RADIATION RESEARCH 176, 678–687 (2011)0033-7587/11 $15.00
g 2011 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR2631.1

Associations of Ionizing Radiation and Breast Cancer-Related Serum
Hormone and Growth Factor Levels in Cancer-Free Female
A-Bomb Survivors
Eric J. Grant,a,1 Kazuo Neriishi,b John Cologne,c Hidetaka Eguchi,d Tomonori Hayashi,e Susan Geyer,fShizue Izumi,g Nobuo Nishi,h Charles Land,i Richard G. Stevens,j Gerald B. Sharpk and Kei NakachieaDepartment of Epidemiology, b Department of Clinical Studies, c Department of Statistics, and e Department of Radiobiology/Molecular
Epidemiology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minamiku, Hiroshima 732-0815, Japan; d Translational ResearchCenter, International Medical Center, Saitama Medical University, 1397-1 Yamane, Hidaka-shi, Saitama 350-1241, Japan; f Department of Internal
Medicine, The Ohio State University, Columbus, Ohio 43210; g Department of Computer Science and Intelligent Systems, Oita University, 700
Dannoharu, Oita, 870-1192 Japan; h Center for Collaboration and Partnership, National Institute of Health and Nutrition, 1-23-1 Toyama,
Shinjuku-ku, Tokyo 162-8636, Japan; i Radiation Epidemiology Branch, National Cancer Institute, Bethesda, Maryland 20892-7335; j Departmentof Community Medicine, University of Connecticut Health Center, Connecticut 06030; and k Epidemiology Branch, Basic Sciences Program,
Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

while they decreased in samples collected from premenopausal
women (211%1Gy, 95% CI: 220%–1% and 212%1Gy, 95% CI:
220%– 22%, respectively). Interactions by menopausal status
were significant (P = 0.003 and P < 0.001, respectively).
Testosterone levels increased with radiation dose in postmenopausal samples (30.0%1Gy, 95% CI: 13%–49%) while theymarginally decreased in premenopausal samples (210%1Gy, 95%
CI: 219%–0%) and the interaction by menopausal status was
significant (P < 0.001). Serum levels of IGF1 increased linearlywith radiation dose (11%1Gy, 95% CI: 2%–18%) and there was asignificant interaction by menopausal status (P = 0.014).
Radiation-associated changes in serum levels of estradiol,bioavailable estradiol, testosterone and IGF1 were modified bymenopausal status at the time of collection. No associations withradiation were observed in serum levels of progesterone,
prolactin, IGFBP-3 or ferritin. g 2011 by Radiation Research Society

Grant, E. J., Neriishi, K., Cologne, J., Eguchi, H., Hayashi,T., Geyer, S., Izumi, S., Nishi, N., Land, C., Stevens, R. G.,
Sharp, G. B. and Nakachi, K. Associations of Ionizing
Radiation and Breast Cancer-Related Serum Hormone and
Growth Factor Levels in Cancer-Free Female A-Bomb Survivors. Radiat. Res. 176, 678–687 (2011).
Levels of exposure to ionizing radiation are increasing for
women worldwide due to the widespread use of CT and otherradiologic diagnostic modalities. Exposure to ionizing radiation
as well as increased levels of estradiol and other sex hormones
are acknowledged breast cancer risk factors, but the effects ofwhole-body radiation on serum hormone levels in cancer-free
women are unknown. This study examined whether ionizing
radiation exposure is associated with levels of serum hormonesand other markers that may mediate radiation-associated breast
cancer risk. Serum samples were measured from cancer-free
women who attended biennial health examinations with a wide
range of past radiation exposure levels (N = 412, ages 26–79).
The women were selected as controls for separate case-control
studies from a cohort of A-bomb survivors. Outcome measures
included serum levels of total estradiol, bioavailable estradiol,
testosterone, progesterone, prolactin, insulin-like growth factor1 (IGF1), insulin-like growth factor-binding protein 3 (IGFBP3), and ferritin. Relationships were assessed using repeatedmeasures regression models fitted with generalized estimating
equations. Geometric mean serum levels of total estradiol andbioavailable estradiol increased with 1 Gy of radiation doseamong samples collected from postmenopausal women (17%1Gy,
95% CI: 1%–36% and 21%1Gy, 95% CI: 4%–40%, respectively),

INTRODUCTION

Exposure to ionizing radiation via diagnostic imaging,particularly CT scans, continues to increase worldwide(1). Numerous studies have proven the association
between exposure to ionizing radiation and breast
cancer (2). Moreover, data from the A-bomb survivors
indicate that the breast is particularly susceptible toionizing radiation exposure, with an attributable fraction for breast cancer of 27% for all women exposed tomore than 5 mGy (3) [a radiation dose roughly twice theyearly background dose (4)]. Breast cancer has also been
associated with levels of endogenous hormones, particularly estradiol (E2) (5–7). Estrogens stimulate cellproliferation (8) and possibly exert a direct genotoxic
effect on breast epithelial cells (9).
Other circulating hormones and proteins, including non-sex hormone-binding globulin (SHBG)-boundNote. The online version of this article (DOI: 10.1667/RR2631.1)
contains supplementary information that is available to all authorizedusers.
1 Address for correspondence: Department of Epidemiology, Radiation
Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku, Hiroshima732-0815, Japan; e-mail: egrant@rerf.or.jp.
678

