Toxicology and Applied Pharmacology 237 (2009) 288–297
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Toxicology and Applied Pharmacology
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p
Differential effect of pure isoflavones and soymilk on estrogen
receptor activity in mice
Gianpaolo Rando, Balaji Ramachandran, Monica Rebecchi, Paolo Ciana, Adriana Maggi ⁎
Center of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milan Via Balzaretti, 9 I-20133 Milan, Italy
a r t i c l e
i n f o
Article history:
Received 5 November 2008
Revised 17 March 2009
Accepted 26 March 2009
Available online 8 April 2009
Keywords:
Estrogen receptor
Estrogenic compound
Phytoestrogen
Nutraceutical
Genistein
Soy
a b s t r a c t
Background: Because of the complexity of estrogen receptor (ER) physiological activity, the interaction of
pure isoflavones or soy-based diets on ER needs to be clearly demonstrated.
Objectives: To investigate the effects of the administration of isoflavones as a pure compound or as a
component of diet on the ER transcriptional activity in adult mice.
Methods: Effects of acute (6 h) and chronic (21 days) oral administration of soy milk, pure genistein and a
mix of genistein and daidzein was studied in living ERE-Luc mice. In this animal model, the synthesis of
luciferase is under the state of ER transcriptional activity. Luciferase activity was measured in living mice by
daily bioluminescence imaging sessions and in tissue extracts by enzymatic assay.
Results: Acute, oral administration of genistein or soymilk caused a significant increase of ER activity in liver.
In a 20 day long treatment, soymilk was more potent than genistein in liver and appeared to extend its
influence on ER transcriptional activity in other tissues, such as the digestive tract. A mixture of pure
genistein and daidzein at the same concentration as in soymilk failed to induce significant changes during
acute and chronic studies suggesting an important, uncharacterized role of the soymilk matrix. Consistent
with this observation, synergistic effects of the matrix plus isoflavones were observed in MCF-7 cells stably
transfected with the ERE-luc construct.
Conclusions: This study underlines the limitations of the analysis of single food components in the evaluation
of their effects on estrogen receptor activity and advocates the necessity to use complex organisms for the
full comprehension of the effects of compounds altering the endocrine balance.
© 2009 Elsevier Inc. All rights reserved.
Introduction
Depending on their individual alimentary regimens, human beings
are differentially exposed to phytoestrogens: chemicals produced by
plants that mimic estrogens by binding to estrogen receptors (ERs)
with high affinity. The phytoestrogen–ER complex may interact with
other nuclear factors and co-activators and regulate the transcription
of selected genes (Mäkelä et al., 1995). Among these compounds, the
isoflavone genistein, commonly found in legumes and other edible
plants, is the most abundant (Fukutake et al., 1996; Liggins et al., 1998;
Setchell et al., 1987). Genistein was shown to bind ERα and ERβ with
an affinity of 20–100-fold lower than estradiol (Kuiper et al., 1997).
Oriental vegetarian diets may provide a daily intake of genistein of
about 20–80 mg/day, while western diets reach about 1–3 mg/day
(Barnes et al., 1995). Ingestion of a meal of soy-nuts (Setchell et al.,
Abbreviations: ATP, adenosine 5′-triphosphate; AUC, area under the curve; BLI,
bioluminescence imaging; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; E2, 17β-estradiol; EDTA, ethylendiaminetetraacetic acid; EGTA, ethylenglycoltetraacetic acid; ER, estrogen receptor; ERE, estrogen-responsive element; FBS, fetal
bovine serum; HBSS, Hanks balanced salt solution; KPO4, potassium phosphate; RLU,
relative light unit; SEM, standard error of the mean.
⁎ Corresponding author. Tel.: +39 02 50318375.
E-mail address: [email protected] (A. Maggi).
0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2009.03.022
2003) or even a single dose of a soy-extract capsule containing 64 mg
of total isoflavones (Joshi et al., 2007), causes a peak of plasma levels
in the micro-molar range, a concentration believed to have the
potential to influence endocrine signaling. Moreover, additive (Kortenkamp, 2007) and synergistic effects (Payne et al., 2001) on
proliferation of ER-positive breast cancer cells were observed when
low concentration of a particular estrogen was combined with other
exogeneous estrogens. All this leads to the belief that the presence of
several diverse phytoestrogens in aliments may have effects unpredictable on the basis of studies carried out using pure compounds
singularly administered.
Several studies in rodents showed an association between
embryonic or neonatal exposure to phytoestrogens and disorders of
the reproductive apparatus including uterine adenocarcinoma (Newbold et al., 2001), altered development of mammary glands (PadillaBanks et al., 2006) and irregularities of the estrous cycles (Jefferson
et al., 2007).
