* Department of Pharmacology and Toxicology, 101C Volker Hall, 1670 University Boulevard, Birmingham, Alabama 35294;
UAB Comprehensive Cancer Center, University of Alabama at Birmingham, 1670 University Boulevard, Birmingham, Alabama 35294; and
Department of Pathology, University of Alabama at Birmingham, 1670 University Boulevard, Birmingham, Alabama 35294
Received August 22, 2001; accepted October 23, 2001
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ABSTRACT |
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Key Words: daidzein; phytoestrogen; bioavailability; toxicology.
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INTRODUCTION |
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Daidzein, like genistein, has been described as a weak phytoestrogen. In B6D2F1 mice, the relative estrogenic potency of daidzein to genistein as measured by uterine weights is 1:4 (Farmakalidis et al., 1985). Daidzein has 1000 times lower affinity for the estrogen receptor (Shutt and Cox, 1972
), and in the Ishikawa cell test, the estrogenic activity is 7700 times less than that of estradiol (Adlercreutz, 1998
). Equol, a metabolite of daidzein, is 1700 times less estrogenic than estradiol in the Ishikawa cell test, but in the estrogen receptor binding assay, the affinity is only 250 times lower than that of estradiol. Recently, researchers cloned a novel estrogen receptor, estrogen receptor ß, from the rat (Kuiper et al., 1996
), mouse (Tremblay et al., 1997
), and human (Mosselman et al., 1996
). The estrogen receptors can form homodimers and heterodimers (Cowley et al., 1997
; Kuiper and Gustafsson, 1997
; Ogawa et al., 1998
; Pace, 1997). Both of these receptors bind daidzein and genistein. Whereas the relative binding affinities of estradiol 17-ß are similar for estrogen receptors
and ß, for daidzein the relative binding affinities are 0.2 and 1% of estradiol, respectively. Hence daidzein, like genistein, preferably binds to the estrogen receptor ß over the estrogen receptor
. Unlike genistein, daidzein does not inhibit protein tyrosine kinase activity (Huang et al., 1999
). Whereas high concentrations of daidzein (> 100 µM) inhibit MCF-7 cells, lower doses (1 µM) stimulate cell growth (Hsu et al., 1999
).
The primary concern of phytoestrogens as dietary supplements stems from the reports that estrogens are associated with perinatal toxicity to the reproductive tract. During the fetal and early postnatal period, when reproductive organs are developing, changes in the hormonal milieu can induce dramatic structural and functional alterations in the reproductive tract of both males and females (Newbold and McLachlan, 1985). Changes in concentrations of estrogens (and androgens) during development alter gene expression and differentiation. Many of these changes are permanent, and some appear later in life. The relation between the potent synthetic estrogen diethylstilbestrol and predisposition of the infant to vaginal adenocarcinoma and reproductive dysfunction later in life is well established (Herbst and Scully, 1970
; McLachlan, et al., 1980
). For the phytoestrogen genistein, it has been shown that injections of neonatal female rats with high doses of genistein (500 µg or 1000 µg/day on days 110 after birth) decreased basal LH levels and pituitary responsiveness to gonadotrophin-releasing hormone (GnRH) and increased the volume of the sexually dimorphic nucleus of the hypothalamus (Faber and Hughes, 1991
). Interestingly, lower doses of genistein (10100 µg) had opposite effects on LH secretion and increased pituitary response to GnRH. More recent developmental toxicology studies have demonstrated that dietary exposure to physiological concentrations of genistein yield little or no toxicity (Flynn et al., 2000a
,b
). Although there is no clear-cut epidemiological evidence to demonstrate that soy or its components have an adverse effect on fertility in humans, this does not rule out potential effects that may go undetected and could impact on other parameters of reproductive health.
The purpose of this work was to investigate the potential of dietary daidzein to cause toxicity to the female reproductive tract and for chemoprevention to the mammary gland, and to study its distribution in rats.
