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Environmental Health Perspectives
•
v o l u m e 117 | n u m b e r 12 | December 2009
1883
Research
Exposure to environmental estrogens during
critical developmental windows has well-
documented adverse consequences on males
and females of many species, including rodents
and humans [National Institutes of Health
(NIH) 1999; Palmlund 1996]. One group
of environmental endocrine disruptors that is
currently receiving significant attention is the
naturally occurring phyto estrogens (for review,
see Rozman et al. 2006a, 2006b). ese com-
pounds are readily available in the diet, par-
ticularly in soy products (Adlercreutz et al.
1999; Lapcik et al. 1998; Whitten et al. 1995).
The major class of phyto estrogens found in
soy is isoflavones; the phyto estrogen in that
class that has received the most attention is
genistein (GEN). GEN is primarily present in
its glycosylated forms, with genistin (GIN), the
4′-β--glucoside, being predominantly found
in most soy products, including soy-based
infant formulas, where GIN makes up > 65%
of the isoflavone content (Setchell et al. 1997).
Infants consuming soy-based formulas have
high circulating levels of GEN, the primary
metabolite and aglycone form of GIN, rang-
ing from 1.4 to 4.5 µM (381–1,224 ng/mL)
(Setchell et al. 1997); a recent study reported
the 25th, 50th, and 75th quartiles in serum
as 405.3, 890.7, and 1455.1 ng/mL (1.5, 3.3,
and 5.4 µM, respectively) (Cao et al. 2009),
indicating that GIN is readily absorbed and
hydrolyzed to the aglycone form, GEN.
Studies using experimental rodent models
have shown that neonatal exposure to GEN
by subcutaneous (sc) injections caused adverse
consequences on the female rodent reproduc-
tive system, including altered ovarian differ-
entiation, altered estrous cyclicity, subfertility/
infertility, and reproductive tract cancer (Chen
et al. 2007; Delclos et al. 2009; Jefferson et al.
2002, 2005, 2006; Kouki et al. 2003; Lewis
et al. 2003; Nagao et al. 2001; National
Toxicology Program 2008; Newbold et al.
2001; Nikaido et al. 2004). After treatment
with 50 mg/kg/day, the maxi mum serum
circulating level (Cmax) of GEN was 6.8 µM
(1,836 ng/mL) (Doerge et al. 2002). Although
this level of GEN in mouse serum was only
slightly higher than reported in infants on soy-
based formulas (Cao et al. 2009; Setchell et al.
1997), we documented adverse effects on the
reproductive system at this dose and at lower
doses of 0.5 and 5 mg/kg GEN (Jefferson
et al. 2002, 2005), suggesting that the levels
found in human infants consuming soy-based
infant formulas have the potential to cause
adverse effects. It was not clear, however,
whether the route (sc) and form of compound
(GEN) administered were appropriate for pre-
dicting human health risks.
Because of limited metabolic capacity
of the neonate, it was hypothesized that GIN
would not be efficiently hydrolyzed to GEN
(Rozman et al. 2006a) and, further, that
phase II metabolism such as glucuronidation
would also be limited (Coughtrie et al. 1988;
Doerge et al. 2001; Onishi et al. 1979). Few
studies have reported exposing neo natal rodents
orally to GIN because of their small size; how-
ever, in a pilot study we demon strated that
oral GIN elicited estrogenic activity (Jefferson
et al. 2007). Neonatal mice treated orally on
days 2–5 with 25 mg/kg GIN showed increased
uterine wet weight gain similar to mice that
received 20 mg/kg GEN sc, thus indicating that
approximately 80% of orally administered GIN
reached sufficient circulating levels of the active
compound to elicit a biological effect com-
pared with sc GEN (Jefferson et al. 2007). is
is important because Rozman et al. (2006a)
argued that orally administered GIN, the form
available to infants consuming soy formula, was
not biologically active as an estrogen and there-
fore could not cause adverse effects associated
with other environmental estrogens.
e purpose of the present study was to
determine neonatal estrogenic activity after
oral exposure to GIN, as determined by
increased uterine wet weight and induction of
Address correspondence to W. Jefferson, NIEHS,
111 Alexander Dr., Research Triangle Park, NC
27709 USA. Telephone: (919) 541-4118. Fax: (919)
541-0696. E-mail: jeffers1@niehs.nih.gov
Supplemental Material is available online (doi:
10.1289/ehp.0900923.S1 via http://dx.doi.org/).
We thank K. Thayer and J. Heindel for helpful
comments.
This research was supported by the Intramural
Research Program of National Institute of
Environmental Health Sciences/National Institutes
of Health. K.A.W. acknowledges support from Oak
Ridge Institute for Science and Education admin-
istered through an interagency agreement between
the U.S. Department of Energy and Food and Drug
Administration.
e views presented do not necessarily reflect those
of the Food and Drug Administration.
The authors declare they have no competing
financial interests.
Received 27 April 2009; accepted 27 July 2009.