It is now well-established that ER expression is not only confined
to reproductive organs; hence, phytoestrogens might regulate a
number of physiological functions and eventually cause undesired
effects in organs other than those involved in reproduction. Indeed,
other studies in adult rodents have associated phytoestrogen exposure
with altered bone development (Migliaccio et al., 2000; Hotchkiss et
G. Rando et al. / Toxicology and Applied Pharmacology 237 (2009) 288–297
al., 2005), obesity (Ruhlen et al., 2008), alteration of thymic functions
(Yellayi et al., 2002), myelotoxicity (Guo et al., 2005) and alterations
in the sexually dimorphic behavior (Flynn et al., 2000).
In spite of all the above, the attitude of the clinical community
towards phytoestrogens has been less negative, and several reports
have suggested that soy or isoflavones may exert a protective activity
towards the incidence of pathologies associated with ageing. Clinical
studies in adults provided evidence of the beneficial effects of
isoflavones on metabolic and skeletal functions: genistein was
shown to have protective effects in bone and decreased the
cardiovascular risk when administered to postmenopausal women
(Atteritano et al., 2007; de Kleijn et al., 2002). In addition, a metaanalysis conducted on adults and children consuming soy protein
showed a significant decrease in the serum cholesterol levels
(Anderson et al., 1995). Soy milk consumption was also reported to
lessen the risk of prostate cancer in men (Jacobsen et al., 1998).
Still debated is the risk to benefit ratio of soy consumption in
infants: recent studies claim no adverse effects (Giampietro et al.,
2004; Merritt and Jenks, 2004; Strom et al., 2001), however in the past
poor immune response (Zoppi et al., 1983) and increased respiratory
infections (Zoppi et al., 1982) have been reported. A better
comprehension of the effects of phytoestrogens in infants would be
advisable because they may be particularly exposed to isoflavones
contained in artificial milk (infant-formula), and the amount of
plasma levels of ingested isoflavones by infants fed with soymilk was
significantly higher than in infants breast-fed or consuming cow milk
and was one order of magnitude higher than in adults consuming soy
food (Setchell et al., 1997).
Considering the multitude of effects that have been associated to
diets containing estrogenic compounds and the complexity of ER
physiological activities a more systemic approach to the study of the
effects of prolonged exposure to phytoestrogen-rich diets is
mandatory. The availability of reporter mice in which the transcriptional effects of estrogenic compounds can be measured globally
and in vivo (Ciana et al., 2001; Lemmen et al., 2004) may provide an
appropriate tool to measure the effects of acute or prolonged
exposure to specific diets on ER transcriptional activity. The aim of
the present study, therefore, was to investigate the extent to which
ER activity is affected by short-term and long-term consumption of
isoflavones as pure compounds or as part of a specific diet. The
results here shown point to the value of the reporter mice in
highlighting differential molecular effects of phytoestrogens when
administered as pure substances or as component of soymilk and
the importance of pursuing these studies to reach a full understanding of the potential benefits and harm of diets rich or enriched
with isoflavones.
Materials and methods
Chemicals used. 17β-estradiol (E2), genistein, daidzein, and ICI
182,780 were purchased from Sigma-Aldrich (Pomezia, Italy),
ketamine (Imalgene 500) from Merial (Tolouse France), xilazine
(Rompun) from Bayer (Shawnee Mission, Kansas, USA), and Dluciferin (Beetle luciferin potassium salt) from Promega (Milan, Italy).
Experimental animals. ERE-Luc mice are transgenic mice engineered
to ubiquitously express a transgene, the firefly luciferase, under the
control of an estrogen-responsive promoter (Ciana et al., 2001); these
mice allow to spatio-temporally monitor ER transcriptional activity by
biochemical or optical imaging technologies (Ciana et al., 2003; Ciana
et al., 2005a, 2005b). In the present study, we used heterozygous
males of 3–4 months of age obtained by mating male homozygous
ERE-Luc mice with C57BL/6 wild-type female mice. A group of four
mice were housed in plastic cages with hardwood chip bedding, fed
ad libitum, and provided with filtered water. The animal room was
maintained within a temperature range of 22–25 °C and relative
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humidity of 50% ± 10%. There was a cycle of 12 h light/dark (lights on
at 07:00 AM). Animals were assigned to a specific experimental group
by measuring baseline photon emission before any treatment.
Animals of the same experimental group were housed in the same
cage. Animal experimentation was carried out in accordance with
European guidelines for animal care and use of experimental animals,
approved by the Italian Ministry of Research and University, and was
controlled by the panel of experts of the Department of
Pharmacological Sciences, University of Milan.
Diet and alimentary habits. Because of our previous observation that
food containing low-phytoestrogens may still activate ERs (Ciana
et al., 2005a, 2005b), animals were maintained with regular lowphytoestrogen diet (4RF21, Mucedola) and switched to AIN-93M diet
(Mucedola) at least 96 h before treatments. The AIN-93M diet was
experimentally selected among other diets, by feeding fasted ERE-luc
mice and scoring the induction of luciferase activity (not shown).