ASSOCIATIONS OF IONIZING RADIATION AND SERUM HORMONES

estradiol, testosterone (7) and insulin-like growth factor 1(IGF1), have been associated with breast cancer (10).
Additional serum markers have been hypothesized to be
associated with breast cancer, including IGFBP-3 (11),progesterone (12), prolactin (13) and ferritin. Ferritin
levels and ionizing radiation are associated with increased
levels of free radicals, and high ferritin levels have beenreported to result in cell transformation and immortalization in mammary glands (14).
The question of whether ionizing radiation exposure
modifies the underlying levels of risk-related circulating
markers has not been addressed, however, and could be
important in mechanistic studies of breast cancer orother hormone-related cancers after ionizing radiation
exposure. The primary purpose of this project is to
examine whether serum levels of risk-related markers
vary with past radiation exposure in a representative
sample of cancer-free female A-bomb survivors.
MATERIALS AND METHODS
Subjects
Study subjects were selected from the Adult Health Study (AHS),
a longitudinal cohort study of A-bomb survivors initiated in 1958
(15). The AHS features biennial clinical examinations with serum
collection and well-characterized radiation doses for numerous bodyorgans (16). Serum samples were selected in the context of concurrent
case-control studies wherein controls were selected by incidence
density sampling from all subjects who met eligibility requirements
and were at risk for a first primary cancer of any type at the time
an index case was diagnosed. Eligibility was defined as women who
were free from various medical conditions (oophorectomy, current
exogenous hormone treatment, prior malignancies, pregnant), werecancer-free for at least 2 years after serum collection, and had
estimated radiation organ doses. Hormone-related diseases/conditions were based on self-reports or, in the case of extreme measured
values, a medical chart review. Samples were matched to an index
case on age at collection (±2 years), sample collection year (samedecade), city (Hiroshima or Nagasaki), and counter-matched using
breast radiation dose strata (low, medium and high with cut-points
at 5 mGy and 790 mGy) (17). Counter-matching assures a wide
exposure range while still providing an appropriate sample forinference of the exposure effects within the underlying cancer-freecohort (18). Because it is not known which organ system might beresponsible for changes to serum markers, we used doses to the colonfor purposes of analyses. The colon is a centrally located organ, andcolon doses have been used as representative doses in other studies(e.g. for overall solid cancer risk estimates), although the results
should be relatively insensitive to the organ chosen since the doses toall organs are highly correlated. Radiation doses to the colon were
adjusted to account for random errors in the individual radiation dose
estimates (19). Menopausal status at the time of sample collection was
assigned using a two-step process. First, we used a conservative
age range to assign menopausal status. If a woman was less than
43 years of age at the time of sample collection, she was designatedpremenopausal (N 5 218); if she was older than 55 years, she wasdesignated postmenopausal (N 5 139). For women in the intermediate age group (43–55 years old), FSH (follicular stimulating
hormone) levels were measured and used to assign menopausal statusprimarily based upon the manufacturer’s test guidelines (premenopausal cutoff: FSH ,515 mIU/ml, postmenopausal cutoff: FSH.523 mIU/ml) (20). Of those in the intermediate age range and679

assigned by FSH values (N 5 152), 75 were designated premenopausal, 60 were postmenopausal, and the others were in between thetwo cutoff values and were designated perimenopausal (N 5 17). Dueto the paucity of samples collected from perimenopausal women, they
were excluded. The final data set included 492 samples collected from
412 women (293 premenopausal and 199 postmenopausal samples).
Of the 80 subjects with two separate samples, 63 had samples thatwere both collected prior to menopause, 6 subjects had samples thatwere both collected after menopause, and 11 had one sample collected
before and one after menopause.
All blood samples were obtained with informed consent, and the
Human Investigation Committee of the Radiation Effects Research
Foundation (RERF) approved the study protocol.
Sample Storage and Serum Assays
Sera were collected in the Hiroshima and Nagasaki laboratories
between 1969 and 1992 and stored in vials at 280uC. Samples for thisstudy were pulled from the freezers simultaneously and assayed for
total E2, bioavailable E2, testosterone, progesterone, prolactin, IGF-1,
IGFBP-3, ferritin, and total protein and protein fractions (albumin
and globulin). All assays were performed blinded to age, city ofcollection and radiation exposure but were not performed in duplicate
due to the limited serum amounts available. Commercial laboratories
with internal quality assurance systems performed all assays.
Supplementary Table 1 (http:dx.doi.org/10.1667/RR2631.1.S1) lists
the assays and their precision as reported by the kits’ manufacturers.
Serum levels of IGF-1, IGFBP-3, FSH, prolactin, testosterone,
progesterone and ferritin were measured at a single laboratory (SRL
Laboratory, Hachioji, Japan). Total protein concentration andprotein fractions were measured by SRL using the Biuret method
and cellulose acetate membrane electrophoresis, respectively. Serumlevels of total E2 and percentage bioavailable E2 were measured at asingle laboratory (Mitsubishi Kagaku Bio-clinical Laboratories,
Tokyo, Japan). Percentage bioavailable E2 (the fraction of serum E2
that is free or loosely bound to albumin) was determined byammonium sulfate precipitation of SHBG-bound E2 (21). Total E2
was multiplied by this percentage to obtain the concentration ofbioavailable E2. Serum ferritin was measured by the conventional
chemiluminescence enzyme immunoassay (CLEIA). When thealiquots of samples were transported to outside laboratories fortesting, they were packed in appropriate protective shipping boxes
with sufficient amounts of dry ice and checked at the time of receipt
by the measuring laboratories.
Methods of Analysis
Due to the skewed distributions of the markers, analyses were
performed using log-transformed values for all of the markers except
IGF1 and IGFBP-3, which did not require transformation. Effects ofradiation exposure were estimated with adjustment only for otherfactors that were significantly related to marker level for statistical
efficiency. Explanatory factors considered were: age at time of sample
collection, year of birth, menopausal status (pre or post), total
protein, body mass index [weight (kg)/height (m2)] and its square,history of exogenous hormone use (yes or no), age at time of radiation
exposure (including dichotomy at 15 years as a surrogate for pre- or
postmenarche at time of exposure), number of full-term pregnancies,
age at first full-term delivery, and radiation dose. Decisions forinclusion of factors other than radiation dose were based on abackward selection process where the least significant explanatory
factor was removed until all factors were significant at a level of P #0.05. The variable selection process was conducted separately for eachmarker. Analyses were performed using a regression method forcorrelated (repeated) measurements (generalized estimating equations; GEE) (22). P values and 95% confidence intervals were basedon the Wald statistic.