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MATERIALS AND METHODS |
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For the toxicology and bioavailability studies, virgin female rats were fed powdered AIN-76A diet (Harlan Teklad, Madison, WI) supplemented with 0, 250, or 1000 mg daidzein/kg diet, starting 2 weeks prior to breeding (10 females/group). Daidzein was chemically synthesized (Roche, Basel, Switzerland) and analyzed by HPLC (98.5% pure, 1.5% methanol). The AIN-76A diet was chosen because it was determined to be free of any phytoestrogens. At 9 weeks of age the females were bred (2 females/male) for 2 weeks. The males were fed standard laboratory diet (Harlan Teklad 16 Rodent Diet, Madison, WI) until breeding, at which time they were placed on the same diet as the females. Offspring were sexed at birth and litters reduced so that each dam had 10 offspring (46 females/dam). At day 21 postpartum, offspring were weaned and fed the same diets for the remainder of the experiments.
For the chemoprevention study, two groups of 10 litters were fed either 0 or 250 mg daidzein/kg diet from birth until weaning at 21 days postpartum. Thereafter, all female offspring were fed control diet (AIN-76A) only. At 50 days postpartum, female offspring were gavaged with 40 mg dimethylbenz(a)anthracene (DMBA, Sigma Chemical Co., St. Louis, MO) in sesame oil per kilogram body weight. Animals were palpated twice a week starting 40 days after DMBA treatment to record the location, size, and date of detection for all tumors. Animals were sacrificed when the tumor diameter reached 2.5 cm, when the animals became moribund, or when they reached 230 days of age. At the time of necropsy, all tumors were removed and weighed. A 1- to 2-mm section was taken from each tumor 1 cm in diameter and was preserved in 4% paraformaldehyde. The section was dehydrated and blocked in paraffin. The paraffin block was cut into 5-µm sections, fixed on slides and stained using hematoxylin and eosin.
For histomorphological analysis of the ovaries, uterus, and vagina, the entire reproductive tract was dissected out and preserved in 4% paraformaldehyde. Cross-sections were then cut and placed vertically in tissue blocks and processed as described above.
Isoflavone analysis.
Serum concentrations of daidzein and its metabolites were analyzed by HPLC-multiple reaction ion monitoring mass spectrometry (MS) (Coward et al., 1993). Tissue samples were digested overnight with 0.1 mg proteinase K/ml (Sigma Chemical Co.) in 10 mM Tris buffer, pH 8.0, containing 100 mM NaCl, 25 mM EDTA, and 0.5% SDS. The SDS was removed by centrifugation after the addition of saturated KCl. Proteinase K was removed by passing the samples over Sep-pak C18 cartridges (Waters, Milford, MA). After a water wash, the isoflavones were eluted with methanol. To determine total isoflavone concentrations (free daidzein and metabolites), samples were incubated with ß glucuronidase/sulfatase (type H-1 from Helix pomatia; Sigma Chemical Co.) after adjusting the pH to 5.0 with ammonium acetate buffer. Daidzein was recovered by diethyl ether extraction. Serum and milk samples were extracted with hexane to remove the lipids, followed by incubation with the ß glucuronidase/sulfatase enzymes and extraction with diethyl ether. For the analysis of free compounds, enzymatic hydrolysis was omitted. Extracted samples were evaporated under nitrogen and redissolved in 80% aqueous methanol prior to analysis by HPLC-MS. Samples were spiked with biochanin A, 4-ethylumbelliferone, and phenolphthalein glucuronide (Sigma Chemical Co.) as internal standards. A daidzein standard curve was run each day.
Extracted samples were analyzed under isocratic conditions (30% acetonitrile in 10 mM ammonium acetate) on a Hewlett Packard 1050 HPLC using a 10 cm x 4.6 mm i.d. Aquapore C8 reversed phase column. Samples were introduced into the Pe-Sciex API III triple quadrupole mass spectrometer via the Heated Nebulizer Atmospheric Pressure Chemical Ionization interface (HN-APCI) in the negative mode. Multiple reaction monitoring was carried out by selection of parent molecular ions and specific daughter ions formed by collision with argon-10% nitrogen gas. Daidzein and metabolite concentrations were quantified by comparison of peak areas with standard curves. The limit of detection was 5 nM.
Milk collection.
Stomach milk from 7-day-old rats was collected as previously described (Fritz et al., 1998). The stomach, including part of the esophagus and duodenum, was removed and frozen in liquid nitrogen. The stomach lining was removed from the frozen milk and discarded. The remaining milk pellet was then analyzed for phytoestrogen concentrations.