Aliquots of the same batch were stored under vacuum at 4 °C until
used. Food and fluid consumption were measured all through the day
and we observed that 70% of the fluids and 75% of the food were
consumed between 7:00 PM and 7:00 AM, with the peak of
consumption around 1:00 AM. Each animal was weighed on the
first and last days of the experiments.
Soymilk isoflavone extraction. 25 mL of analytical grade EtOH 80%
(Sigma), were added to 10 mL of soymilk and the mixture was kept at
37 °C for 2 h under continuous stirring. After centrifugation (10 min at
2500 ×g), the supernatant was separated from the precipitate and
stored at 4 °C; the precipitate was further extracted with 25 mL of
EtOH 80%. The supernatant was removed and combined with the first
one; the precipitate was washed twice with bidistilled water. The
precipitate (containing soy matrix) and the alcoholic supernatants
(containing isoflavones) were dried under rotavapor and the resulting
powder was solubilized in 10 mL of water. Aliquots were stored in
glass vials at −20 °C in the dark.
Pharmacological treatments. Stock solutions of estradiol (E2), ICI
182,780, genistein and daidzein were made by dissolving the
compounds in 99% v/v ethanol to a concentration of 10− 2 M in
glass vials, and stored at − 20 °C in the dark. For gavage, compounds
were generally diluted in saline and administered in a single bolus of
100 μl. Before filling the gavage syringe, solutions were carefully
vortexed to maximize homogeneity.
Chronic treatments were done every 24 h, right after each imaging
session. Genistein was administered at 1 or 5 mg/kg/day. The mixture
mimicking soymilk composition was prepared with genistein and
daidzein at the relative ratio of 3:1 (Nahas and Nahas-Neto, 2006; Di
Lorenzo, D., personal communication). To ensure that the treatment
with the two compounds reproduced the dosage found in consumed
soymilk, we prepared solutions based on the averages of soymilk
consumed in chronic experiments. Animals thus received daily 100 μl
of a solution containing 3.8 mg/ml of genistein and 12.7 mg/ml
daidzein (the final daily dose administered was 11.5 mg/kg genistein
and 3.8 mg/kg daidzein). When lyophilized soymilk was administered, due to its reduced solubility, we increased the total amount of
fluid to be administered to 900 μl (divided in three administrations of
300 μl each at 45 min distance from the previous).
Bioluminescence imaging. Bioluminescence imaging (BLI) sessions
were carried out at 3:00 PM. For bioluminescence analyses, mice
where anesthetized by subcutaneous injection of a solution of
ketamine (78 mg/kg) and xilazine (6 mg/kg); the luciferase substrate
luciferin (25 mg/kg) was administered intraperitoneally 20 min
before bioluminescence quantification: previous kinetic studies
demonstrated that this time length is sufficient to ensure distribution
of the substrate at a saturating concentration (Biserni et al., 2008).
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G. Rando et al. / Toxicology and Applied Pharmacology 237 (2009) 288–297
Bioluminescence was measured by a Night Owl imaging unit
(Berthold Technologies, Bad Wildbad, Germany), consisting of a
Peltier cooled charge-coupled device slow-scan camera equipped
with a 25 mm/f 0.95 lens. The camera was operated by the WinLight
software (Berthold Technologies). For photon emission measurements, mice were placed in a light-tight chamber. Gray-scale
images of the animals were first taken with dimmed light, and then
photon emission was registered for 5 min. Merging of the pictures
enabled to visualize the body areas where photon emission occurred
(luciferase signal was transformed in pseudo-colors: blue—low, white
—high). For quantification, photon emission was measured in selected
body areas by superimposing manually a standardized electronic grid
(showed in Fig. 1A) over the regions of interest and integrating the
signals from these areas (counts per second, cts/s). Quantifications
were done using WinLight32 imaging software (Berthold
Technologies). Normalization was performed using an external source
of photons to measure the instrumental efficiency of photon counting
(Glowell, Luxbiotech, Edinburgh, UK). To evaluate the Area under the
Curve (AUC), we used GraphPad Prism (5.01).
Cell studies. A reporter MCF-7 line was generated in the lab by
stable transfection of the construct used for the generation of the ERELuc mouse (MAR-dimerized ERE-TK-Luciferase-MAR, Ciana et al.
2001). For the analysis of phytoestrogen effects, cells were plated
into 96-well plates (Costar, Cambridge, MA, USA) at a density of
40,000 cells per well in 5% FBS in DMEM. Cells were allowed to attach
for 24 h, then the medium was removed and the cells were maintained
over-night in HBSS medium without serum. To evaluate the effects of
the estrogenic compounds, the starving medium was then replaced
with 150 μL of HBSS containing the test compounds at a different
concentration. After 6 h the medium was removed and the cells
washed with ice-cold PBS before adding 30 μL of the passive lysis
buffer (Promega). Plates were then stored at − 80 °C until assayed for
luciferase content.