Oral Exposure to Genistin, the Glycosylated Form of Genistein, during
Neonatal Life Adversely Affects the Female Reproductive System
Wendy N. Jefferson,1,2 Daniel Doerge,3 Elizabeth Padilla-Banks,1,2 Kellie A. Woodling,3 Grace E. Kissling,4
and Retha Newbold 2,5
1Laboratory of Reproductive and Developmental Toxicology, and 2Laboratory of Molecular Toxicology, National Institute of
Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park,
North Carolina, USA; 3National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, USA;
4Biostatistics Branch, and 5National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of
Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA
Background: Developmental exposure to environmental estrogens is associated with adverse
con sequences later in life. Exposure to genistin (GIN), the glycosylated form of the phytoestrogen
genistein (GEN) found in soy products, is of concern because approximately 20% of U.S. infants are
fed soy formula. High circulating levels of GEN have been measured in the serum of these infants,
indicating that GIN is readily absorbed, hydrolyzed, and circulated.
oBjectives: We investigated whether orally administered GIN is estrogenic in neonatal mice and
whether it causes adverse effects on the developing female reproductive tract.
Me t h o d s : Female CD-1 mice were treated on postnatal days 1–5 with oral GIN (6.25, 12.5, 25, or
37.5 mg/kg/day; GEN-equivalent doses), oral GEN (25, 37.5, or 75 mg/kg/day), or subcutaneous
GEN (12.5, 20, or 25 mg/kg/day). Estrogenic activity was measured on day 5 by determining uterine
wet weight gain and induction of the estrogen-responsive gene lactoferrin. Vaginal opening, estrous
cyclicity, fertility, and morphologic alterations in the ovary/reproductive tract were examined.
results: Oral GIN elicited an estrogenic response in the neonatal uterus, whereas the response to
oral GEN was much weaker. Oral GIN altered ovarian differentiation (i.e., multio ocyte follicles),
delayed vaginal opening, caused abnormal estrous cycles, decreased fertility, and delayed parturition.
conclusions: Our results support the idea that the dose of the physiologically active compound
reaching the target tissue, rather than the administered dose or route, is most important in model-
ing chemical exposures. is is particularly true with young animals in which phase II metabo lism
capacity is under developed relative to adults.
ke y w o r d s : development, diethylstilbestrol, endocrine disruptors, environmental estrogen, isoflavone,
ovary. Environ Health Perspect 117:1883–1889 (2009). doi:10.1289/ehp.0900923 available via http://
dx.doi.org/ [Online 27 July 2009]
Jefferson et al.
1884
v o l u m e 117 | n u m b e r 12 | December 2009
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Environmental Health Perspectives
the estrogen-regulated gene lactoferrin (LF),
and then compare this response with that of
sc GEN exposure. Further, we determined
long-term consequences on the developing
reproductive system after orally administered
GIN, including multio ocyte follicles (MOFs),
timing of vaginal opening, estrous cyclicity,
and reproductive function. The findings of
this study show that oral administration of
GIN, the route and chemical found in soy
products, including soy-based infant formulas,
adversely affects the developing reproductive
system in mice.
Materials and Methods
Animals. Adult female CD-1 [Crl:CD-1
(ICR) BR] mice were obtained from Charles
River Breeding Laboratories (Raleigh, NC)
and bred to male mice of the same strain at the
National Institute of Environmental Health
Sciences (NIEHS). Vaginal plug detection
was considered day 0 of pregnancy. Pregnant
mice were individually housed in ventilated
polysulfone cages (Technoplast, Inc., Exton,
PA) with hardwood chip bedding under con-
trolled lighting (12/12-hr light/dark cycle) and
temperature (21–22°C) conditions. Mice were
fed NIH 31 mouse chow (Zeigler Brothers,
Gardners, PA)—which was assayed for phyto-
estrogen content as previously described
(Thigpen et al. 1999)—and provided fresh
water ad libitum. All animals were treated
humanely and with regard for alleviation of
suffering, and all animal procedures complied
with NIEHS/NIH animal care guidelines.
Female pups were pooled together, sepa-
rated by sex, and then randomly standardized
to eight female pups per dam; male pups were
untreated and used as breeders after reaching
adulthood. Female pups were treated by one of
two protocols on post natal days (PNDs) 1–5:
a) by sc injection with corn oil or GEN (12.5,
20, or 25 mg/kg/day; 98% pure; Sigma
Chemical Co., St. Louis, MO) suspended
in corn oil (0.02 mL/pup); or b) orally with
corn oil, GEN (25, 37.5, or 75 mg/kg/day),
or GIN (10, 20, 40, or 60 mg/kg/day; 98%
pure; Sigma) suspended in corn oil (2.5 µL/g).
Oral doses were adminis tered using a pipette
inserted inside each pup’s mouth. Pups con-
sumed the dose easily and did not show weight
loss, stress, or any other gross toxic effect.
Chemical structures of GIN and GEN are
shown in Figure 1. Structurally, GIN is com-
posed of GEN and a large sugar group that
accounts for 37.5% of its molecu lar weight.