Mammary gland analysis.
Whole mounts of the fourth abdominal mammary glands were prepared as previously described (Murrill et al., 1996; Russo and Russo, 1978
). Mammary glands were removed at the time of sacrifice and spread on a microscope slide. The slides were then placed in neutral buffered formalin for 8 h, followed by acetone overnight to remove fat. The glands were placed in 70% alcohol for 30 min, then rehydrated in water for 15 min. The glands were stained using alum carmine for 8 h. After staining, the slides were dehydrated in an increasing gradient of alcohol concentrations from 35 to 100%. The slides were placed in xylene and then compressed between two glass slides for 24 h. The glands were allowed to expand for 8 h and were then mounted using glass cover slips and Permount (Fisher Scientific, Atlanta, GA) for preservation.
Glands were analyzed for the quantity of terminal end buds, terminal ducts, lobules type I, and lobules type II as previously described (Murrill et al., 1996; Russo and Russo, 1978
). Terminal end buds were characterized as elongated ductal structures that contained 36 epithelial cell layers and were greater than 100 µm in diameter. Terminal ducts had 13 epithelial cell layers and were less than 100 µm in diameter. Lobules type I were identified as having 510 alveolar buds; lobules type II had 1020 alveolar buds.
Statistics.
Data were analyzed by one-way analysis of variance (ANOVA) using the Sigma Stat computer program (Jandel Scientific, San Rafael, CA).
Other analysis.
Serum estrogen and progesterone concentrations were determined (Azziz et al., 1995) using commercially available kits (Pantex, Santa Monica, CA). Histomorphological evaluations of coded slides of the female reproductive tract and mammary tumors were carried out by Dr. Isam-Eldin Eltoum, a board-certified pathologist.
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RESULTS |
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In the mammary chemoprevention study with daidzein, we measured daidzein and metabolites from mammary glands of neonatal rats nursed by dams fed the 250 mg daidzein/kg diet to demonstrate that daidzein could be transported to this organ (Table 9). Interestingly, mostly daidzein (407 nM) was detected in the mammary glands of 7-day-old offspring exposed to 250 mg daidzein/kg diet, and 98% of the daidzein was in the aglycone form. Only low nanomolar concentration of equol was detected in the mammary gland.
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DISCUSSION |
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These dietary formulations of daidzein resulted in very high daidzein concentrations in the blood of these rats compared to those reported in humans. Adlercreutz et al. (1993) have reported that Japanese persons eating a traditional diet high in soy had an average circulating daidzein concentration of 107 nM. In our studies with young adult female rats receiving 250 mg and 1000 mg daidzein/kg diet, serum daidzein concentrations were 630 nM and 1.4 µM daidzein, respectively. Hence, we are dealing with supraphysiological concentrations of this isoflavone in rats, i.e., 6- and 13-fold higher daidzein concentrations.
Developmental Toxicology
To determine if daidzein would adversely affect fertility in females, we initiated dietary daidzein exposure 2 weeks prior to the beginning of mating. To minimize the potential of the studs being affected, they were maintained on standard laboratory rat chow until they were mated with the females. One day after birth, we determined the number and weight of male and female offspring. There was no significant changes in percent of litters, number of male and female offspring, and respective anogenital distances from exposure to daidzein in the diet.
The 1000 mg daidzein/kg, but not the 250 mg/kg, diet resulted in a significant decrease in body weights at 2 days postpartum and continuing through 50 days. Ovarian and uterine weights and mammary gland size were not significantly altered in 50-day-old female offspring. When organ to body-weight ratios were calculated, these differences were not statistically significant, indicating that the changes in organ weights occurred as a consequence of changes in body weights, and not because of direct daidzein toxicity. In addition, there were decreases in circulating estrogen and progesterone concentrations, especially in animals eating the high daidzein-containing diet. This is consistent with the report by Lu et al. (2001), whereby daily consumption of a soy-containing diet in women reduced daily circulating levels of estradiol and progesterone by 20 and 33%, respectively.
Measurement of feed intake revealed that rats fed the high daidzein-containing diet ate less (16.5%) than controls, accounting for the decrease in body weight (18.5%) and possibly for the other biomarkers. The sex-differentiated end points, anogenital distances and number of male and female offspring, were not significantly altered, suggesting that perinatal exposure to daidzein via the diet does not cause developmental alterations. Histomorphological evaluations did not reveal any alterations to vaginal, uterine, ovarian, and mammary tissues.