Compounds were tested in triplicate with 1:10 serial dilution of:
17β-estradiol (10− 13–10− 8 M), pure soymilk, soymilk precipitate
after alcoholic extraction, alcohol-extracted isoflavones and reconstituted soymilk by addition of the precipitate with the alcoholic
extract.
The amount of isoflavone content in the soy milk used for the
experiments was 87.5 mg/L (as measured by Di Lorenzo, D., personal
communication, including conjugated forms). Thus in the dilutions
tested (1:500,000–1:500), we calculated that the isoflavone concentration was in a range between 4.8 × 10− 10 and 4.8 × 10− 7 M
(isoflavone mean molecular weight: 360 g/mol).
Luciferase enzymatic assay.
Tissues were homogenized in 200 μl of
lysis buffer (100 mM KPO4, 1 mM DTT, 4 mM EGTA, 4 mM EDTA, pH
7.8) with a 5 mm inox bead in a Tissuelyser (Qiagen), undergone one
freezing–thawing cycle, centrifuged for 30 min at 4900 ×g at 4 °C
(Rotanta 460R, Hettich Zentrifugen) and supernatants containing
luciferase were collected in ice. In samples containing luciferase,
protein concentration was measured by the Bradford assay, following
the reagent's manufacturer instructions (Pierce Biotech). Luciferase
enzymatic activity was then assayed with a commercial Luciferase
assay buffer (Promega). Light intensity was measured with a
Fig. 1. Time-course of the effects of E2 and genistein administration in ERE-Luc mice. (A) In vivo imaging of representative ERE-Luc male mice treated orally with either vehicle
(control), genistein (5 mg/kg), or E2 (50 μg/kg). Stock solutions of estradiol (E2) and genistein were made by dissolving the compounds in 99% v/v ethanol to a concentration of
10− 2 M in glass vials. For oral administration the compounds were suspended in saline at the desired concentrations and administered by gavage in a single bolus of 100 μl.
Between each gavage, solutions were carefully vortexed to maximize homogeneity. Bioluminescence imaging analyses were performed 0, 3, 6, and 17 h after treatment and
photon emission was measured in specific body areas with the aid of the electronic grid shown in the first panel (from top: thymic area, chest, abdomen, genital area, limb).
(B) Photon emission as measured in the selected body areas. Empty bars indicates the mean of photon emission measured in mice before treatments (baseline). (C) Luciferase
enzymatic activity in tissues representative of body areas selected for in vivo imaging. Bars (n = 4–6) represents mean +/− SEM; ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 as assessed by
two-way ANOVA followed by Bonferroni post hoc analysis.
G. Rando et al. / Toxicology and Applied Pharmacology 237 (2009) 288–297
luminometer (Glomax, Promega) and expressed as relative light units
over 10 s/μg protein (RLU/μg prot).
Statistical analysis. Statistical analyses (explained in figure legends)
were performed using GraphPad Prism version 5.01 for Windows,
GraphPad Software (San Diego, California, USA).
Results
Genistein induces a rapid activation of ER in liver of ERE-Luc mice after
oral administration
First, we tested the extent to which oral administration of
genistein at a nutritional dose induced ER transcriptional activity in
the ERE-Luc model. As positive control we administered E2 at a
dosage known to fully activate ERs (50 μg/kg). Thus, 4-month-old
ERE-Luc male mice were treated by gavage with either pure genistein
(5 mg/kg) or E2 and the state of transcriptional activation of ERs was
measured by bioluminescence imaging (BLI) at 3, 6 and 17 h after
treatment. Photon emission in selected body areas was measured as
described in the Materials and methods section. Oral administration
of E2 induced a significant activation of ERs in all the body areas taken
into consideration (Figs. 1A and B) with a peak of activity at 6 h
(chest: +3600%; genital area: +80%; thymic area: +120%; limb:
+50%); this was in line with previous observations by ours and other
laboratories (Ciana et al., 2003; Lemmen et al., 2004) where the
hormone was administered either intraperitoneally or subcutaneously. After the treatment with genistein, the only area where we
observed a change in photon emission was the chest, with an
increased photon emission that was gradual in time and was highest
6 h after gavage (+160%). To better quantify the effects of the
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treatments, we repeated the experiment in a second group of animals.
Mice were euthanized at 0, 3, 6 and 17 h after gavage and tissues were
dissected for luciferase enzymatic assay (Fig. 1C). Luciferase enzymatic assay proved that genistein increased ER activity in liver
(+300% 3 h and 6 h after treatment) but not in thymus, femur and
testis. In both experiments E2 administration caused a significant
increase of luciferase in all organs taken in consideration and the
effect of E2 was maximal at 6 h after treatment. We found, in
agreement with previous reports (Di Lorenzo, et al. 2008), a good
correlation between measurements made by BLI and luciferase
enzymatic analysis indicating the reliability of photon emission
measurement. Testis was an exception; possibly because of the high
conversion of local testosterone to estradiol, luciferase content in this
organ was high at time 0 and was not augmented by the treatment,
the lack of detection of luciferase present in testis by in vivo imaging
was attributed to the significant differences in the body area taken
into consideration for BLI.