680

GRANT ET AL.

TABLE 1
Characteristics of Study Cohort and Collected Samples by Menopausal Status
Variable
City (Hiroshima/Nagasaki)

Premenopausal samples (N 5 293)

Postmenopausal samples (N 5 199)

P valuea

187/106

145/54

0.036

Mean (range)

Mean (range)

13.4 (1.0–24.0)
40.3 (26.3–51.9)
0.54 (0.0–3.2)
22.4 (15.6–32.4)
2.7 (0–11)
1971.9 (1969–1991)
116.0 (0.0–320.0)
2.53 (0.4–4.3)

28.2 (12.0–49.0)
57.7 (43.0–78.9)
0.49 (0.0–2.8)
22.8 (14.6–39.3)
3.6 (0–11)
1975.4 (1970–1992)
94.6 (62–130)
2.37 (2.1–2.8)

Median (interquartile rangec)

Median (interquartile rangec)

86.65 (59.2–116.0)
39.48 (27.3–53.7)
15.7 (10.4–23.3)
1.60 (0.40–10.0)
6.20 (4.0–9.2)
21.50 (7.5–41.0)

18.50 (13.1–25.2)
9.19 (5.8–13.2)
10.5 (6.7–18.3)
0.25 (0.19–0.30)
3.60 (2.4–5.1)
50.00 (29–94)

b

Age ATB (years)
Age at collection (years)
Radiation dose (weighted gray)
BMI (kg/m2)
Number of full-term pregnancies
Year of sample collection
IGF-1 (ng/ml)
IGFBP-3 (mg/ml)

Total E2 (pg/ml)
Bioavailable E2 (pg/ml)
Testosterone (ng/dl)
Progesterone (ng/ml)
Prolactin (ng/ml)
Ferritin (ng/ml),0.001,0.0010.472
0.267,0.001,0.001,0.001
0.002,0.001,0.001,0.001,0.001,0.001,0.001

Notes. The upper section is the count of samples by city. The middle section shows the averages and ranges of non-transformed variables. The
bottom section shows the median and interquartile ranges of natural log-transformed variables.
aCounts tested with x2 test for independence, means tested with t test for equality, medians tested with the Wilcoxon rank-sum test forindependent samples.
b
412 subjects (Hiroshima, N 5 267/Nagasaki, N 5 145). 80 subjects had multiple samples (63 subjects with two premenopausal samples,
6 subjects with two postmenopausal samples, 11 subjects with one premenopausal sample and one postmenopausal sample).
c25th percentile to the 75th percentile.
Radiation effects were assessed using linear dose–response functions. Dose responses were estimated with and without regard toeffect modification by menopausal status at the time of bloodcollection and ionizing radiation exposure before or after age 15. For
markers that were transformed prior to analyses, the radiation effects
were reported on the untransformed scale by exponentiating theregression coefficient obtained using the log-transformed data andrepresent the association between 1 Gy radiation dose and thepercentage change in geometric mean of the marker level. IGF1 and
IGFBP-3, which were not transformed, were also reported on arelative scale by dividing the dose coefficient by the regression
constant. To test the strength of the observed radiation effects, we
identified and removed the most influential observations using thesize-adjusted criterion 2/sqrt(n) for the t-statistic-like standardized
deletion diagnostic DFBETAS of Belsley et al. (23). To alleviate
concerns about misclassification of menopausal status at the time ofsample collection due to possible nonspecificity of the FSH results, we
conducted a sub-analysis after restricting samples to those collectedfrom women at ages less than 43 or greater than 55 (135 samples
omitted) and compared results to the full analyses.
Statistical comparisons between samples collected pre- and postmenopause were performed using x2 tests (for counts), t tests forequality (for means) and Wilcoxon rank-sum tests for independent
samples (for medians). Interactions of radiation effects by menopausal status or exposure before or after age 15 were tested using the
likelihood ratio. To compare fits of statistically significant non-nested
interaction models, the quasi-likelihood/independence criterion (QIC)
was used (24). All results were obtained using Stata (25).

RESULTS

Table 1 shows the demographics of the study cohortand marker values for samples separated by menopausal
status at the time of collection. The distribution of theradiation dose is broader than what would be expectedfrom a random sampling of all cancer-free women since
they were counter-matched (low, medium, high) against
the dose status of the case to ensure a broad representation of doses. The average year of sample collection
is somewhat later for postmenopausal samples because
the AHS study is a fixed cohort; more women were
postmenopausal in later calendar years.
Table 2 shows the agreement of assays for the 63 pairsof samples collected from women who were premenopausal at each collection. Comparisons of multiple
samples collected from the same women while postmenopausal were not reported (N 5 6 pairs). The earlier
sample was collected at average age 39.7 years and theaverage interval between sample collections was 2.8 years.
Correlation coefficients ranged from 0.11 for progesterone to 0.75 for ferritin. A paired t test comparing thedifference of the paired values with zero was not
significant for any marker except for serum prolactin,
where the earlier value proved to be significantly higher
than the second value.
The first column of Table 3 shows the associations ofradiation with marker levels with no allowance for effectmodification but with adjustment for other significant
factors. No significant radiation associations wereobserved in any of the markers when ignoring menopausal status. Columns two through five in Table 3
show the radiation associations separately by menopausal status at the time of sample collection or by age

681

ASSOCIATIONS OF IONIZING RADIATION AND SERUM HORMONESTABLE 2Comparison of Assay Values from Women Donating Multiple Samples
Assayed biomarker