Mammary Cancer Chemoprevention?
We have reported that injections of a pharmacologic dose (500 mg/kg body weight) of genistein during the prepubertal period conferred long-lasting protection against DMBA-induced mammary cancer (Lamartiniere et al., 2000; Murrill et al., 1996
). We and others (Fritz et al., 1998
; Hilakivi-Clarke et al., 1999
; Lamartiniere et al., 2000
) subsequently demonstrated that dietary genistein administered during the prepubertal period could also suppress DMBA-induced mammary tumor development. This protective effect of genistein is consistent with the epidemiological data showing that Asians eating a diet high in soy have reduced incidence of mammary cancer (Lee et al., 1991
; Wu et al., 1996
). Furthermore, it has been reported that there is a 6-fold gradient in breast cancer risk by migration history between recent Asian migrants to the United States and American-born Asian Americans with at least three grandparents also born in the West (Ziegler et al., 1993
). More recently, using the Shanghai Cancer Registry, a case-control study (Shu et al., 2001
) reported an inverse relationship (50%) between adolescent (1315 years) soy food intake and breast cancer incidence later in life. The authors ruled out adult soy as making a contribution.
Armed with this epidemiological and laboratory data, we chose to investigate if daidzein, the other major soy phytoestrogen, would contribute to protecting against chemically induced mammary cancer in one of the same protocols used for genistein (Lamartiniere et al., 2000). We chose dietary prepubertal exposure only because this protocol used the least amount of daidzein, a compound not readily available in large quantities. Prepubertal administration of daidzein did not suppress mammary tumor development in rats. This is unlike genistein (Fritz et al., 1998
), pregnancy (Grubbs et al., 1983
), human chorionic gonadotrophin (Russo et al., 1990
), and estrogen and progesterone (Grubbs et al., 1985
), which have been shown to protect against chemically induced mammary cancer by enhancing mammary gland differentiation. As demonstrated in Table 3
, daidzein does not enhance mammary gland differentiation. However, this data only demonstrates that daidzein does not confer a chemopreventive effect in a manner like genistein, i.e., enhancing mammary gland differentiation during the prepubertal period. It is possible that daidzein may possess other mechanisms by which it may protect against mammary cancer, for example, a direct effect in adulthood on sex steroid receptor signaling and/or signal transduction pathways. Particularly interesting is the potential of isoflavones to differentially modulate estrogen receptor coregulators (An et al., 2001
; Wong et al., 2001
), and the ability of daidzein to modulate the estrogen receptor ß as a potential tumor suppressor gene (Horvath et al., 2001
). Investigations into daidzein mechanism of action are currently underway.
Bioavailability
For a dietary component to have a biological impact, it must be bioavailable. Using HPLC-MS, we have measured the concentrations of daidzein and metabolites in the blood, milk, and mammary glands of rats exposed to daidzein via the diet. Increasing concentrations of daidzein in the diet resulted in a dose response in blood concentrations. A 4-fold increase of daidzein in the diet resulted in 3- to 4-fold total daidzein and metabolite concentrations in the blood of pregnant females and the offspring. In the blood of all animals fed daidzein in the diet, the primary metabolite was equol. In rats fed the high and low daidzein-containing diets, equol concentrations were highest in the blood of adult female offspring (Table 8: 7.2 µM and 3.8 µM), followed by 21-day-old females (Table 8
: 6.5 µM and 1.2 µM), pregnant females (Table 5
: 4.5 µM and 529 nM), and 7-day-old neonates (Table 8
: 1.0 µM and 163 mM). The least amount of circulating equol was detected in 21-day-old postconception fetuses (Table 6
: 358 nM and 59 mM from litters exposed to the high- and low-dose daidzein diets, respectively). Most of the equol was in the conjugated form as opposed to the free (aglycone) form. Equol was increasingly conjugated with age, i.e., from 89% in 7-day neonates to 99% in 50-day-old female offspring, and 99% conjugated in pregnant rats. This is consistent with the ontogeny of the UDP-glucuronosyltransferases. UDP-glucuronosyltransferases increase postnatally (Lucier et al., 1979
), and this is evident for the conjugation of daidzein and its primary metabolite, equol, culminating in conjugation of 99% of the equol in the adult female. In the neonatal females, only 14% of the equol is conjugated, but at that age only 28 nM and 107 nM equol were measured in offspring of dams eating the low and high daidzein-containing diets, respectively. Although equol is the most estrogenic of the metabolites, it is approximately 1/1700 as potent an estrogen as estradiol 17-ß (Adlercreutz, 1998
). It is noteworthy that virtually all of 7 µM concentration of equol from the 1000 mg daidzein/kg AIN-76A diet was conjugated. This demonstrates the high capacity of the UDP-glucuronoslytransferase for equol. Isoflavones have been reported to increase UDP-glucuronosyltransferase (Sun et al., 1998
), hence daidzein could be regulating its own metabolism and excretion.
