RADIATION RESEARCH 181, 172–176 (2014)0033-7587/14 $15.00
Ó2014 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR13446.1

Fetal Irradiation of Rats Induces Persistent Translocations in Mammary
Epithelial Cells Similar to the Level after Adult Irradiation, but not in
Hematolymphoid Cells
Mimako Nakano,a Mayumi Nishimura,b Kanya Hamasaki,a Shuji Mishima,a Mitsuaki Yoshida,c Akifumi Nakata,cYoshiya Shimada,b Asao Noda,a Nori Nakamuraa and Yoshiaki Kodamaa,1aDepartment of Genetics, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Miami-ku, Hiroshima 732-0815, Japan; b Radiobiology forChildren’s Health Research Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa,Inage-ku, Chiba, 263-8555, Japan; and c Department of Radiation Biology, Institute of Radiation Emergency Medicine, Hirosaki University,
Hirosaki 036-8564, Japan

does not contribute to the increased risk of cancer since it has
been reported that irradiation of fetal rats of the SD strain
does not increase the risk of mammary cancers. Possible
reasons for this discrepancy are discussed. Ó 2014 by Radiation

Nakano, M., Nishimura, M., Hamasaki, K., Mishima, S.,
Yoshida, M., Nakata, A., Shimada, Y., Noda, A., Nakamura, N.
and Kodama, Y. Fetal Irradiation of Rats Induces PersistentTranslocations in Mammary Epithelial Cells Similar to theLevel after Adult Irradiation, but not in Hematolymphoid
Cells. Radiat. Res. 181, 172–176 (2014).

Research Society

In both humans and mice, fetal exposure to radiation fails
to induce a persistent increase in the frequency of chromosome aberrations in blood lymphocytes. Such a low-level
response to radiation exposure is counterintuitive in view ofthe generally accepted belief that a fetus is sensitive toradiation. To determine if this is a general phenomenon, bothmammary epithelial cells and spleen cells were studied inrats. Fetuses of 17.5 days postcoitus were irradiated with 2
Gy of gamma rays, and mammary tissues were removed 6–45
weeks later. Subsequently, short-term cultures were established to detect translocations using the two-color FISH
method. The results showed that translocation frequencies
were not only elevated in rats irradiated as fetuses, but werealso almost as high as those in rats that were irradiated as
adults (12 weeks old, pregnant mothers or young virgins) andexamined 6–45 weeks later. There was no evidence of higher
sensitivity in fetal cells with respect to the induction oftranslocations. In contrast, translocation frequencies in
spleen cells were not elevated in adult rats irradiated as
fetuses but were increased after irradiation of adults aspreviously seen in mouse spleen cells and human T
lymphocytes. In the case of irradiation of adult rats, theinduced translocation frequencies were similar between
spleen cells and mammary epithelial cells. If we taketranslocation frequency as a surrogate marker of potential
carcinogenic effect of radiation, the current results suggestthat fetal irradiation can induce persistent potential carcinogenic damage in mammary stem/progenitor cells but thisINTRODUCTION

It is generally believed that the fetus is sensitive toradiation exposure. This belief is derived from two main
sources. The first is the classic observation in the early 20th
century by Bergonié and Tribondeau that the degree ofradiation sensitivity is inversely related to the degree ofdifferentiation of a cell; e.g., bone marrow or male germ
cells which consist of actively dividing stem and progenitor
cells can be sterilized with relatively small doses ofradiation while terminally differentiated cells such asmuscle and nerve cells require much larger doses (1). A
fetus must contain a proportionately larger number of
immature, actively dividing cells than an adult, and may
well be sensitive to the killing effects of radiation. This inturn leads to the supposition that a fetus is also sensitive tosome of the adverse effects of radiation such as cancer
induction. The second source is derived from epidemiologic
case-control studies that indicated diagnostic low-dose
exposures to the pelvic region of pregnant women (therefore
to the fetus as well) on the order of 10 mGy increase the riskof childhood leukemia and cancer by 30–50% above
background in the irradiated offspring (2, 3). This result
corresponds to a huge relative risk of 30–50 per Gy if alinear dose response is assumed.
However, despite these observations, it was previously
observed that atomic bomb (A-bomb) survivors exposed toradiation as a fetus did not show elevated frequencies oftranslocations in blood lymphocytes when examined atabout 40 years of age, although they had clear mothers