N

Sample 1 averagevalue (SD)Sample 2 averagevalue (SD)

Correlation

Sample 1 2 Sample 2
average value (SD)P value

Log total E2 (pg/ml)
Log bioavailable E2 (pg/ml)
Log testosterone (ng/dl)
Log progesterone (ng/ml)
Log prolactin (ng/ml)
Log ferritin (ng/ml)
IGF-1 (ng/ml)
IGFBP-3 (mg/ml)

63
63
57
54
57
56
57
57

4.47 (1.02)
3.65 (0.94)
2.74 (0.59)
0.49 (1.71)
1.86 (0.85)
3.12 (1.30)
129.0 (69.7)
2.51 (0.55)

4.34 (0.54)
3.64 (0.54)
2.74 (0.59)
0.53 (1.64)
1.47 (0.61)
3.11 (1.35)
149 (67.9)
2.54 (0.43)

0.14
0.16
0.52
0.11
0.28
0.75
0.16
0.36

0.13 (1.10)
0.014 (1.03)
0.0066 (0.54)
20.042 (2.3)
0.39 (0.91)
0.0082 (0.83)
220.3 (88.1)
20.029 (0.60)

0.10
0.42
0.55
0.54
0.001
0.93
0.19
0.96

Notes. Samples are confined to multiple premenopausal samples (there were only six persons who donated multiple postmenopausal samples).
The average age at the time of Sample 1 collection was 37.9 years and the average year of collection was 1970.9. The average age at the time ofcollection for the second sample was 40.8 years and the average year of collection was 1973.7. Values in parentheses (SD) after averages are
standard deviations. The P value in the last column is a paired t test (Ho: Sample 1 – Sample 2 5 0).

at time of bombing (#15 or .15 years; ATB15).
Because menopausal status at blood donation was
highly correlated with ATB15 in this study sample(particularly for a woman’s first/only serum sample), thelatter two columns largely mirror the results displayed in
the former two. Total and bioavailable E2, testosterone
and IGF-1 demonstrated statistically significant associations with radiation dose in one or both periods.
Total E2 (Fig. 1) decreased with radiation dose in thepremenopausal period (relative change 0.89 at 1 Gy;
95% CI: 0.80, 0.99; P 5 0.027), or in other words, thelevel of total E2 changed as k*0.89dose, where k is the levelof total E2 when the dose was zero (results from otherlog-transformed variables are interpreted in the sameway). The level of total E2 increased with dose in the
postmenopausal period (relative change 1.17 at 1 Gy;
95% CI: 1.01, 1.36; P 5 0.037). Effect modification bymenopausal status was highly significant (P 5 0.003)
and remained significant after removing the mostinfluential values (P 5 0.038). The association of total
E2 with radiation dose was similar when categorizing
subjects based on ATB15 (rather than menopausal
status at the time of collection); relative change for a
1-Gy dose at age less than 15 years was 0.86 (95% CI:
0.76, 0.96; P 5 0.010) and relative change for exposure
after age 15 was 1.12 (95% CI: 0.99, 1.26; P 5 0.063).
Models used to estimate the radiation effects on total E2
included menopausal status, age at sample collection,
total protein, BMI and a BMI*postmenopause interaction. The QIC values for the two models were very
similar, but the value for the ATB15 interaction model
was slightly lower (i.e. better fit) than for the model withinteraction by menopausal status.
Bioavailable E2 (Fig. 2) showed a significant decrease
with radiation dose in the premenopausal period, with arelative change of 0.88 at 1 Gy (95% CI: 0.80, 0.98; P 5
0.022) and a significant increase with radiation dose in the
postmenopausal period with a relative change of 1.21 at
1 Gy (95% CI: 1.04, 1.40; P 5 0.011). Effect modification
by menopausal status was highly significant (P , 0.001)

and remained so after exclusion of influential observations(P 5 0.003). As with total E2, bioavailable E2 also demonstrated similar radiation effects according to whether
exposure was before or after age 15 (0.87 at 1 Gy; 95% CI:
0.77, 0.98; P 5 0.019 and a relative increase of 1.13 at 1 Gy;
95% CI: 1.00, 1.27; P 5 0.045, respectively). The model
used to estimate the radiation effects on bioavailable E2
included menopause status, age at sample collection andtotal protein. Again the QIC value was slightly lower forthe model with ATB15 interaction.
Testosterone (Fig. 3) exhibited an increase with radiation dose in the postmenopausal period (relative change
1.30 at 1 Gy; 95% CI: 1.13, 1.49; P , 0.001) and amarginally significant decrease with radiation dose in thepremenopausal period (0.90 at 1 Gy; 95% CI: 0.81, 1.00; P
5 0.049). Estimates of effect modification by menopausal
status were highly significant when estimates were derived
using all observations (P , 0.001) and after excluding
outliers (P 5 0.003), whereas no effect modification wasevident with ATB15. The testosterone model included
menopause status, age at sample collection, total protein
and a total protein*postmenopause interaction, and BMI.
IGF-1 (Fig. 4) displayed a significant increase withradiation dose in the premenopausal period (relativechange 1.11 per Gy; 95% CI: 1.02, 1.18; P 5 0.024). Since
the values from this assay were not log-transformed, theresults are interpreted as the percentage change in marker
levels per gray. No significant change with dose wasobserved in the postmenopausal period (relative change
0.91 per Gy; 95% CI: 0.75, 1.04; P 5 0.18). Similar effects
were seen when categorizing the women by age at
exposure. Effect modification by menopausal status was
significant (P 5 0.014) and remained so after removing
the most influential observations (P 5 0.017). Comparing
QIC values indicated that the model with menopausal
interaction model was preferable to the one with ATB15
interaction. Estimates of the radiation effects were
adjusted for age at sample collection, year of birth, total
protein*premenopause interaction, and number of fullterm pregnancies.