While we did not assay for free (aglycone) equol in fetal blood, the total equol concentration was relatively low compared to the other age groups (1/20 to 1/65 of adult females). In pregnant rats, 99% of the circulating equol was conjugated. In previous work with genistein, only 7% of the genistein in fetal blood was in the aglycone form (Fritz et al., 1998).
On the other hand, circulating daidzein concentrations were higher in prepubertal (3.3 µM and 1.4 µM), and 7-day-old rats (3.8 µM and 982 nM) than in young adult females (1.4 µM and 630 nM), and pregnant females (407 nM and 303 nM) exposed to the high and low daidzein-containing diets. Except for pregnant females, where the conjugation rate was 9299%, conjugation of daidzein occurred less readily than did equol. The conjugation of daidzein was 8185% in adult female offspring, 7073% in 21-day-old offspring, and 3437% in 7-day-old neonates.
The highest concentration of daidzein (7.3 µM), all of which was unconjugated, was found in the milk of the 7-day-old nursing offspring. This was 5-fold higher than that found in the blood of adult female rats eating a diet high in daidzein. Likewise, there were high concentrations of equol in stomach milk of neonates. Forty-four to 94% of the equol was in the aglycone form. This implies that milk daidzein and equol are either transferred as unconjugated compounds from the dam to the neonates, or that the conjugated forms are readily hydrolyzed to free daidzein and equol in the offspring's stomach.
Daidzein and equol concentrations in neonatal stomach milk are not in equilibrium with blood concentrations. Circulating daidzein and equol concentrations were 2- to 3-fold lower than stomach milk concentrations. Furthermore, bioavailability of daidzein, but not equol, to the mammary gland is evident by the 407-nM daidzein concentration found in mammary tissue of 7-day-old female offspring fed the 250 mg daidzein/kg diet. Ninety-eight percent was unconjugated daidzein, suggesting that the rat mammary gland does not conjugate this phytoestrogen. Similar results were previously noted with dietary genistein and analysis of neonatal mammary glands. In fact, similar concentrations of genistein (440 nM) were measured in animals exposed to 250 mg genistein/kg diet (Fritz et al., 1998).
Comparison of blood genistein concentrations from our previously published data (Fritz et al., 1998; Lamartiniere et al., 2000
) to blood daidzein concentrations in animals of the same age treated with similar concentrations of these two soy phytoestrogens provided insight into bioavailability. Although circulating genistein and daidzein concentrations were similar in an age-specific manner in lactating, neonatal, and prepubertal rats exposed to 250 mg genistein or daidzein/kg diet, total phytoestrogen concentrations in the blood were higher from daidzein in the diet because of one of its metabolites, equol. The combined daidzein and equol concentrations were approximately 2-fold higher than genistein blood levels. It is also noteworthy to point out that the half-life of daidzein and genistein in men and women fed soy milk has been reported to be 15.7 and 8.9 h, respectively (Zhang et al., 1999
). In studies of women receiving soy milk, and rats receiving soy extract, daidzein was found to be more bioavailable and to have a longer half-life than genistein (King et al., 1998; Xu et al., 1994
, 1995
). Hence, our daidzein and genistein measurements are consistent with these reports.