Editor’s note. The online version of this article (DOI: 10.1667/
RR13446.1) contains supplementary information that is available to
all authorized users.
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.
172

TRANSLOCATION INDUCTION AFTER FETAL IRRADIATION OF RATS

evidence of dose response (4). These findings wereconfirmed in subsequent studies using mice, which showedthat not only irradiated fetuses, but also neonates, wererefractory to preserving or expressing a record of radiation
damage. The low yield of translocations was seen not onlyin blood T cells, but also spleen cells and bone marrow cells(5). A cautionary note should be sounded here as newborn
mice are relatively immature when compared to humans,and consequently experimental results from mouse neonates/infants may not necessarily apply to humans. Because
those studies were restricted to hematolymphoid cells inboth human and mouse studies, the question arises ofwhether the findings were common to different types ofcells or restricted to limited cell types. In view of this,testing this model on epithelial cells appeared to beimportant. Among candidate animals and tissue models,mammary tissues were chosen because breast cancer shows
one of the highest relative risks among malignancies in Abomb survivors after radiation exposures (6). Because
mammary tumorigenesis in mice has been indicated to
involve mammary tumor virus (7), which is not relevant in
humans, rats are frequently utilized for such investigations.
In addition, culture methods of rat mammary epithelial cells
are well established (8), and rats would provide data as thethird species after humans and mice in regard toinvestigation of the refractory nature of hematolymphoidcells in recording radiation damage when irradiated as a
fetus.
MATERIALS AND METHODS
All animals were treated in accordance with the Safety and HealthRegulations compiled by the Animal Care and Use Committee at the
National Institute of Radiological Sciences (NIRS). Sprague Dawley
strain male and female rats (10 weeks old) were purchased from
CLEA Japan, Inc. (Tokyo, Japan) and housed in a rat facility at the
NIRS. Virgin or pregnant female rats [17.5 days postcoitus (dpc)]
were exposed at the NIRS to 2 Gy of 137Cs gamma rays (0.5 Gy/min)when they were 12 weeks old. Adult animals and the offspring weremaintained for 6, 9 or 45 weeks after irradiation and a total of 35
female rats were examined. Specifically, 23 mice were irradiated as
fetuses, seven were irradiated as adults (i.e., five as mothers and two as
virgins), and five were nonirradiated controls. Animals were thensacrificed under anesthesia with ether and mammary glands were
removed. Removed tissues were minced with surgical scissors andtreated overnight with collagenase (0.2% type II/III: [(Worthington)dissolved in DMEM/F-12 (50:50) medium in the presence of 5% fetal
bovine serum (FBS) and 50 lg/ml Gentamicin]. Incubation times ofapproximately 14 h in a humidified CO2 incubator at 378C were
utilized. After digestion, cells were passed through stainless mesh(pore size: 77 lm) to remove undigested tissues or clumps, and weresuspended in a culture medium, which consisted of DMEM/F-12
supplemented with 10% FBS and other factors (insulin, apotransferrin, prolactin, mouse EGF, progesterone, hydrocortisone andascorbic acid) as specified by Darcy et al. (8). Cells were inoculated
into 10 cm culture dishes at a density of 1 to 4 3 106 cells with 10 mlof medium. Within 2–9 days (usually within 2–5 days) of incubation,
the cultures were treated overnight with colcemid (0.02 lg/ml) andthen removed with trypsin. Chromosome slides were preparedfollowing standard hypotonic treatment and fixation with Carnoy’s
solution (5).