Based on this, we used actual GEN (aglycone)
equivalents for dosing with GIN; therefore,
10 mg/kg/day GIN = 6.25 mg/kg/day GEN,
20 mg/kg/day GIN = 12.5 mg/kg/day GEN,
40 mg/kg/day GIN = 25 mg/kg/day GEN,
and 60 mg/kg/day GIN 60 = 37.5 mg/kg/
day GEN. roughout the remainder of this
article, we refer to these GEN-equivalent doses
as GIN 6.25, 12.5, 25, and 37.5 mg/kg.
Uterotropic bioassay for estrogenicity in
neonates. Female pups were treated with oral
GIN or GEN or with sc GEN on PNDs 1–5;
4 hr after the last treatment, individual body
weights were taken and pups were euthanized
by decapitation. Uteri were carefully collected
using an Olympus SZX16 dissecting scope
(Olympus America, Center Valley, PA), and
uterine wet weight was obtained (eight mice
per treatment group). Uterine weight was not
adjusted for body weight because body weights
were not significantly different across treat-
ment groups. Uteri were frozen on dry ice and
stored at –80°C.
RNA was isolated from uteri (minimum
of four per group) using the RNeasy Mini
Kit (Qiagen, Valencia, CA) and then reverse
transcribed into cDNA using the First Strand
cDNA Synthesis Kit (Invitrogen, Carlsbad,
CA). We determined lactoferrin (LF) expres-
sion by real-time reverse transcriptase poly-
merase chain reaction (RT-PCR) as verification
of estrogenic activity, as previously reported
(Newbold et al. 2007); 18S ribosomal RNA
was used for normalization. We determined
expression using the mathematical model
described by Pfaffl (2001):
Expression = 2 (Ct18S – CtLF) × 105,
where Ct is the cycle threshold.
Serum levels of GEN in neonates. Female
pups were treated orally with GIN 37.5 mg/kg
or GEN 37.5 mg/kg on PNDs 1–5. On
PND5 at each time point (0, 0.5, 1, 2, 4, 8,
24, and 48 hr) after the last treatment, four to
six individual pups were decapitated and trunk
blood was collected. After clotting at room
temperature, serum (20–40 µL) was prepared
immediately by centrifugation, frozen on dry
ice, and stored at –80°C. We determined
total GEN content by liquid chromatography
with electrospray tandem mass spectrometry
(LC-ES/MS/MS) after enzymatic deconjuga-
tion and for GEN (aglycone) without decon-
jugation using aliquots of 10 µL as described
previously (Doerge et al. 2002). e limit of
detection was approximately 0.03 µM (signal/
noise, 3); the inter- and intra day precision and
accuracy were 3–8% and 93–96%, respectively
(Twaddle et al. 2002). Model-independent
pharmaco kinetic analysis was performed as
previously described (Doerge et al. 2002).
Levels of daidzein and equol were also evalu-
ated by LC-ES/MS/MS and found to be con-
sistently undetectable (data not shown).
Ovarian histology. To determine effects on
ovarian development, ovaries were collected
prepubertally, after secondary follicle forma-
tion but before corpora lutea formation, as
previously reported (Jefferson et al. 2002).
Female mice treated orally with GIN (0,
6.25, 12.5, 25, or 37.5 mg/kg/day) on PNDs
1–5 were sacrificed at 19 days of age by CO2
asphyxiation (eight mice per treatment group).
Ovaries were collected and fixed in 10% cold
neutral buffered formalin overnight and then
changed to cold 70% ethanol. Tissues were
then processed for histology, embedded in
paraffin, and cut at 5 µm. ree sections from
each of three different levels for both ovaries
were scored for the presence and numbers of
MOFs and any alterations in ovarian histology
(total of 18 sections scored per mouse).
Vaginal opening and estrous cyclicity.
Female mice treated orally with GIN (0, 6.25,
12.5, 25, or 37.5 mg/kg) on PNDs 1–5 were
weaned at 22 days of age, housed four per
cage, and followed daily for vaginal opening
(16 mice per treatment group). At 2 months
of age, vaginal smears were obtained daily for
2 weeks from half of the mice in each treat-
ment group (eight mice per group). Smears
were collected on positively charged slides
(Superfrost Plus; Fisher Scientific, Pittsburgh,
PA), sprayed with Spraycyte fixative (Fisher),
and stained with hematoxylin and eosin
(Sigma) to determine the stage of the estrous
cycle, as previously described (Champlin et al.
1973). Mice with 3 consecutive days in either
diestrus or estrus were considered to have
abnormal cycles.
Fertility assessment. At 2, 4, and 6 months
of age, female mice that were used to determine
vaginal opening and estrous cyclicity were bred
to proven control males of the same strain for a
2-week period (16 mice per treatment group).
Females were checked in the morning for vagi-
nal plugs, removed from the male cages, and
individually housed until delivery. All females
that were not “vaginal plug positive” were
removed and then returned to the male cages
Figure 1. Chemical structures of GIN (oral exposure) and GEN (sc exposure). MW, molecular weight.