This first experiment showed that orally administered genistein, at
5 mg/kg was able to activate ER activity in mouse liver and the
potency of genistein effect at this dosage was about 10% of the effect of
50 μg/kg estradiol. This was expected because it is well known that
the potency of genistein on ER is lower than E2 (Kuiper et al., 1997)
and because of the relatively high dosage of E2 used in our
experiment.
Soymilk is known to contain high concentrations of isoflavones
and genistein in particular (Nahas and Nahas-Neto, 2006), therefore
we next investigated the extent to which phytoestrogens present in
soymilk were able to modulate the ER activity. To this aim, ERE-Luc
mice were given free access to soymilk or water (controls) for 24 h;
this treatment significantly increased luciferase content in liver, as
measured by BLI (+ 260%) (Figs. 2A and B) and luciferase enzymatic
Fig. 2. Soymilk induces hepatic ER transcriptional activity in ERE-Luc male mice. For the treatment, soymilk was substituted to water and mice had full access to either soymilk or
water (controls). To prevent clotting or changes in the organolectic properties, soymilk was changed twice a day. The amount of liquid consumed was on average 18.1 g/day. (A) In
vivo imaging of whole-body ER activation in single representative mice before and after administration of soymilk as free drinking fluid. (B) Photon emission in selected body areas.
(C) Luciferase enzymatic activity in representative tissues of previously examined body areas. Data in (B) and (C) were obtained from the same experiment; bars (n = 10) represents
mean +/− SEM; ⁎p b 0.05 as assessed by two-way ANOVA followed by Bonferroni post hoc analysis (B) and by unpaired t-test (C).
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G. Rando et al. / Toxicology and Applied Pharmacology 237 (2009) 288–297
assay (+159%) (Fig. 2C). We found no significant accumulation of the
reporter in testis, thymus and femur.
We concluded that ER activity in liver may be regulated either by
oral administration of the free isoflavone genistein, or by exposure to a
food rich in isoflavones.
Effect of a long-term treatment with soymilk or genistein
Isoflavones being components of natural food, exposure to their
effects is generally prolonged in time. We therefore asked what would
have been the effects of an extended exposure to either the pure
compound genistein or soymilk. ERE-Luc animals were allowed to
drink soymilk (or water for controls) ad libitum for 20 days. During
this length of time luciferase activity was measured daily by BLI. At the
end of treatment, animals were euthanized and luciferase content was
measured in tissue extracts. Fig. 3 shows the daily recording of photon
emission in discrete body areas. In the chest of the soymilk group (Fig.
3B, continuous line) luciferase activity clearly increased at the
beginning of the treatment and remained significantly higher than
in controls for the entire duration of the experiment. The extent of
photon emission was of the same order of magnitude (800 cts/s, Fig.
3B) as the acute treatment with E2 (800–1200 cts/s, Fig. 1B). No major
changes were observed in thymus, limb, and genital area (not shown).
Interestingly, this study revealed that the treatment affected photon
emission in abdomen where the effect slightly increased over time
and was highest around day 15. The increased bioluminescence was
directly associated to estrogen receptor transcriptional activity, as
demonstrated by a second group of mice consuming soymilk that
were treated with the antiestrogen ICI 182,780 (6.6 mg/kg, s.c.) 24 h
and 6 h before the imaging session of the 5th day of treatment. ICI
administration clearly decreased photon emission in liver and abdomen to levels comparable to controls (Fig. 3C). With this experiment,
we concluded that photon emission reports faithfully on ER
transcriptional activity after soymilk consumption.
As a parameter of the transcriptional effect of soymilk consumption during the 20 days, we calculated the Area Under the Curve (AUC)
(Fig. 4A). AUC of soymilk-treated mice was significantly increased in
chest (+520%) and in abdomen (+110%), but not in other body areas.
Quantitative measurement of luciferase enzymatic activity in tissues
excised at the end of the chronic treatment (Fig. 4B), confirmed that
the prolonged soymilk consumption increased luciferase content in
liver (+300%), but not in testis, thymus and femur.
These results provide clear evidence that in selected organs such as
liver, the continuous exposure to the estrogens contained in milk
maintains high levels of ER activity: this phenomenon was unanticipated, because an uninterrupted stimulation of ER was expected to
cause receptor down-regulation (Ciana et al., 2006, Berry et al., 2008).