173

For FISH analysis, whole-chromosome probes for chromosome 2(FITC) and chromosome 4 (Rhodamine) were used (Applied Spectral
Imaging, Carlsbad, CA). These were the probes that were commercially available for the two largest chromosomes. For each sample,
800 cells were examined bearing at least a pair of chromosomes 2 and4. The FISH technique measures the frequency of translocations with
one breakpoint in a painted chromosome (5).
Spleen cells were also examined in some animals. Culture methods
have been described previously (5) and were used with minor
modifications. Briefly, spleen cells were suspended in RPMI 1640
medium supplemented with 20% FBS, phytohemagglutinin (PHA;
0.18 mg/ml, Murex Diagnostics, Inc.), 2-mercaptoethanol (50 lM),
Concanavalin A (5 lg/ml), and lipopolysaccharide (LPS; 10 lg/ml).
They were cultured for 48 h. The cultures were treated with colcemid(0.02 lg/ml) for the final 24 h before preparation of slides. Because
both PHA and LPS were used, T and possibly B cells were expected torespond to the stimuli and were subjected to FISH analysis. A total of
800 cells were scored for each sample.
When apparent clonal aberrations were detected, cells wereanalyzed again after treatment of the slides for Q-banding analysis(9). This was to identify the unpainted chromosome involved in thetranslocation by referring to the rat karyotype (10). When the results
failed to identify the unpainted chromosome, FISH examinations wereconducted anew with probes specific to the suspected (unpainted)
chromosome involved in the translocation. For calculation oftranslocation frequencies, clonal aberrations were counted as single
events.
To determine the histologic origin of cells in the mammary cultures,a portion of the cell population was isolated in a few cell cultures at
the time of slide preparation and the cells subjected to immunohistochemical staining. Anti-human cytokeratin antibody (aCK18 Ab,
Abcam, Tokyo, Japan) was used as the primary antibody to stain
epithelial cells (11). Anti-vimentin antibody (Dako Japan, Inc., Tokyo,
Japan) and anti-smooth muscle Actin antibody (aSMA Ab, Abcam,
Tokyo) were used to stain mesenchymal or myoepithelial cells. For thesecondary antibody, Histofinet Simple Staine Rat MAX PO(MULTI) Ab (Nichirei Biosciences Inc., Tokyo, Japan) was usedwith a DAB substrate kit (Nichirei, Biosciences Inc., Tokyo). It has
been confirmed, using formalin-fixed paraffin-embedded sections of
rat mammary tissues, that aCK18 Ab stains luminal epithelial cells,
aSMA Ab stains myoepithelial cells and anti-vimentin Ab stains
stromal fibroblasts but not cells of the ducts (Mishima, unpublishedresults). Under the current study conditions, about 70% of the cells
used for FISH examinations were aCK18 Ab positive, often roundshaped and were therefore considered as epithelial cells. In contrast,
cells that were positively stained with anti-vimentin Ab were scatteredand fibroblast-like, and aSMA Ab positive cells were mostly large andelongated. These cells were probably mesenchymal cells or fibroblasts(results not shown).

RESULTSThe frequency of translocations in primary cultures ofmammary cells from rats exposed as a fetus varied from
2.7–5.5%. The mean individual frequency was 3.74% (SD
0.78%) at the 6th week after irradiation (n ¼ 10). The
corresponding means were 3.79% (SD 1.35%) at the 9th
week (n ¼ 5) and 3.36% (SD 1.03%) at the 45th week (n ¼8) (Fig. 1, closed circles and see Supplementary materials;
http://dx.doi.org/10.1667/RR13446.1.S1). The differences
between these means were not statistically significant(Student’s t test, results not shown). Thus, the frequency
did not seem to change with increasing time intervals
between the exposures and the cytogenetic tests. These

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FIG. 1. Observed translocation frequencies involving painted chromosomes 2 or 4 in mammary cell cultures
or spleen cells in rats gamma irradiated with 2 Gy: Fetus ( ); mother, 12 weeks old (u); virgin, 12 weeks old(^); and nonirradiated control rats (n). The time intervals between irradiation and FISH examinations areshown at the top of the figure. Each point was derived from scoring 800 cells.

