-type aberrations (as wellas unstable-type aberrations) are actually induced after
misrepair of radiation-induced DNA double-strand breaks(DSBs), but they are subsequently negatively selectedagainst. For example, translocation-bearing cells may carry
persistent subtle abnormality and be outcompeted by
nonaberrant cells, as was reported by Bondar andMedzhitov between irradiated and nonirradiated HSCs
(28). A priori, replicative disadvantage of the aberrant cells

as well as defective interactions between stem cells and theniche can be the cause. In the second model, DNA breaks
are induced, but efficient DNA DSB repair, which may leadto formation of exchange-type aberrations, does not takeplace effectively in the immature precursors or stem cells. In
this scenario, persistent DNA breaks can cause either
mitotic death after cell division or apoptotic death without
division. This possibility is supported by the following
reported findings. In one study, early mouse embryoniccells (E6.5 and E7.5) but not extra-embryonic cells
underwent ATM- and p53-dependent apoptosis after lowdose irradiation (,0.5 Gy) (29). And in another study,
mouse HSCs from fetal liver (E14.5) expressed relatively
low levels of the ATM gene product, one of the key players
involved in the repair of DNA DSBs, compared to HSCs
from the bone marrow of postnatal mice (30).
The seemingly dissimilar findings between the epidemiological data (high radiosensitivity for cancer induction) andanimal data (low yield of chromosome aberrations) requires
an explanation. One possible explanation is that acytogenetic end point was not appropriate to indicate
susceptibility to developing leukemia after irradiation
because the underlying mechanisms leading to aberrations
may be different from those that lead to somatic mutations
relevant to carcinogenesis. Alternatively, fetal lymphoidstem or progenitor cells that had acquired translocations
spontaneously, specific to childhood leukemia (i.e., preleukemic cells) (31), are under altered physiological conditions
compared to their normal counterparts and are notsusceptible to negative selection after radiation-induced
DNA breaks. A third possibility is that a small fraction of
HSCs is already active in an early stage of embryogenesis.
We have previously reported on the presence of clonaltranslocations observed in adult mice that were irradiated as
fetuses on E15.5 (32). In the current study, we confirmedthat irradiation of E15.5 fetuses gives rise to clonaltranslocations, but this was also the case after irradiationof E6.5 fetuses (Table 2).2 A fourth possibility is that theapparently increased risks seen in the OSCC study and otherrelated studies may not have been caused by low-dose X ray
exposure, but by other unknown confounding factors.

TABLE 2
Summary Results of Mice Mice 2 Gy X-Ray Irradiated as Fetuses in Spleen Lymphocyte StudyAge at 2 Gy irradiationb

Number of mice
Number of cells
Number of stable events
Stable frequency (CI 95%)

Fetuses (E6.5)

Fetuses (E15.5)

Adultsa (9–15 week)

No irradiation (controls)

7
5,550
6c
1.1 (0.4, 2.2)

11
8,763
30d
3.4 (2.3, 4.8)

4
3,097
151e
48.3 (41.0, 56.5)

3
2,400
0
0.0 (0.0, 0.9)

Note. Frequencies of stable-type aberrations (translocations and insertions) were estimated by the Bayesian model and are expressed per 1,000
cells.
aMothers of irradiated fetuses.
b
All mice used for the spleen study were part of the group used for thyroid cell cultures shown in Table 1.
c
These include three clonal events, each of which was found in 14–20 cells out of 800.
d
These include eight clonal events, each of which was found in 3–5 cells except for one which was found in 18 cells out of 800.
e
These include 17 clonal events, each of which was found in 3–18 cells out of 800.

FETAL IRRADIATION AND TRANSLOCATION FREQUENCY

While the etiology of childhood leukemia has yet to beclarified, it is well established that risks are affected byvarious factors including ethnic group, socioeconomic
status, birth order and maternal age, among others (33,34). Among the potential confounding factors, birth weight
is of greatest interest. While large fetal size has traditionallybeen viewed as a strong indicator of healthy development,
the combination of large fetal size and small pelvis size ofthe mother may have compelled obstetricians to obtainpelvimetric X-ray photographs as they decided on approaches to Caesarean section. Later on, heavy birth weight
was associated with an increased risk of developingchildhood leukemia (35, 36) and probably solid cancers aswell (37), which can be attributed to higher levels ofmaternal growth factors such as IGF.
In at least one reported study, birth weight was taken into
account in estimating radiation risks after fetal X-ray
exposure and the conclusion remained the same (38).
Nevertheless, the issue is complicated and knowledge of thebirth weights from the original OSCC data would be ofsignificant help to better understand the biological mechanisms involved in increased cancer risks after low-dose
irradiation of the fetus. This data collection process iscurrently ongoing (39).
SUPPLEMENTARY INFORMATION

Description of the Bayesian analyses for this study.
ACKNOWLEDGMENTS
The authors are grateful to Ms. K. Muramoto, T. Matsumoto and Mr. S.
Mishima for excellent technical assistance, to Dr. K. Hamatani and Mr. K.
Koyama for valuable advice for thyroid cultures, to Mr. N. Kajitani forhelp with animal care, to Drs. R. Ullrich and L. Kapp for careful reading of
the manuscript and to Ms. M. Utaka for manuscript preparation. Dr. R.
Wakeford and one anonymous reviewer are also acknowledged for theirinsightful comments. The Radiation Effects Research Foundation (RERF),
Hiroshima and Nagasaki, Japan is a public interest foundation funded by
the Japanese Ministry of Health, Labor and Welfare (MHLW) and the U.S.
Department of Energy (DOE). This research was also funded in partthrough DOE award no. DE-HS0000031 to the National Academy of
Sciences. This publication was supported by RERF Research Protocol
RP6-11. The views of the authors do not necessarily reflect those of thetwo governments. This work was supported in part by JSPS KAKENHI
grant no. 24710067: Grant-in-Aid for Young Scientists (B).
Received: January 18, 2016; accepted: June 17, 2016; published online:
September 14, 2016

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