682

GRANT ET AL.

TABLE 3
Relative Radiation Associations with Adjusted Mean Marker Values and Effect Modification By Menopausal Status
and Age at Radiation Dose
Relative radiation
association at 1 Gy no
effect modification(95% confidence intervals),
P value

Markerb,d0.97(0.89, 1.06)
P 5 0.52

Bioavailable E2b,e0.98(0.93, 1.06)
P 5 0.84

Testosteroneb,f

1.02(0.93, 1.12)
P 5 0.70

Progesteroneb,g1.08(0.90, 1.30)
P 5 0.39

Prolactinb,h1.01(0.91, 1.11)
P 5 0.905

IGF-1c,i

1.04(0.96, 1.10)
P 5 0.29

IGFBP-3c,j1(0.97, 1.03)
P 5 0.89

Ferritinb,k

0.97(0.83, 1.13)
P 5 0.72

Total E2

Relative radiation association at 1 Gy Separately by effect-modifier categorya(95% confidence intervals), P value
Menopause status at time of sample collection
Pre (N 5 293)

Post (N 5 199)

0.89
1.17
(0.80, 0.99)(1.01, 1.36)
P = 0.027
P = 0.037
Interaction P = 0.003
0.88
1.21
(0.80, 0.98)(1.04, 1.40)
P = 0.022
P = 0.011
Interaction P <0.001
0.90
1.30
(0.81, 1.00)(1.13, 1.49)
P = 0.049
P < 0.001
Interaction P < 0.001
1.08
1.10
(0.86, 1.35)(0.81, 1.50)
P 5 0.52
P 5 0.54
Interaction P 5 0.905
0.97
1.07
(0.86, 1.09)(0.91, 1.26)
P 5 0.641
P 5 0.384
Interaction P 5 0.313
1.11
0.91
(1.02, 1.18)(0.75, 1.04)
P = 0.024
P 5 0.18
Interaction P = 0.014*
1.01
0.98
(0.97, 1.05)(0.92, 1.05)
P 5 0.48
P 5 0.44
Interaction P 5 0.28
0.98
0.96
(0.81, 1.18)(0.73, 1.27)
P 5 0.81
P 5 0.78
Interaction P 5 0.92

Age at time of the bomb
#15 (N 5 190)

.15 (N 5 302)

0.86
1.12
(0.76, 0.96)(0.99, 1.26)
P = 0.010
P 5 0.063
Interaction P = 0.002*
0.87
1.13
(0.77, 0.98)(1.00, 1.27)
P = 0.019
P = 0.045
Interaction P = 0.002*
0.97
1.06
(0.84, 1.10)(0.93, 1.20)
P 5 0.617
P 5 0.387
Interaction P 5 0.337
1.00
1.21
(0.77, 1.29)(0.93, 1.58)
P 5 0.992
P 5 0.16
Interaction P 5 0.313
0.94
1.08
(0.82, 1.08)(0.95, 1.24)
P 5 0.413
P 5 0.230
Interaction P 5 0.161
1.11
0.98
(0.99, 1.20)(0.85, 1.07)
P 5 0.061
P 5 0.850
Interaction P = 0.091
1.00
0.93
(0.97, 1.04)(0.97, 1.03)
P 5 0.81
P 5 0.82
Interaction P 5 0.98
0.87
1.07
(0.70, 1.09)(0.86, 1.32)
P 5 0.22
P 5 0.55
Interaction P 5 0.19

Notes. Numbers in parentheses are 95% confidence intervals. The first column shows the radiation associations without adjustment formenopause status at the time of sample collection or age at the time of the exposure from the atomic bombings. The right two sets of columns
show the radiation associations by menopause status (columns 2, 3) and age less than or greater than 15 at the time of the bombing (columns 4,5). The ‘‘Interaction P’’ values test whether the results differ significantly by menopausal status or age at the time of the bombing. Note that there
were no detected effects when effect modification was ignored. The models were developed using a backwards elimination process for statistical
efficiency as described in the text. Covariates for each model are listed in the footnotes. All effect modification models included the main effectvariable regardless of whether it was selected in the elimination process. *QIC value indicates that this interaction model was better than thealternative interaction model. This score was compared only between the two interaction models and only when both models were significant.
aEffects of radiation are displayed separately for each of two categories of either menopausal status at time of serum collection (pre- or
postmenopause) or age at time of exposure (up to age 15 or after age 15). The main effect was always included when testing the interaction.
b
Log-transformed variable (relative change is calculated using Xdose, where X is the reported coefficient).
c
Non-transformed variable (relative change is calculated using X * dose, where X is the reported coefficient).
dAdjusted for age and menopause status at sample collection, total protein, BMI and BMI*postmenopause interaction.
e
Adjusted for age and menopause status at sample collection, total protein.
fAdjusted for age and menopause status at sample collection, total protein, total protein*postmenopause interaction, BMI.
g
Adjusted for menopause status at sample collection, total protein.
hAdjusted for age and menopause status at sample collection.
i
Adjusted for age at sample collection, year of birth, total protein*premenopause interaction, number of full-term pregnancies.
jAdjusted for year of birth, total protein*premenopause interaction, BMI, BMI2.
k
Adjusted for age and menopausal status at sample collection, BMI, number of full-term pregnancies.

ASSOCIATIONS OF IONIZING RADIATION AND SERUM HORMONES

683

FIG. 1. Log total estradiol (E2) by menopausal status and radiation dose. The relative change is thepercentage change at a dose of 1 Gy. The regression lines and P values are based on the final models obtained
for the markers (Table 3). The slopes of the two regression lines by menopausal status are significantly different(P 5 0.003).