frequencies were nearly the same or slightly higher thanthose in the irradiated mothers (2.91%, SD 0.54%, n ¼ 5)
(Fig. 1, open squares). Two virgin females irradiated as
young adults (16 weeks old) showed translocation frequencies of 1.8 and 3.8% when examined at the 6th and 45th
weeks, respectively, after irradiation (Fig. 1, open diamonds). These do not seem to be higher than the means
detected in the mother rats that raised the offspring. The
results are notable in that there does not appear to beevidence for higher radiation sensitivity in fetal cells.
In sharp contrast to the observations in mammary
epithelial cells, spleen cells from rats irradiated as fetuses
and examined at 9th and 45th weeks after irradiation did notshow an increased frequency of translocations, which is inline with previous observations in mice (5). Translocation
frequencies in spleen cells from two mother rats were 3.0
and 3.8% at the 9th week, and 3.9% at the 45th week inanother mother rat after irradiation. The translocation
frequencies in spleen cells of irradiated adults rats weresimilar to those found in mammary epithelial cells
regardless of the time interval since exposure (9 vs. 45
weeks) (Fig. 1).
Identical translocations were occasionally observed in 2
or more cells (clonal translocations) among the 800 cells
scored; these cells arise as a result of proliferation of stem/
progenitor cells in vivo and hence were counted as single
events. In the mammary cell cultures, 12 rats among the 23
rats irradiated at the fetal stage showed 16 different clonaltranslocations (individual cases are shown in the Supplementary materials; http://dx.doi.org/10.1667/RR13446.1.
S1). Specifically, the number of clonal cells varied from
2–8 out of 800 cells scored (0.3–1%). Among five mothers
and one virgin female, one mother was found to show 2translocation clones (2 and 3 cells among 800 cells,
respectively, 0.3–0.4%). In contrast, in the spleen cells,
only one rat irradiated as a fetus had a clonal translocation(7 cells out of 800 cells, 0.9%).
DISCUSSION

The current study demonstrated that radiation exposure
induces translocations in fetal stem/progenitor cells that
may later develop into mammary glands. It further
demonstrated that the frequency of translocations after
irradiation at the fetal stage is not higher than that resulting
from adult exposures and the frequency does not change
during 6–45 weeks after irradiation. In contrast, radiationinduced cytogenetic damage to hematolymphoid cells did
not persist after fetal irradiation but it did persist after adultexposures in humans (4), mice (5) and rats (current study).
Therefore, in both hematolymphoid cells and mammary
epithelial cells, no evidence was found of higher radiosensitivity in fetal cells leading to the induction of chromosome
aberrations. One may ask which tissue response is common
after fetal irradiation, full recording (mammary epithelial
cells) or no recording (hematolymphoid cells) of radiation-