Quickly hydrolyzed
in digestive system
Glucosides-O
OH OH OH OH
HO
OO
162.5 g/mol
37.5%
270.24 g/mol
62.5%
270.24 g/mol
100%
GIN (genistein glucoside)
MW = 432.4 g/mol
GEN (aglycone)
MW = 270.24 g/mol
Oral exposure to genistin
Environmental Health Perspectives
•
v o l u m e 117 | n u m b e r 12 | December 2009
1885
in the afternoon for a total of six attempts at
achieving vaginal plug–positive status. Fertility
assessment included the following end points:
number of plug-positive females, number of
mice with live litters, average number of live
pups per litter, and time to delivery.
Statistical analysis. e data were analyzed
using JMP 7 and SAS 9.1 software (both from
SAS Institute Inc., Cary, NC). For uterine
wet weight and real-time RT-PCR data, we
performed analysis of variance, followed by
Dunnett’s test. For MOFs, estrous cyclicity,
and fertility end points, statistical significance
was determined using the non parametric
Mann-Whitney tests or Fisher’s exact test,
as appropriate. e Cochran-Armitage trend
test was used to test for dose trends in quan-
tal responses such of MOFs. p-Values < 0.05
were considered statistically significant.
Results
Estrogenic activity in neonates. Female mice
treated with sc GEN at 20 and 25 mg/kg/day
had increased uterine wet weights at 5 days of
age compared with controls (Figure 2A). We
also observed increased uterine wet weight
in mice treated orally with GIN at 25 and
37.5 mg/kg/day (Figure 2A). Compared
with sc GEN, approximately 20–33% more
oral GIN was needed to elicit similar uterine
wet weight increase compared with controls.
We observed no increase after oral GEN 25
or 37.5 mg/kg and only a slight increase at
75 mg/kg/day (Figure 2A), suggesting that
much more oral GEN is needed to elicit an
estrogenic response compared with either sc
GEN or oral GIN.
To investigate functional estrogen activity
after sc or oral exposures, we determined
mRNA levels of the estrogen-regulated pro-
tein LF by real-time RT-PCR. Mice treated
by sc GEN 12.5, 20, and 25 mg/kg showed
increased LF mRNA compared with controls,
correlating with increased uterine wet weight
at higher doses, resulting in higher expression
of LF (Figure 2B). Oral exposure to GIN 12.5,
25, and 37.5 mg/kg showed similar increases
in LF mRNA compared with controls, which
also correlated with increased uterine wet
weight (Figure 2B).
Serum levels of GEN in neonates. Figure 3
shows the total and aglycone levels of GEN
measured in serum after oral treatment with
37.5 mg/kg GIN. ese data suggest that the
glucoside moiety of GIN is readily cleaved to
the aglycone form, GEN, which can either
be absorbed into the circulation as the agly-
cone (Cmax = 5.6 µM) or conjugated by UDP-
glucuronosyl transferases in the gut and secreted
as conjugated forms into circulation (total Cmax
= 19.2 µM). These levels of GEN are higher
than those previously reported after sc GEN
50 mg/kg (Cmax = 2.3 µM aglycone and 5.0 µM
for total) (Doerge et al. 2002). However, the
dose-adjusted area under the curve (AUC)
for total GEN is slightly lower after oral GIN
(83% of sc GEN) and lower than the AUC for
aglycone adjusted for dose (48% of sc GEN;
Table 1). ese differences are captured by the
difference in percent aglycone AUC for sc ver-
sus oral administration (22% vs. 13% of total
AUC), which reflects the bypass of phase II
conjugation in the gut after injection.
e internal exposures after orally admin-
istered GEN were much lower compared with
oral GIN, measured either as Cmax (~ 1 µM)
or as AUC (3.6 µM-hr; Table 1). erefore,
it was not surprising that orally administered
GEN did not result in a robust estrogenic
response in the neonate; because results of sc
administration of GEN have been previously
reported (Doerge et al. 2002), in the present
study we followed only mice treated by oral
GIN for ovarian histology, timing of vaginal
opening, estrous cyclicity, and fertility.
Ovarian histology. To determine the
effect of oral GIN on the developing ovary,
we evaluated the presence of MOFs in imma-
ture mice at 19 days of age. e percentage
of MOFs increased with dose of oral GIN
(p < 0.05, Cochran-Armitage trend test) and
were significantly higher than in controls in
all dose groups except the lowest dose [see
Supplemental Material, Figure 1A, available
online (doi:10.1289/ehp.0900923.S1 via
http://dx.doi.org/)].
Vaginal opening and estrous cyclicity. Mice
exposed to the highest dose of GIN (37.5 mg/
kg) had delayed vaginal opening compared
with their age-matched control counterparts,
with 50% of the GIN-treated mice achieving
vaginal opening 2 days later [see Supplemental
Material, Figure 2 (doi:10.1289/ehp.0900923.