The effect of chronic treatment with pure genistein was then
studied using two doses 1 and 5 mg/kg/day of the compound. AUC
analysis of the chest (Fig. 5A) showed that administration of the pure
compound increased ER activity in chest in a dose-dependent manner
but not in the other body areas. However, the potency of genistein was
much lower with respect to soymilk (AUCgenistein 2800; AUCsoymilk
12,500). The low AUC value was consistent with the daily analysis of
photon emission in liver (Fig. 5C) which showed that in genisteintreated mice the extent of photon emission was low during all
treatment and had a tendency to decrease with time. Indeed, at the
end of treatment, photon emission in chest as well as liver enzymatic
activity (Fig. 5B) were very similar in treated and control mice.
Genistein treatment did not induce changes in ER activity in abdomen.
Fig. 3. Whole-body ER activity in mice consuming soymilk for 20 days. (A) In vivo imaging of a single, representative mouse, drinking either water (control) or soymilk analyzed daily
for 20 days. (B) Plot of the daily photon emission in selected body areas versus time. For each experimental group, lines represent the mean of the trend lines obtained by calculating a
mobile average of the photon emissions of single animals (each point is obtained by averaging 3 days). Data represent the average of observations carried out on 6 animals. (C) Photon
emission was measured daily in controls (open bars) and in mice consuming soymilk (filled bars). After 4 days of treatment mice were injected with the ER antagonist ICI 182,780
(6.6 mg/kg i.p.) 24 and 6 h before the imaging section. Bars represent mean +/− SEM of 5 animals.
G. Rando et al. / Toxicology and Applied Pharmacology 237 (2009) 288–297
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entire longitudinal study and luciferase enzymatic activity in tissues
obtained from animals at the end of treatment (Fig. 6C) showed that
the soymilk-mimic mixture was unable to increase ER activity;
conversely, a non-significant tendency toward decrease was observed
in most organs.
The daily measurement of food and fluid consumption and the
measure of animal weight before and after the experiment (Table 1),
failed to highlight changes in the nutritional habits of the animals.
Hence, the nutritional behavior did not influence the differential ER
activity observed upon chronic treatment with genistein, soymilk or
soymilk-mimic.
Differential ER activation by soymilk and soymilk-mimic isoflavone
solution
In the chronic experiment, the soymilk-mimic was administered
once a day while the soymilk was consumed all throughout the day,
indeed when we measured soymilk consumption we found that 75%
of the daily consumption occurred at night-time, however about 25%
was through the day. Thus to directly compare the effects of the
treatments, we measured ER activity of ERE-Luc mice treated by
gavage with 0.8 g lyophilized soymilk reconstituted in water or with
the equivalent amount of genistein and daidzein (38 μg genistein and
12.7 μg daidzein). In a time-course study, a significant increase of
photon emission was observed in chest (+350%) and abdomen
(+200%) 6 h after the treatment with lyophilized soymilk; once more,
the soymilk-mimic solution did not have significant effects on ER
activity (Fig. 7A). Consistent with BLI, measurement of luciferase
enzymatic activity in liver showed a + 150% increase in soymilktreated mice and just a minor tendency to increase in mice treated
with the soymilk-mimic solution (data not shown). In conclusion, this
experiment, where soymilk and isoflavone solutions were administered at the same dosages and at the same time, showed that in
soymilk elements other than phytoestrogens were necessary to fully
induce ER transcriptional activities.
Synergistic ER activation by isoflavones and soymilk matrix in
MCF-7 cells
Fig. 4. Soymilk maintains hepatic ER activation during and after 20-day treatment.
(A) The global AUC was obtained by the integer of photon emission over time in
selected body areas. (B) Luciferase enzymatic activity in tissues dissected on the 20th
day of treatment. Bars (n = 6) represents mean +/− SEM; ⁎p b 0.05, ⁎⁎⁎p b 0.001 as
assessed by unpaired t-test.
In this organ fluctuations in photon emission were observed in both
controls and treated mice: the origin of such changes associated to the
prolonged treatment with gavage are unknown.
This last experiment indicated that, in chronic exposure, soymilk
was significantly more potent than genistein on modulating ER
activity. This differential effect could be due to: i.) the fact that
genistein was administered once a day and photon emission was
measured 24 h after treatment or ii.) the presence in soymilk of
factors, other than genistein, contributing to activate ERs.
These two questions were addressed by administering to ERE-Luc
mice once a day, a solution mimicking the composition of phytoestrogens measured in soymilk. The two main characterized isoflavones
present in soymilk are genistein and daidzein (Nahas and Nahas-Neto,
2006). Other compounds endowed of estrogen-like activity like
gliceitin might be present at lower concentrations but have not been
fully characterized. We therefore prepared a mixture of genistein–
daidzein containing the amounts of the two compounds ingested daily
by animals consuming soymilk (genistein 11.5 mg/kg/day; daidzein
3.8 mg/kg/day), as measured in our previous experiments and this
mixture was administered once a day for 21 days. Fig. 6A shows the
daily photon emission measured in chest, thymic area, limb, and
abdomen. The soymilk-mimic solution failed to affect ER activity in all
the body areas examined, including liver. The AUC (Fig. 6B) of the
To finally test that in soymilk factors other than phytoestrogens are
involved in ER transcriptional activation, we carried out an alcoholic
extraction of soymilk (as described in the Materials and methods
section) and compared the effect of alcohol-extracted compounds
with the hydrophilic compounds present in milk in MCF-7 cells stably
transfected with an ERE-LUC reporter.