TRANSLOCATION INDUCTION AFTER FETAL IRRADIATION OF RATS

induced damage. It appears that the former is more likely forthe following reasons: Our recent study indicated that irradiation of fetal mice
could increase the frequency of translocations in thyroid
cells when the animals were examined as adults(Hamasaki et al., unpublished results). The results are
similar to the observations made in the current study in
rat mammary epithelial cells; Hematopoietic stem cells (HSCs) remain in fetal liver atbirth and start to migrate into final niches in bonemarrow during a few weeks after birth in the mouse (12),which closely corresponds to the period when radiationinduced translocations start to persist (5); Finally, fetal hematopoietic stem cells express only avery low level of ATM gene (13), which indicates that
initial recognition processes of DNA damage response
may be defective exclusively in fetal HSCs.
In our experiments, clonal translocations were observed,
and the results can be used to estimate the number ofsurviving mammary stem cells in a fetus after irradiation
with 2 Gy, assuming that clonal expansion occurs by chanceand does not accompany a growth advantage. In addition, it
was previously shown that the number of surviving stem
cells is inversely related to the clone size (14). Thus, a clone
size of 0.3–1% as observed in the current study indicatesthat the total number of surviving stem cells is 1/0.003–1/
0.01 or 300–100.
Taken together these results raise an interesting question:
if cytogenetic damage is a surrogate marker of the
carcinogenic effect of radiation, will the translocation
frequency be directly relevant to the carcinogenic risks
after irradiation? With regard to hematolymphoid cells, theanswer is yes, e.g., Upton et al. irradiated RF mice ofvarious ages with 3 Gy and followed them to observe thedevelopment of acute myeloid leukemia (AML) (15). The
results showed that fetuses are the least sensitive to the
induction of AML after radiation exposure and thesensitivity steadily increases as the age of the animals
increases at the time of irradiation up to 100 days. The
results are in close agreement with our previous study on the
induction of persistent translocations in the hematolymphoid cells of mice varying from fetal to young adult stages(5). Specifically, the frequency was nearly zero for mice
irradiated as fetuses or neonates (cytogenetic examinations
were made when the animals reached 20 weeks of age),while the frequency steadily increased with an increase in
the mouse age at the time of irradiation. The close
correlation between the induction of AML and translocations in hematolymphoid cells is reasonable if it can beassumed that both end points are involved with the fate ofdamaged hematopoietic stem cells.
With regard to induction of tumors other than AML,
however, the answers are much less clear, specifically, it
appears that fetal radiosensitivity differs depending on thetissues (e.g., 16, 17) and there also appears to be no general175

trend suggesting that fetuses are more sensitive thanneonates or young adults. It should be cautioned, however,that currently available information is so limited that ageneral conclusion cannot be derived on this issue; e.g., theradiation effect may be affected not only by the fetal stageof development at the time of irradiation but also by theradiation dose employed since the dose-response curve canbe humped at low doses (17). As for the current study on
mammary glands, cytogenetic damage to fetal mammary
stem/progenitor cells is not correlated with risk oftumorigenesis because fetal irradiation of SD rats is reported
to not increase the risk of mammary tumors (18, 19) eventhough translocations were induced as effectively as inadults. Several possible explanations, not mutually exclusive, may reconcile this discrepancy.
 The number of surviving target cells at risk after 2 Gy
exposure is too small to preserve the carcinogenic effectof radiation, e.g., the results of clonal translocations
indicated that after 2 Gy exposure the number would be
only 100–300 surviving stem cells. These cells cannot
record radiation damage quantitatively if we assume that
irradiation adds oncogenic mutations to the cells, because
mutation induction rates at a specific gene are on theorder of 105–104 per Gy (20, 21).
 Exposure to 2 Gy can partly destroy the oocyte pool in a
fetus (22), leading to subsequent low estrogen levels or
earlier menopause and resulting in reduced promotion of
precancerous cells.
 Stem cells and their descendants in mammary glands
might escape cytogenetic tests. This may happen when invitro tissue culture conditions are inadequate and forexample, stimulate stroma cells to grow rather thanepithelial cells. However, this explanation appears tohave a low probability because about 70% of the cells
examined were aCK18Ab positive and are hence
regarded as epithelial cells.
 The possibility, as described by Degregori (23), thatcertain mutations required for a carcinogenic process
may actually impair rather than stimulate growth ofhematopoietic stem cells. In any event, further studies are
required to understand the refractory nature of a fetus forthe development of mammary cancers after radiation
exposure.
In humans, mammary glands are among the mostsensitive tissues in terms of developing malignancies after
postnatal exposure to radiation. Specifically, after radiation
exposure in infancy, increased risk levels were observed instudies of patients treated with radiation for hemangioma(24) or enlarged thymus (25, 26) and in studies ofindividuals exposed to A-bomb radiation (6, 27). After
fetal exposure, however, even the total cancer risk is notwell established in the A-bomb survivors because theepidemiologic cohorts of A-bomb survivors are fairly small
and the number of subjects with known doses is about 2,500

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NAKANO ET AL.