The other markers (IGFBP-3, progesterone, prolactin
and ferritin) demonstrated no associations with radiation dose, either with or without effect modification. No
effect modification of the radiation effects was observedin relation to the sample storage period, time since
exposure, or the number of full-term pregnancies,
although E2 levels did increase at a marginally significant level for each additional full-term pregnancy(results not shown). Model fits generally improved when
including the number of full-term pregnancies (as either
an effect modifier or a main effect), but the estimatedcoefficients for the number of pregnancies were neverstatistically significant and therefore the results were notreported. No improvements in model fit were detectedby the addition of a quadratic dose term. Point estimates
were generally unchanged by the removal of samples
retrieved when women were aged 43 to 55.
DISCUSSION

The present study was designed to investigate theassociations of ionizing radiation exposure with sex andgrowth hormones in cancer-free A-bomb survivors using
stored sera. Increasing levels of ionizing radiation exposure
led to disparate changes by menopausal status in a numberof serum markers. Levels of E2 and bioavailable E2
increased in postmenopausal samples while they decreased

in premenopausal samples. Caution must be used wheninterpreting the results in premenopausal samples, especially E2 levels, which are highly variable in premenopausal
women due to the menstrual cycle. Table 2 illustrates thehigh variability of serum marker levels among premenopausal women. Controlling for the day of the menstrual
cycle was not possible, because the clinical examination
protocol did not attempt to record these values. It should
be noted, however, that the point estimates for theradiation findings were largely unchanged after separately
removing the most influential measurements (e.g., those
associated with the E2 peak at ovulation) and excluding
those in the perimenopausal age range 43–55. Also, it isunlikely that there is any association with the timing ofblood sample collection and radiation dose, eliminating
most concerns of confounding. Nevertheless, chancefindings for the premenopausal hormone associations
cannot be completely ruled out.
The magnitude of change in total E2 levels was about
10% at 1 Gy of radiation (negative in premenopausal
women and positive in postmenopausal women). It is notclear that either of these are clinically significant changes,
particularly among premenopausal women where there isso much variation. However, if E2 levels were assigned toquartiles in samples taken from postmenopausal women,
a 10% change in any particular result would generally be
sufficient to increase its quartile assignment by one

684

GRANT ET AL.

FIG. 2. Log bioavailable estradiol (E2) concentration by menopausal status and radiation dose. The relativechange is the percentage change at a dose of 1 Gy. The regression lines and P values are based on the final
models obtained for the markers (Table 3). The slopes of the two regression lines by menopausal status are
significantly different (P , 0.001).

category. Interpolating the results from Fig. 1 of themeta-analysis performed by Key et al. (7), this increase intotal E2 would roughly correspond to a 25% increase inbreast cancer risk.
The significance of IGF-1 and IGFBP-3 levels in preor postmenopausal breast cancer has been under debate.
The most recent evidence indicates that high levels of
IGF-1 confer an increased risk of breast cancer (10). In
our study, radiation exposure appeared to increase thelevel of IGF-1 in premenopausal women but had noeffect on levels of IGF-1 in postmenopausal women.
Again, however, large variations were observed in thepremenopausal IGF-1 levels. IGFBP-3 was not associated with radiation exposure.
If the results are correct, questions arise regarding
the radiation-related biological pathways that may
affect sex hormone levels, particularly the most potent
hormone E2. In premenopausal women, E2 is produced
primarily by the ovaries whereas after ovarian involution, estrogen is primarily produced in adipose tissue by
converting androgen to estrogen via aromatase. A
number of speculative mechanisms for an association
between radiation and sex steroid alterations can besuggested, including damage to primordial ovarianfollicles leading to decreased ovarian aromatase anddepressed E2 production as observed in the samples
taken from women who were exposed at less than

15 years of age (26, 27). Ataya et al. reported similarfindings with decreased ovarian size and long-term
decreased serum E2 levels in rhesus monkeys withovarian exposure to ionizing radiation (28). Larsen et al.
reported decreased ovarian volume in spontaneously
ovulating women who had received chemotherapy andradiotherapy for cancers diagnosed at mean age 5.4 years;
however, E2 levels were reported to be higher in thosereceiving (any type of) therapy compared to a control
group of similar age (29).
For the results seen among older birth cohorts (i.e.
postmenopausal samples), radiation-induced upregulation of inflammatory cytokines, specifically TNFa, canlead to increased estrogen biosynthesis (30–33); radiation can also alter lipid metabolism (34–36), possibly
leading to hyperandrogenism through hypothalamuspituitary-adrenal axis dysregulation (37). Either of theselatter mechanisms would be consistent with the observed
increased E2 and testosterone levels among the postmenopausal women.
No effect modification by the number of full-term
pregnancies was evident in the radiation effects on anyof the markers. The number of women reporting any
history of exogenous hormone intake was less than 10%,and that variable was not a significant risk factor in anyof the tested models. Therefore, we do not believe that
hormone treatment had a meaningful impact on ourASSOCIATIONS OF IONIZING RADIATION AND SERUM HORMONES

685

FIG. 3. Log testosterone by menopausal status and radiation dose. The relative change is the percentagechange at a dose of 1 Gy. The regression lines and P values are based on the final models obtained for themarkers (Table 3). The slopes of the two regression lines by menopausal status are significantly different(P , 0.001).