Fig. 8 shows that 6 h after the treatment, alcohol-extracted
compounds (mainly isoflavones) and soymilk matrix had a mild
estrogenic activity (+30% and + 26%, respectively), but when tested
together had a dose-dependent, synergistic effect in inducing
luciferase expression (+200%). The combined alcohol-extracted and
hydrophilic compounds of soymilk showed a potency comparable to
17β-estradiol. In conclusion, this study, clearly points to a major role of
the soymilk matrix on ER transcriptional activity.
Discussion
Several studies have demonstrated that isoflavones largely present
as natural constituents in the human diet (particularly genistein and
daidzein), may have estrogenic activities in cells or whole organisms
even at the low concentrations found in food. The present study
supports and significantly expands previous findings by showing the
complexity and variety of isoflavone effects on ER transcriptional
activity when the compound is taken as a pure substance or in a more
complex mixture (e.g. in soymilk). Indeed the present study shows
major differences in response to pure genistein alone or in combination with daidzein or as component of soymilk. Both in short- and in
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G. Rando et al. / Toxicology and Applied Pharmacology 237 (2009) 288–297
Fig. 5. Long-term administration of genistein results in reduced sensitivity of ERs to the administration of the compound. (A) AUC analysis in selected body areas over a 20-day length
of time. (B) Luciferase enzymatic activity after 20-day treatment fails to find any significant difference between groups. (C) Longitudinal monitoring of daily photon emission with
trend line analysis is helpful to explain the differences observed comparing data in A and B.
Fig. 6. Soymilk-mimic fails to induce ER transcriptional activity in liver and abdomen after long-term administration. (A) The mean trend line analysis obtained in mice treated for
20 days with soymilk-mimic was compared with the trend line obtained in animals treated with soymilk. (B) AUC analysis in selected body areas over a 20-day length of time.
(C) Luciferase enzymatic activity in tissues dissected after 20 days of treatment. Bars (n = 4–7) represents mean +/− SEM; ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 as assessed by one-way
ANOVA plus Bonferroni post hoc. For easy reference the soymilk dataset reported is the same of Figs. 3B and 4.
G. Rando et al. / Toxicology and Applied Pharmacology 237 (2009) 288–297
295
Table 1
Animal weight before and after chronic exposure to different treatments.
Animal treatment
Control
Soymilk
Genistein (1 mg/kg)
Genistein (5 mg/kg)
Soymilk mimic (Gen:Dad 11.5: 3.8 mg/kg)
a
Mouse weight (grams)a
day 0
day 20
Δ%
33 ± 1.1
34 ± 1.6
32 ± 1.0
35 ± 1.6
32 ± 1.5
32 ± 1.3
32 ± 1.3
33 ± 0.9
34 ± 1.6
33 ± 1.3
− 4%
− 5%
+ 2%
− 5%
+ 4%
Mean +/− SEM; n = 10 mice/experimental group.
long-term treatments, soymilk was more potent than genistein alone
particularly in activating hepatic ER. The major finding of this study is
that in soymilk, factors other than phytoestrogens (the food matrix)
contribute to the overall estrogenicity of soymilk.
At the present time we can only speculate on the mechanisms
responsible for the findings here reported: i.) differential receptor
regulation: administration of free isoflavones is likely to cause a
sudden increase of their plasmatic concentrations, with concomitant
induction of a peak of ER activity that, repeated for some times, leads
to desensitization of the receptor to the ligand or to its decreased
transcriptional capacity. Conversely, the continuous consumption of
low concentration of phytoestrogens with soymilk may cause a
permanent status of ER activity without causing ER down-regulation.
Preliminary western analysis did not show a clear decrease in ER
protein in liver after long-term treatment with genistein (data not
shown), however it is conceivable that, in spite of the presence of ERs,
the activity of the receptor could be blunted at the level of its
interactions with co-regulators. ii.) Differential bioavailability: concomitant administration of genistein and daidzein failed to increase
ER activity in both acute and chronic studies. A potential explanation
could be that daidzein or its metabolites modulate liver catabolic
enzymes leading to a higher catabolism/excretion of the compounds
administered (Atherton et al., 2006; Chen et al., 2005; Ronis et al.,
2006). It is conceivable that the presence of the soy matrix, by limiting
the kinetics of absorption, distribution, metabolism and excretion of
isoflavones, could optimize/synchronize their permanence in blood or
in other tissues with the availability of ER to be transcriptionally
activated. iii.) The presence in soymilk of estrogenic compounds other
than isoflavones: recent evidence suggests that soy proteins may have
a direct effect on human liver participating in the changes in LDL
receptor activity (reviewed in Sirtori et al., 2009). Intriguingly, hepatic
LDL receptor is still widely accepted as an estrogen-target gene (Parini
et al., 1997). Hence, we may speculate that dietary proteins regulate
estrogen receptor activity, similarly to what was shown for other
nuclear factors with other macro-nutrients such as fatty acids (Jansen
et al., 2004) or glucose (Mitro et al., 2007).