S1)]. In addition, a few mice in the top two
dose groups did not have definitive vaginal
opening, even 5 days after the last control
mouse exhibited opening. Mice in all other
groups appeared to achieve vaginal opening at
a similar time as controls.
To determine the effect of oral GIN on
estrous cyclicity, vaginal smears were taken
for 2 weeks beginning at 2 months of age.
None of the mice in the control group had
abnormal estrous cycles, whereas 38% of GIN
12.5 mg/kg, 62% of GIN 25 mg/kg, and 88%
of GIN 37.5 mg/kg mice had abnormal cycles
(p < 0.001, Cochran-Armitage trend test), and
the highest two groups had cycles significantly
different from those of controls [Fisher’s exact
test, p < 0.05; see Supplemental Material,
Figure 1B (doi:10.1289/ehp.0900923.S1)].
Abnormal cycles exhibited by mice were pre-
dominantly prolonged time in estrus (with
a few mice exhibiting prolonged time in
diestrus).
Fertility assessment. e percentage of oral
GIN-treated females that were vaginal plug
positive was similar among dose groups at
2, 4, and 6 months of age [Figure 4; see also
Supplemental Material, Table 1 (doi:10.1289/
ehp.0900923.S1)]. We observed a significant
reduction in the number of mice delivering
live pups after oral exposure to GIN 25.0
and 37.5 mg/kg at 6 months of age. In addi-
tion to fewer pregnant females, mice that did
deliver live pups in the GIN 37.5 mg/kg dose
had fewer pups at 2 and 6 months of age; the
GIN 25 mg/kg group also had fewer pups at
6 months of age.
We also combined all the data across the
time points (Figure 4C, Table 2). e percent-
age of plug-positive females that resulted in
litters with live pups was significantly reduced
in the oral GIN 12.5, 25.0, and 37.5 mg/kg
groups. In addition, the total number of pups
produced per dam over the three time points
and the average number of pups per litter were
Figure 2. Effects of neonatal treatment with GEN and GIN on female mice on PND5. C, corn oil control.
(A) Uterotropic bioassay after sc or oral GEN or oral GIN; values shown are mean ± SE uterine wet weight
for each treatment group (n = 8 mice per group). (B) Real-time RT-PCR for LF shown as mean ± SE of LF
expression normalized to cyclophilin × 10,000.
*p < 0.05, compared with controls, Dunnett’s test.
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0.000
160
140
120
100
80
60
40
20
0
Uterine weight (g)
LF expression
normalized to 18S
**
*
*
*
*
*
CCCCC20 2025 25 25 257537.5 37.5 37.525
sc GEN sc GENOral GIN Oral GIN
Neonatal treatment (mg/kg) Neonatal treatment (mg/kg)
Oral GEN
12.5 12.5 12.5 12.5
Figure 3. Serum levels of GEN (mean ± SE) after oral
exposure to GIN 37.5 mg/kg on PNDs 1–5. Serum
was collected at 0.5, 1, 2, 4, 8, 24, and 48 hr after the
last treatment (n = 4–6 samples per group).
25
20
15
10
5
0
0246
Time (hr)
82448
Aglycone GEN
Total GEN
Serum level (µM)
Jefferson et al.
1886
v o l u m e 117 | n u m b e r 12 | December 2009
•
Environmental Health Perspectives
significantly reduced in the 37.5 mg/kg group
compared with the control group. e average
numbers of pups per litter were borderline sig-
nificantly reduced in the 12.5 and 25.0 mg/kg
GIN groups, as was the total number of pups
produced in the 12.5 mg/kg group.
The timing of delivery after achieving
vaginal plug–positive status is summarized
in Figure 5. All control mice (treated with
corn oil) delivered their pups early in the
morning of gestation day (GD) 19, before
0900 hours. All oral GIN groups had some
mice that delivered late in the afternoon of
GD19 (between 1400 and 1800 hours) or
delivered 1–2 days late, on GD20 or GD21.
One mouse in the GIN 6.25 mg/kg group was
visibly pregnant but did not deliver by 2 days
past expected delivery; after the mouse was
euthanized, 16 fully formed pups were found
dead. On-time deliveries were significantly
reduced after oral GIN 25.0 and 37.5 mg/kg
at all time points and oral GIN 12.5 mg/kg at
4 and 6 months of age (p < 0.05, Fisher’s exact
test). In addition, the percentage of mice with
on-time deliveries decreased with increasing
dose and increasing time (p < 0.05, Cochran-
Armitage trend test). Figure 5B shows the per-
centage of mice with late deliveries (afternoon
or 1–2 days late) and no live pups per treat-
ment group over time. More than half of the
mice at 4 and 6 months of age in the 12.5,
25, and 37.5 mg/kg groups had either late
deliveries or no live pups. We also observed
this effect in GIN 6.25 mg/kg mice, with 27%
exhibiting late delivery at 6 months of age.