findings. Interaction models categorizing exposures
before or after age 15 more often had a better fit thanmodels categorized by menopausal status at the time ofserum collection. However, this may be due tomisclassification of menopausal status as opposed toan important biological effect involving menarche status
at the time of radiation exposure. McDougall et al.
found no association of breast cancer risk and menarche
status at the time of radiation exposure but did notconsider serum marker levels (38).
Associations of radiation and markers modified bymenopausal status were tested in the results of eightbioassays (the two parallel analyses of modification byeither menopause status or ATB15 are effectively thesame), which may raise concerns of finding associations
due to multiple testing. Using the conservative Bonferroni
correction for multiple testing (a 5 0.05/8 5 0.00625), allof the tests for interaction remain significant with the
exception of IGF-1 (P 5 0.014).
The study boasts a number of strengths, including
well-characterized radiation doses to a representative
population of women, and data collected prospectively
from cancer-free women as part of a long-term follow-up
regimen, including medical history data on hormonerelated conditions. However, there are also several weaknesses. The day of blood collection in relation to themenstrual cycle was not available, which is problematicfor premenopausal estradiol measurements in particular.
The use of FSH to assign menopausal status has beenshown to be not fully specific (39). It is also likely thatmisclassification occurred when assigning menarche
status by age. Due to the limited number of samples,
duplicate assays for quality control were not attempted. A
limited number of women did have multiple samples
collected while premenopausal, for which the correlation
between marker assays was generally poor, probably due
to different times in the menstrual cycle. Despite theselimitations, no major changes in the interactions between
the measured radiation associations and menopausal
status were detected after separately removing the mostinfluential observations and those observations drawn
from women in the perimenopausal age range, whichhelps confirm the robustness of the associations.
Conclusions
Previous studies have proven the potency of ionizing
radiation as a carcinogen via direct damage effects to
DNA while separate lines of research have establishedserum levels of E2, testosterone and IGF1 as risk factors
for breast cancer. The results of this study suggest thationizing radiation may lead to changes in serum levels ofcancer-related hormones and proteins in cancer-free
women and that these changes are dependent upon

686

GRANT ET AL.

FIG. 4. IGF-1 by menopausal status and radiation dose. The relative change is the percentage change at adose of 1 Gy. The regression lines and P values are based on the final models obtained for the markers(Table 3). The slopes of the two regression lines by menopausal status are significantly different (P 5 0.014).

either the menopausal status at the time of collection or
the menarche status at the time of exposure. Several
mechanisms for these changes are suggested, but they
are all speculative. Researchers of cancer etiology after
whole-body ionizing radiation exposure should be awareof possible underlying changes in the levels of serum
markers, which may confer independent carcinogenic
risks.
ACKNOWLEDGMENTS AND FUNDING
We deeply thank the participating members of the AHS. The
Radiation Effects Research Foundation (RERF), Hiroshima andNagasaki, Japan is a private, non-profit foundation funded by theJapanese Ministry of Health, Labor and Welfare (MHLW) and the U.S.
Department of Energy (DOE), the latter in part through the National
Academy of Sciences. This publication was supported by RERF
Research Protocol RP 06-02 and by US National Cancer Institutecontract NO1-CP-31012 and the Japanese Ministry of Education,
Culture, Sports, Science and Technology grants 14031227 and 15026220.
Received: March 27, 2011; accepted: May 24, 2011; published online:
June 30, 2011

REFERENCES
1. Brenner DJ, Hall EJ. Computed tomography – an increasing
source of radiation exposure. N Engl J Med 2007; 357:2277–84.
2. Preston DL, Mattsson A, Holmberg E, Shore R, Hildreth NG,
Boice JD. Radiation effects on breast cancer risk: a pooled
analysis of eight cohorts. Radiat Res 2002: 158:220–35.

3. Preston DL, Ron E, Tokuoka S, Funamoto S, Nishi N, Soda M,
et al. Solid cancer incidence in atomic bomb survivors: 1958–1998.
Radiat Res 2007; 168:1–64.
4. UNSCEAR. Sources and effects of ionizing radiation. United
Nations Scientific Committee on the Effects of Atomic
Radiation: UNSCEAR 2000 Report to the General Assembly,
with scientific annexes; 2000.
5. Hankinson SE, Willett WC, Manson JE, Colditz GA, Hunter DJ,
Spiegelman D, et al. Plasma sex steroid hormone levels and riskof breast cancer in postmenopausal women. J Natl Cancer Inst
1998; 90:1292–9.
6. Toniolo PG, Levitz M, Zeleniuch-Jacquotte A, Banerjee S,
Koenig KL, Shore RE, et al. A prospective study of endogenous
estrogens and breast cancer in postmenopausal women. J NatlCancer Inst 1995; 87:190–7.
7. Endogenous Hormones and Breast Cancer Collaborative Group,
Key T, Appleby PN, Reeves GK, Roddam AW. Endogenous sex
hormones and breast cancer in postmenopausal women: reanalysis
of nine prospective studies. J Natl Cancer Inst 2002; 94:606–16.
8. Henderson BE, Feigelson HS. Hormonal carcinogenesis.
Carcinogenesis 2000; 21:427–33.
9. Russo J, Russo IH. Genotoxicity of steroidal estrogens. Trends
Endocrinol Metab 2004; 15:211–4.
10. Endogenous Hormones and Breast Cancer Collaborative Group,
Key T, Appleby PN, Reeves GK, Roddam AW. Insulin-likegrowth factor 1 (IGF1), IGF binding protein 3 (IGFBP3), andbreast cancer risk: pooled individual data analysis of 17
prospective studies. Lancet Oncol 2010; 11:530–42.
11. Renehan AG, Zwahlen M, Minder C, O’Dwyer ST, Shalet SM,
Egger M. Insulin-like growth factor (IGF)-I, IGF binding
protein-3, and cancer risk: systematic review and metaregression analysis. Lancet 2004; 363:1346–53.
12. Kaaks R, Berrino F, Key T, Rinaldi S, Dossus L, Biessy C, et al.
Serum sex steroids in premenopausal women and breast cancer