(28, 29). Therefore, the risk of breast cancer after fetal
exposure to radiation is still unclear.
In summary, the current results suggest that ordinary
cytogenetic tests using blood lymphocytes will not work forbiological dose evaluations of irradiated fetuses. However,
it is anticipated that cells from solid tissues (e.g., skinfibroblasts) could be an alternative source for biodosimetry.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Tatsuhiko Imaoka at the NIRS for his
valuable comments, Dr. Leon Kapp for his careful reading of themanuscript, Ms. K. Muramoto, T. Matsumoto, M. Miura and H.
Nagahashi for their excellent technical assistance and Ms. M. Utaka formanuscript preparation. The Radiation Effects Research Foundation(RERF), Hiroshima and Nagasaki, Japan, is a private, non-profit
foundation funded by the Japanese Ministry of Health, Labor andWelfare (MHLW) and the U.S. Department of Energy (DOE). The
research was also founded in part through DOE award DE-HS0000031 to
the National Academy of Sciences. This publication was supported by the
Radiation Effects Research Foundation, Research Protocol RP 8–93. The
views of the authors do not necessarily reflect those of the two
governments.
Received: August 27, 2013; accepted: October 9, 2013; published online:
February 10, 2014

REFERENCES
1. Bergonié J, Tribondeau L. Interpretation of some results fromradiotherapy and an attempt to determine a rational treatment
technique. Radiat Res 1959; 11:587–588 (Reproduced in Yale J
Biol Med 2003; 76:181–2).
2. Doll R, Wakeford R. Risk of childhood cancer from fetal
irradiation. Br J Radiol 1997; 70:130–139.
3. Wakeford R, Little MP, Kendall GM. Risk of childhood leukemia
after low-level exposure to ionizing radiation. Expert Rev Hematol
2010; 3:251–4.
4. Ohtaki K, Kodama Y, Nakano M, Itoh M, Awa AA, Cologne J, etal. Human fetuses do not register chromosome damage inflicted byradiation exposure in lymphoid precursor cells except for a small
but significant effect at low doses. Radiat Res 2004; 161:373–9.
5. 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.
6. Preston DL, Ron E, Tokuoka S, Funamoto S, Nishi N, Soda M, etal. Solid cancer incidence in atomic bomb survivors: 1958–1998.
Radiat Res. 2007 Jul; 168:1–64.
7. Pitot HC. Fundamentals of oncology. 3rd ed. New York: Marcel
Dekker; 1986. p.58.
8. Darcy KM, Zangani D, Lee PPH, Ip MM. Isolation and culture of
normal rat mammary epithelial cells. In: Ip MM, Asch BB, editors.
Methods in mammary gland biology and breast cancer research.
New York: Kluwer Academic/Plenum Publishers; 2000. p.163–75.
9. Yoshia MC, Ikeuchi T, Sasaki M. Differential staining of parental
chromosomes in interspecific cell hybrids with a combined
quinacrine and 33258 Hoechst technique. Proc Jap Acad 1975;
51:185–187.
10. Committee for a standardized karyotype of Rattus norvegicus.
Standard karyotype of the Norway rat, Rattus norvegicus.
Cytogenet Cell Genet 1973; 12:199–205.
11. Zarbo RJ, Visscher DW, Crissman JD. Two-color multiparametricmethod for flow cytometric DNA analysis of carcinomas using