After observing this effect at 2 months of age,
we took some litter weights at 4 and 6 months
of age and calculated the average pup weight
per litter. Although there were too few pup
weights per treatment group per age to evalu-
ate statistically, the averages combined by cate-
gory of delivery time suggest increased pup
weight with delayed parturition. All litters that
were on time (regardless of treatment) had an
average pup weight of 1.64 ± 0.03 g (n = 28
litters), and litters that were born late in the
afternoon were no different, with an average
pup weight of 1.65 ± 0.03 g (n = 8 litters);
however, litters that were born 1 or 2 days
late had a significantly increased average pup
weight of 1.82 ± 0.04 g (n = 7 litters; p < 0.05,
Student’s t-test).
Discussion
e present study shows that oral exposure to
GIN, the glycosylated form of GEN found
in soy products, results in estrogenic activity
in PND5 female neonates similar to the
response to sc GEN. We measured estrogenic
activity by increases in uterine wet weight
and induction of the estrogen-responsive gene
LF. is is significant because experimental
studies using sc exposure have been criticized
for not modeling oral exposure with respect
to metabo lism and kinetics and were thus
dismissed as offering no value for human risk
assessment (Rozman et al. 2006a, 2006b). In
addition, animal studies using oral dosing of
GEN to nursing dams produced very little
circulating GEN in neonates, demonstrating
that indirect exposure through the dam’s milk
is not sufficient to produce levels of GEN that
approach levels in human infants consum-
ing soy formula (Doerge et al. 2006; Lewis
et al. 2003). Perinatal deficiencies in phase II
conjugating activity have also been shown
to play a major role in determining inter-
nal exposures of rodent fetuses and neonates
to the active aglycone form (GEN) (Doerge
et al. 2001, 2002). Therefore, questions of
relative bioavailability of aglycone versus glu-
coside forms of isoflavones persist, particularly
because soy-based foods and formula con-
tain predominately glucosides (Rozman et al.
2006a, 2006b).
The relative bioavailability of isoflavone
glucosides versus aglycones has been studied
extensively in rodents and humans, but results
have conflicted (Izumi et al. 2000; King et al.
1996; Kwon et al. 2007; Richelle et al. 2002;
Rufer et al. 2008; Sepehr et al. 2007; Setchell
et al. 2001; Steensma et al. 2006; Zubik and
Meydani 2003). It is generally accepted that
the aglycone form of isoflavones has the high-
est estrogenic activity. However, glucosides
are quickly hydrolyzed to produce the agly-
cone form, so administration of either the
glucoside or aglycone leads to absorption of
the biologically active aglycone. us, expo-
sure to GEN is theoretically the sum of the
aglycone and respective glucoside concen-
trations converted on the basis of molecular
weight (Rozman et al. 2006a). Absorption
of GIN has been demon strated by showing
glucuronidated metabolites of GEN and other
GEN metabolites in urine of infants fed soy-
based formulas (Hoey et al. 2004). Our study
confirms that oral GIN is rapidly hydrolyzed
and absorbed into neonatal circulation, which
is similar to metabolism in human infants
because high circulating levels of GEN are
seen after oral ingestion of soy-based infant
formulas containing predominantly GIN.
We compared internal exposures to GEN
from oral GIN and GEN in the present study
with results for sc GEN from our previous
study (Doerge et al. 2002). e dose-adjusted
AUCs for aglycone and total GEN for oral
GIN and sc GEN were remarkably similar
(Table 1). e dose-adjusted AUC for GEN
aglycone after oral GIN were approximately
half those for sc GEN, which reflected the
difference in percent aglycone (22% for sc
GEN and 13% for oral GIN). Despite this
difference, similar magnitudes of effects were
Table 1. Comparison of serum circulating levels of GEN after sc or oral GEN or oral GIN exposure.
Treatment (mg/kg)
Total GEN Aglycone
AUC
Dose corrected
(AUC/dose) Percent sc AUC
Dose corrected
(AUC/dose) Percent sc
sc injection
GEN 50a147 2.9 100 33 0.66 100
Oral exposure
GIN 37.5 90.4 2.4 83 12.1 0.32 48
GEN 37.5 12.8 0.34 12 3.6 0.10 15
aData from Doerge et al. (2002) were adjusted for the dose differences in the present study.
Figure 4. Fertility assessment of mice treated neonatally with oral GIN. C, corn oil control. (A) Percentage of vaginal plug–positive mice delivering live pups by age
(2, 4, and 6 months). (B) Number of live pups per litter (mean ± SE) by age (2, 4, and 6 months). (C) Percentage of mice delivering live pups for all ages combined
(mean ± SE).
*p < 0.05, compared with controls by Fisher’s exact test. **p < 0.05, compared with controls by Mann-Whitney test.