ASSOCIATIONS OF IONIZING RADIATION AND SERUM HORMONES

risk within the European Prospective Investigation into Cancer
and Nutrition (EPIC). J Natl Cancer Inst 2005; 97:755–65.
13. Tworoger SS, Eliassen AH, Sluss P, Hankinson SE. A
prospective study of plasma prolactin concentrations and riskof premenopausal and postmenopausal breast cancer. J Clin
Oncol 2007; 25:1482–8.
14. Russo J, Hu YF, Silva ID, Russo IH. Cancer risk related tomammary gland structure and development. Microsc Res Tech
2001; 52:204–23.
15. Yamada M, Wong FL, Fujiwara S, Akahoshi M, Suzuki G.
Noncancer disease incidence in atomic bomb survivors, 1958–
1998. Radiat Res 2004; 161:622–32.
16. (Young RW, Kerr GD, editors. Reassessment of the atomic
bomb radiation dosimetry for Hiroshima and Nagasaki–
Dosimetry System 2002. Hiroshima, Japan: Radiation Effects
Research Foundation; 2005. 998 p.
17. Cologne JB, Sharp GB, Neriishi K, Verkasalo PK, Land C,
Nakachi K. Improving the efficiency of nested case-control
studies of interaction by selecting controls using countermatching on exposure. Int J Epidemiol 2004; 33:485–92.
18. Biesheuvel CJ, Vergouwe Y, Oudega R, Hoes AW, Grobbee DE,
Moons KG. Advantages of the nested case-control design indiagnostic research. BMC Med Res Methodol 2008; 8:48.
19. Pierce DA, Stram DO, Vaeth M. Allowing for random errors inradiation dose estimates for the atomic bomb survivor data.
Radiat Res 1990; 123:275–84.
20. Tokyo Special Reference Laboratories (SRL), Inc [Internet].
(FSH)[Follicle-stimulating hormone(FSH) reference levels]. [cited 2010 Jun 7]. Available from: http://
www.srl.info/srlinfo/news/2003-14.htm. Japanese.
21. Pearce S, Dowsett M, Jeffcoate SL. Three methods compared for
estimating the fraction of testosterone and estradiol not bound to
sex-hormone-binding globulin. Clin Chem 1989; 35:632–5.
22. Liang KY, Zeger SL. Longitudinal data analysis using
generalized linear models. Biometrika 1986; 73:13–22.
23. Belsley DA, Kuh E, Welsch RE. Regression diagnostics:
identifying influential data and sources of collinearity. New
York: Wiley; 1980.
24. Pan W. Akaike’s information criterion in generalized estimating
equations. Biometrics 2001; 57:120–5.
25. StataCorp. Stata Statistical Software: Release 11. College Station,
TX: StataCorp LP; 2009.
26. Lee CJ, Yoon YD. Gamma-radiation-induced follicular degeneration in the prepubertal mouse ovary. Mutat Res 2005; 578:
247–55.

687

27. Lee YK, Chang HH, Kim WR, Kim JK, Yoon YD. Effects of
gamma-radiation on ovarian follicles. Arhiv za higijenu rada i
toksikologiju 1998; 49:147–53.
28. Ataya K, Pydyn E, Ramahi-Ataya A, Orton CG. Is radiationinduced ovarian failure in rhesus monkeys preventable byluteinizing hormone-releasing hormone agonists?: Preliminary
observations. J Clin Endocrinol Metab 1995; 80:790–5.
29. Larsen EC, Müller J, Schmiegelow K, Rechnitzer C, Andersen
AN. Reduced ovarian function in long-term survivors ofradiation- and chemotherapy-treated childhood cancer. J Clin
Endocrinol Metab 2003; 88:5307–14.
30. Frasor J, Weaver A, Pradhan M, Mehta K. Synergistic upregulation of prostaglandin E synthase expression in breast
cancer cells by 17b-estradiol and proinflammatory cytokines.
Endocrinology 2008; 149:6272–9.
31. Salama S, Kamel M, Diaz-Arrastia C, Xu X, Veenstra TD, Salih
S, et al. Effect of tumor necrosis factor on estrogen metabolism
and endometrial cells: potential physiological and pathologicalrelevance. J Clin Endocrinol Metab 2008; 94:28–93.
32. Hayashi T, Morishita Y, Kubo Y, Kusunoki Y, Hayashi I, Kasagi
F, et al. Long-term effects of radiation dose on inflammatory
markers in atomic bomb survivors. Am J Med 2005; 118:83–6.
33. Nakachi K, Hayashi T, Imai K, Kusunoki Y. Perspectives oncancer immuno-epidemiology. Cancer Sci 2004; 95:921–9.
34. Bezemer ID, Rinaldi S, Dossus L, Gils CH, Peeters PH, Noord
PA, et al. C-peptide, IGF-I, sex-steroid hormones and adiposity:a cross-sectional study in healthy women within the European
Prospective Investigation into Cancer and Nutrition (EPIC).
Cancer Causes Control 2005; 16:561–72.
35. Akahoshi M, Amasaki Y, Soda M, Hida A, Imaizumi M,
Nakashima E, et al. Effects of radiation on fatty liver andmetabolic coronary risk factors among atomic bomb survivors in
Nagasaki. Hypertens Res 2003; 26:965–70.
36. Wong FL, Yamada M, Sasaki H, Kodama K, Hosoda Y. Effectsof radiation on the longitudinal trends of total serum cholesterollevels in the atomic bomb survivors. Radiat Res 1999; 151:736–46.
37. Pasquali R. Obesity and androgens: facts and perspectives. Fertil
Steril 2006; 85:1319–40.
38. McDougall JA, Sakata R, Sugiyama H, Grant E, Davis S, Nishi
N, et al. Timing of menarche and first birth in relation to riskof breast cancer in A-bomb survivors. Cancer Epidemiol
Biomarkers Prev 2010; 19:1746–54.
39. Henrich JB, Hughes JP, Kaufman SC, Brody DJ, Curtin LR.
Limitations of follicle-stimulating hormone in assessing menopause
status: findings from the National Health and Nutrition Examination
Survey (NHANES 1999–2000). Menopause 2006; 13:171–7.