Fig. 8. Comparative analysis of the isoflavones extracted from soymilk, and soymilk
matrix in MCF-7 cells stably transfected with a luciferase reporter. Cells were
exposed to increasing concentrations of isoflavones (1:10 serial dilution in the range
4.8 × 10−10–4.8 × 10−7 M), soy matrix, isoflavones + soy matrix, and 17β-estradiol
(10−13–10−8M). Bars represent mean ± SEM of the luciferase enzymatic activity
measured 6 h after the addition of the estrogenic compounds.
The long-term administration of soymilk seems to suggest that this
regimen may affect also other ER-positive organs such as thymus and
bone: the tendency (not significative) toward increased luciferase
activity at the end of the treatment in these two organs seems to point
to a delayed effect which may become statistically significant at times
longer than those here investigated. Differential effects observed with
soymilk administered for short (Figs. 2 and 7) or prolonged (Fig. 3)
lengths of time need to be underlined. The results presented show
that treatment with this common food generates a state of activation
of ERs which is much more widespread than the effect observed
during acute response. In view of the increasing usage of soymilk in
diet, it is important to better establish the long-term effects of this
food. Interestingly, soymilk administered to MCF-7 cells at different
concentrations resulted in a diverse state of activation of the ERs,
further demonstrating that the state of ER activity is directly
proportional to the amount of given soymilk.
Within the ERE-Luc, the luciferase is under concurrent transcriptional control of both ERalpha and ERbeta. Hence, in tissues expressing
both receptors, the photon emission recorded is the resultant of both
activities which may be in opposition with each other (i.e., yin-yang
conjecture by Lindberg et al., 2003). Interestingly, genistein has a
higher affinity for ERβ than ERα (Kuiper et al., 1997). However, the
present study, by showing a strong effect of genistein in liver, an organ
mainly expressing ERα, demonstrates that this phytoestrogen in vivo
may act on both receptor subtypes.
Fig. 7. Comparative analysis of activity of lyophilized soymilk and soymilk-mimic on ER transcriptional activity. Photon emission in mice treated with lyophilized soymilk or the
solution of genistein and daidzein mimicking soymilk. Bars represent mean +/− SEM of 4–7 mice; ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 as assessed by two-way ANOVA followed by
Bonferroni post hoc analysis.
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G. Rando et al. / Toxicology and Applied Pharmacology 237 (2009) 288–297
The present study shows the power of reporter mouse technology:
the introduction of a surrogate marker (ERE-driven luciferase expression) offers the possibility to obtain: i.) global view of the tissues
affected by the compound of interest for the recognition of the organs
potentially affected; ii.) unequivocal assessment of dosage and timing
necessary to elicit the activity of the receptor; iii.) longitudinal studies
in a single individual during repeated exposure, unravels the sites of
compound accumulation and activity, or the dynamics of the response
of target to the treatment. The data presented here further
demonstrate the applicability of the ERE-Luc model to the study of
the in vivo effects of endogenous, dietary and environmental estrogens
completing a series of previously published reports (Ciana et al.,
2005a, 2005b; Di Lorenzo et al., 2002; Mussi et al., 2005; Penza et al.,
2007, Di Lorenzo et al. 2008). Considering that most tissues in
mammals express ERs and that each estrogen has unpredictable
tissue-specific actions on ERs, the ERE-Luc mouse model represents, to
the best of our knowledge, the most appropriate model to obtain a
realistic, systemic view of the transcriptional effects of endogenous or
exogenous estrogens.
In conclusion, the present study provides further ground to the
growing skepticism concerning the true potential of phytoestrogens
as pure compounds to beneficially affect cardiovascular and skeletal
systems (Sirtori et al., 2005), by demonstrating the limited activity
that these compounds have in activating the ER, particularly when
administered for prolonged lengths of time. In addition, the study
demonstrates the strong estrogenic action of diets based on soymilk,
thus underscoring the potential risk of the use of such diets in infants.
Finally, the study underlines the necessity to generate appropriate
model systems in which the dietary effects of these compounds may
be systematically analyzed.
Conflict of interest statement
Adriana Maggi and Paolo Ciana are the founders of TOP S.r.l., a spin-off of the University
of Milan owning the ERE-Luc model.
Acknowledgments
This work was supported by the European Union (Network of
Excellence CASCADE FOOD-CT-2004-506319, STREPs EXERA LSHB2006-037168 and EWA, LSHM-CT-2005-518245; IP CRESCENDO LSHCT-2005-018652).
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