100
80
60
40
20
0
100
80
60
40
20
0
16
14
12
10
8
6
4
2
0
Percent mice with
live pups
Percent mice with
live pups
Live pups/litter
** ** **
**
** **
**
2
C
GIN 6.25
GIN 12.5
GIN 25.0
GIN 37.5
4 6 2 4 6
Age (months) Age (months) Treatment
Oral exposure to genistin
Environmental Health Perspectives
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v o l u m e 117 | n u m b e r 12 | December 2009
1887
observed on uterine weight gain, ovary histol-
ogy, estrous cyclicity, and fertility. e 50%
reduction in aglycone after oral GIN com-
pared with sc GEN, with only a 20% reduc-
tion in uterine weight gain, poses interesting
questions about the dynamics of circulating
levels of GEN and estrogenic activity in tar-
get tissues. Whether the peak concentration
of GEN or the total exposure to GEN (e.g.,
AUC) or some combination of the two is
the driving factor for estrogenic activity is
unknown, but the present data suggest that
both might be important. In addition, the
glycosylated form can be passively transported
across the small intestinal membrane and
enter circulation by the sodium-dependent
glucose transporter (SGLT1), unlike the
mechanism by which the aglycone form is
absorbed (i.e., passive diffusion) (Kwon et al.
2007). Detailed information on the metabo-
lism, transport, and absorption of these com-
pounds in neonates is necessary to further
understand these results. e lack of measur-
able levels of daidzein or equol in serum elimi-
nates those compounds as potential sources of
estrogenic activity in this model.
Oral exposure to GEN, which has known
estrogenic activity in the human adult (Setchell
et al. 2001) and in pre pubertal and mature
rodents (Diel et al. 2004; van Meeuwen et al.
2007), does not have a robust estrogenic activ-
ity in the mouse neonate. In the uterotropic
bioassay, the highest dose of oral GEN was
75 mg/kg/day, twice the sc GEN or oral GIN
dose, yet there was only a slight increase in
estrogenic activity in the mouse neonate.
Serum circulating levels of GEN after oral
exposure to GEN support this lack of biologi-
cal effect, with very little GEN found in the
circulation (only 12% of the sc dose). Lewis
et al. (2003) demonstrated similar results in
7 rats. e total AUC for the oral route was
approximately 10 times less than the sc treat-
ment, meaning the oral route resulted in only
9% of the circulating levels compared with the
sc route.
Although many studies have described
a role for phytoestrogens, such as GEN, in
influencing hormone-dependent states in
adults, there is limited information, espe-
cially regarding long-term effects, on infant
exposure to soy-based formulas or prod-
ucts. In an epidemiologic study, Cruz et al.
(1994) reported increased cholesterol syn-
thesis rates in human infants fed soy-based
formulas. Strom et al. (2001) concluded that
there was no statistically significant differ-
ence in > 30 outcomes meas ured in young
adult women and men (20–34 years of age)
who were fed soy-based formula or cow-based
formula as infants. However, in women fed
soy-based formula, these authors found a
significant increase in the number of twin
births, duration of the menstrual cycle, and
pain associated with the cycle, despite the
small cohort size (129 soy-based formula–
fed women vs. 269 cow-based formula–fed
women). A recent study of human infants
fed soy formula, cow milk formula, or breast
milk, Bernbaum et al. (2008) found that
female infants fed soy formula have re-estro-
genization of vaginal cells at 6 months of age;
this effect was not observed in the two non soy
groups. In another recent epidemiology study,
Zung et al. (2008) reported that breast tissue
is more prevalent in the second year of life
in female infants fed soy-based formula than
in infants who were breast-fed or fed dairy-
based formula. ese studies support the idea
that soy-based infant formulas exert biological
effects, including estrogenic activity.
The present study clearly demonstrates
that oral exposure to GIN has adverse con-
sequences on the female reproductive system
when exposure occurs during neonatal life.
Furthermore, these effects are similar to sc GEN
at similar doses (Jefferson et al. 2005). For com-
parison, the incidence of MOFs after exposure
to oral GIN is similar to that after sc GEN,
with 37.5% after oral GIN 6.25 mg/kg, 25%
after sc GEN 5 mg/kg, and 75% after both
oral GIN 37.5 mg/kg and sc GEN 50 mg/kg.
Disruptions in estrous cyclicity were also simi-
lar after both treatment routes; 88% of the
mice in both sc GEN 50 mg/kg and oral GIN
37.5 mg/kg groups exhibited abnormal cycles.
We recently demonstrated that the major con-
tribution to infertility in neonatal GEN-treated
mice is the oviductal and uterine environment
(Jefferson et al. 2009). During the preimplan-
tation period, half of the embryos were lost
before the four-cell stage in the oviduct, and
the other half were unable to develop properly
in the uterus, leading to complete infertility.
Table 2. Statistical analysis of fertility end points after oral exposure to GIN (all ages combined).