staining for cytokeratin and leukocyte-common antigen. Anal
Quant Cytol Histol 1989; 11:391–402.
12. Bowie MB, McKnight KD, Kent DG, McCaffrey L, Hoodless PA,
et al. Hematopoietic stem cells proliferate until after birth and show
a reversible phase-specific engraftment defect. J Clin Invest 2006
Oct; 116:2808–16.
13. Bowie MB, Kent DG, Dykstra B, McKnight KD, McCaffrey L,
Hoodless PA, Eaves CJ. Identification of a new intrinsically timed
developmental checkpoint that reprograms key hematopoietic stem
cell properties. Proc Natl Acad Sci U S A 2007 Apr 3; 104:5878–
82.
14. Nakamura N, Nakano M, Kodama Y, Ohtaki K, Cologne J, Awa
AA. Prediction of clonal chromosome aberration frequency inhuman blood lymphocytes. Radiat Res 2004; 161:282–9.
15. Upton AC, Odell TT Jr, Sniffen EP. Influence of age at time ofirradiation on induction of leukemia and ovarian tumors in RF
mice. Proc Soc Exp Biol Med 1960; 104:769–772.
16. Sasaki S. Influence of the age of mice at exposure to radiation onlife-shortening and carcinogenesis. J Radiat Res 1991; 32 Suppl 2:
73–85.
17. Di Majo V, Coppola M, Rebessi S, Covelli V. Age-related
susceptibility of mouse liver to induction of tumors by neutrons.
Radiat Res 1990; 124:227–234.
18. Inano H, Suzuki K, Onoda M, Yamanouchi H. Susceptibility offetal, virgin, pregnant and lactating rats for the induction of
mammary tumors by gamma rays. Radiat Res 1996; 145:708–13.
19. Imaoka T, Nishimura M, Daino K, Kokubo T, Doi K, Iizuka D, etal. Influence of age on the relative biological effectiveness ofcarbon ion radiation for induction of rat mammary carcinoma. Int J
Radiat Oncol Biol Phys 2013 Mar 15; 85:1134–40.
20. Cox R, Thacker J, Goodhead DT, Munson RJ. Mutation andinactivation of mammalian cells by various ionising radiations.
Nature 1977; 267:425–7.
21. Liang L, Mendonca MS, Deng L, Nguyen SC, Shao C, Tischfield
JA. Reduced apoptosis and increased deletion mutations at Aprt
locus in vivo in mice exposed to repeated ionizing radiation.
Cancer Res 2007 Mar 1; 67:1910–17.
22. Mazaud-Guittot S, Guigon CJ, Coudouel N, Magre S. Consequences of fetal irradiation on follicle histogenesis and earlyfollicle development in rat ovaries. Biol Reprod 2006; 75:749–59.
23. Degregori J. Challenging the axiom: does the occurrence of
oncogenic mutations truly limit cancer development with age?
Oncogene 2013; 32:1869–75.
24. Holmberg E, Holm LE, Lundell M, Mattsson A, Wallgren A,
Karlsson P. Excess breast cancer risk and the role of parity, age atfirst childbirth and exposure to radiation in infancy. Br J Cancer
2001; 85:362–6.
25. Hildreth NG, Shore RE, Dvoretsky PM. The risk of breast cancer
after irradiation of the thymus in infancy. N Engl J Med 1989;
321:1281–4.
26. Adams MJ, Dozier A, Shore RE, Lipshultz SE, Schwartz RG,
Constine LS, et al. Breast cancer risk 55 þ years after irradiation
for an enlarged thymus and its implications for early childhood
medical irradiation today. Cancer Epidemiol Biomarkers Prev
2010; 19:48–58.
27. Land CE, Tokunaga M, Koyama K, Soda M, Preston DL,
Nishimori I, et al. Incidence of female breast cancer among atomic
bomb survivors, Hiroshima and Nagasaki, 1950–1990. Radiat Res
2003; 160:707–17.
28. Delongchamp RR, Mabuchi K, Yoshimoto Y, Preston DL. Cancer
mortality among atomic bomb survivors exposed in utero or as
young children, October 1950-May 1992. Radiat Res 1997;
147:385–95.
29. Preston DL, Cullings H, Suyama A, Funamoto S, Nishi N, Soda
M, et al. Solid cancer incidence in atomic bomb survivors exposed
in utero or as young children. J Natl Cancer Inst 2008; 100:428–36.