Phytoestrogens circulate in soy formula-fed infants at concentrations that are 13,00022,000 times higher than plasma estrogen concentrations, which range from 40 to 80 pg/ml in early life (Setchell et al., 1997). In fact, mean plasma concentrations of daidzein and genistein in seven infants fed soy-based formulas have been reported to be 295 ng/ml (1.18 µM) and 443 ng/ml (1.64 µM), respectively (Setchell et al., 1997
). This is an order of magnitude higher than typical plasma concentrations of adults consuming soy foods. Yet, there are few reports of toxicity to infants and adults fed soy-based diets. Hence, our present dietary daidzein study and previous dietary genistein animal studies by our laboratory (Fritz et al., 1998
) and Flynn et al. (2000a,b) put a perspective on the potential of these isoflavones to exert toxicity. As shown in this report, very high dietary daidzein concentrations (1000 mg/kg) and genistein concentrations (1250 mg/kg; Flynn et al. 2000a
) in the diet resulted in reduced feed intake, but 250 mg daidzein or genistein/kg diets do not alter fertility, body weights, sexually dimorphic and nursing behavior, or developmental maturation, and do not cause toxicity to the male and female reproductive tracts (Fritz et al., 1998
, in press; Flynn et al., 2000a
,b
).
This is in contrast to the report that prenatal injections of genistein to rats resulted in increased incidence of DMBA-induced mammary cancer in the offspring (Hilakivi-Clarke et al., 1999). However, there are no epidemiological reports to support this data. In fact, as pointed out earlier, Asians eating a diet high in soy have reduced incidence of breast cancer (Lee et al., 1991
; Wu et al., 1996
; Ziegler, et al., 1993
). We believe that the explanation lies in the animal model and experimental protocol that delivers the genistein. In those experiments, bolus injections of genistein were administered, resulting in significant concentration of the genistein delivered as the aglycone as opposed to being conjugated. In a recent publication (Cotroneo and Lamartiniere, 2001
), it was pointed out that injections of genistein resulted in 4448% of the circulating genistein being unconjugated, even 1618 h after being injected. In contrast, dietary genistein at 250 mg genistein/kg diet is 8896% conjugated (Fritz et al
., in press). In fact, dietary genistein given only during gestation to rats did not alter the incidence and multiplicity of DMBA-induced mammary tumors (Lamartiniere et al., 2000
). Likewise, in human plasma, under steady-state conditions, daidzein and genistein aglycone forms account for a relatively constant 3 and 2%, respectively, of the total isoflavones in plasma (Setchell et al., 2001
). Hence, mode of nutrient administration can determine bioavailability in a laboratory experiment and is extremely important in evaluating the potential for health effect.
It has also been reported that uterine adenocarcinomas developed in mice injected neonatally with genistein (Newbold et al., 2001). However, Lian et al. have shown that genistein and daidzein had an inhibitory effect on N-methylnitrosoureainduced/estrogen-promoted endometrial carcinogenesis in mice (Lian et al., 2001
). Consistent with the second publication is the epidemiological report that Asians have a lower incidence of uterine cancer (Mant and Vessey, 1994
).
To summarize, we fed 250 mg and 1000 mg daidzein/kg diet to rats, starting prior to conception and continuing until the offspring were 50 days old. These daidzein-containing diets resulted in micromolar concentrations of daidzein and equol in pregnant female rats and postnatal female offspring, but lower concentrations in fetal blood (nanomolar). Conjugation of isoflavones is demonstrated and may play a significant role in disposition and bioavailability. Micromolar concentrations of daidzein and equol were measured in the stomach milk of neonates. In the neonatal mammary gland exposed to 250 mg daidzein/kg diet, daidzein concentrations were 407 nM, 98% of that being unconjugated. The 1000-mg daidzein/kg diet resulted in significantly reduced body weights, a fact that can be explained on the basis of reduced feed consumption. Circulating progesterone, but not estrogen, levels were statistically reduced with the high daidzein-containing diet only. Otherwise, neither daidzein dose had a significant effect on the weights and histomorphology of the female reproductive tract. Prepubertal exposure to 250 mg daidzein/kg diet did not alter the ontogeny of chemically induced mammary cancer in female offspring. We conclude that supraphysiologic concentrations of daidzein administered via the diet did not cause significant toxicity to the female reproductive tract or provide a protective effect against chemically induced mammary cancer in rats.
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ACKNOWLEDGMENTS |
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NOTES |
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