Outcome, GIN dose (mg/kg) No.Mean ± SE
Mann-Whitney
p-value
No. of litters with live pups per dam
0 15 1.8 ± 0.2
6.25 14 1.9 ± 0.2 0.6192
12.5 15 1.4 ± 0.2 0.1114
25.0 15 1.6 ± 0.2 0.3326
37.5 12 1.3 ± 0.2 0.0631
Percent plug positive with live pups
0 15 100.0 ± 0.0
6.25 14 89.3 ± 7.7 0.2241
12.5 15 84.4 ± 7.7 0.0498
25.0 15 66.7 ± 8.9 0.0004
37.5 12 63.9 ± 11.0 0.0009
Total no. of live pups per dam
0 15 23.5 ± 2.6
6.25 13 26.5 ± 2.6 0.7974
12.5 14 17.9 ± 2.2 0.0570
25.0 13 21.2 ± 2.1 0.3882
37.5 10 13.8 ± 1.9 0.0040
Average no. of live pups per litter
0 15 13.3 ± 0.5
6.25 13 13.2 ± 0.4 0.3966
12.5 14 12.0 ± 0.8 0.0540
25.0 13 11.8 ± 0.8 0.0597
37.5 10 9.3 ± 1.0 0.0007
End points for each mouse (all three breeding ages) were combined to examine the overall effect on fertility (number of
mice with litters out of 16).
Figure 5. Timing of delivery of live pups. C, corn oil control. (A) Percentage of vaginal plug–positive mice
delivering on time for each group by age (2, 4, and 6 months). (B) Percentage of mice per treatment group by
age (2, 4, and 6 months) that delivered in the late afternoon or 1–2 days late, or that delivered no live pups.
*p < 0.05 compared with controls by Fisher’s exact test.
100
80
60
40
20
0
100
80
60
40
20
0
Percent mice delivering on time
Percent vaginal plug–positive
2
2 months
No litter
Late ≥ 1 day
Late ≥ afternoon
C
GIN 6.25
GIN 12.5
GIN 25.0
GIN 37.5
4
4 months
Oral GIN (mg/kg)Age (months)
6
6 months
*
**
*
*
*
*
*
C25 37.56.25 12.5C25 37.56.25 12.5C25 37.56.25 12.5
Jefferson et al.
1888
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•
Environmental Health Perspectives
Although we did not see complete infertility
at any dose examined in the present study,
observed reduced fertility may result from an
insufficient uterine environment. Interestingly,
oral GIN 12.5 mg/kg did not exhibit an estro-
genic response in the uterus on PND5, yet
adverse effects on the reproductive system were
seen later in life. Further studies are under way
to elucidate mechanisms involved in decreased
embryo survival and how developmental expo-
sure to estrogenic chemicals may permanently
alter gene expression necessary for maintaining
pregnancy.
Delayed parturition was another impor-
tant finding in the present study. The bio-
logical signals for parturition are not fully
understood, but several factors, including
progesterone levels, placental prostaglandins,
and uterine oxytocin receptors, have been
identified as playing roles (Cook et al. 2003).
us, potential reasons for late parturition in
neonatal GIN-exposed mice include lack of
signaling from the pup (e.g., prostaglandins),
elevated progesterone during late pregnancy,
or lack of response or signaling from the
uterus (e.g., oxytocin receptors) (Cook et al.
2003). Because our previous study showed
that high doses of sc GEN render the uterus
incapable of supporting pregnancy, it seems
reasonable that lower doses might also impair
uterine signaling. Studies are under way to
understand mechanism(s) responsible for late
parturition in these mice.
Although care must be taken in extrapolat-
ing data from rodents to humans, important
information can be gained from experimental
animal studies. In summary, our study demon-
strates that exposure to oral GIN during
neonatal development results in adverse conse-
quences in the adult female mouse reproduc-
tive system. Reduced fertility is seen at GIN
doses of 12.5, 25, and 37.5 mg/kg. By com-
parison, estimated human infant consump-
tion is 6–11 mg/kg isoflavones and 4–7 mg/kg
GEN (Setchell et al. 1997, 1998). Although
the doses used in our study are slightly higher,
similar circulating concentrations of total
GEN resulted in mice and humans. To date,
the pharmaco kinetics of GEN in the serum of
infants on soy formulas has not been accurately
evaluated; only single-point estimates of the
steady-state concentration measured at some
unknown time since last feeding have been
evaluated [e.g., 2.5 µM (675 ng/mL) mean
total GEN in plasma (Setchell et al. 1997),
3.6 µM (972 ng/mL) median total GEN in
whole blood (Cao et al. 2009)]. Data on adult
human pharmaco kinetics suggest that these
steady-state estimates are probably much lower
than peak levels of GEN. e small margin of
difference between circulating GEN levels in
infants consuming soy-based infant formula
and the levels in neo natal mice that we found
to produce significant increases in reproductive
toxicity suggest that risks to reproductive
health should be carefully considered, espe-
cially because an estimated 20–25% of infants
in the United States consume soy-based infant
formulas and the Committee on Nutrition of
the American Academy of Pediatrics found
“very limited indications for its use” (Bhatia
and Greer 2008). Thus, further studies on
reproductive end points in the human popula-
tion are warranted. Our data also show that
peri natal deficiencies in metabolism after oral
exposure to GEN can affect internal exposures
in ways similar to that of injection, so only the
dose of the active chemical that reaches the
target tissue is important. e present study
further validates the use of sc-injected GEN as
a suitable model for oral exposure to GIN in
neonatal mice.
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