2000 Progress Report: Developmental Effects of Dietary Soy PhytoestrogensEPA Grant Number: R825721
Title: Developmental Effects of Dietary Soy Phytoestrogens
Investigators: Hughes, Claude L. , Davis, Vickie , Tyrey, Lee
Institution: Duke University
Current Institution: Duke University Medical Center
EPA Project Officer: Carleton, James N
Project Period: November 1, 1996 through October 30, 1999
Project Period Covered by this Report: November 1, 1999 through October 30, 2000
Project Amount: $574,747
RFA: Endocrine Disruptors (1996) RFA Text | Recipients Lists
Research Category: Endocrine Disruptors , Health , Safer Chemicals
Objective:This project is determining whether intake of dietary soy phytoestrogens by rodents from mid-gestation throughout lactation to weaning will affect the progeny's sexually dimorphic development of various hormone-dependent target tissues.
There is widespread concern that developmental exposure to endocrine active chemicals (EACs) may adversely affect reproductive, neurobehavioral, and immunological development or to predispose those exposed towards various cancers later in life. If these concerns about developmental exposures to EACs are provable, then there must be evidence of effects in animal models of EACs at properly scaled levels of exposure during sensitive time frames, and demonstration that exposure of the human fetus to comparable quantities of these same EACs occurs during biologically comparable sensitive time frames. Our recent studies have continued to show effects that can be produced by EACs in a rodent model and a new approach to quantitating in utero exposure of the human fetus to these types of chemicals.
First Study. This study focuses on the effects of diethylstilbestrol (DES) at marginally effective doses and genistein (GEN) at levels comparable to the upper range of potential dietary intake on some markers of sexual development in Long-Evans hooded rats. Dams, sires, and pups were maintained on a purified casein based diet throughout the study. Dams were randomly assigned to each of the five treatment (daily doses) groups: Control (Corn oil); Low DES (0.5 mg/kg body weight [BW]); High DES (5.0 mg/kg BW); GEN (15 mg/kg BW ); and GEN + DES (GEN at 15 mg/kg BW and DES at 0.5 mg/kg BW). Mating generated 43 control pups, 47 pups exposed to low DES, 29 pups exposed to high DES, 43 pups exposed to GEN, and 43 pups exposed to the combination of GEN + DES. Dams were gavaged from Gestation Day (GD) 14 through weaning on Post-Natal Day (PND) 21 with either corn oil alone (unexposed controls) or the estrogen agent in corn oil, with the exception that DES was not administered GD 20 through birth. On PND 1, litter size, sex, birth anogenital distance (bAGD), and birth body weight (bBW) were recorded, and individual pups were tattooed for identification. At weaning (PND 21), weaning anogenital distance (wAGD) and weaning body weight (wBW) were documented. The age of puberty was determined by inspecting the offspring for the day of vaginal opening or preputial separation. Upon vaginal opening, vaginal cytology was assessed by daily saline lavage to monitor initial cyclicity. The reproductive organs of male pups age 50-70 days were removed and weighed. At birth, no treatments affected duration of gestation, litter size, or birth anogenital distance/birth body weights (bAGD/bBW) of pups of either sex. The ratios of bAGD/cube root of bBW were calculated and with the data expressed in this fashion, no effects of treatments were found. At weaning, the ratio of weaning AGD to weaning body weight (wAGD/wBW) differed significantly between the control group and each of the estrogenic treatments in both sexes with larger wAGD/wBW values associated with each of the estrogenic treatments. Expression of the data in the form of the ratio of wAGD/cube root of wBW did not change the finding of effects in any of the treatment groups in either sex, except for the one group exposed to the high dose of DES, in which case the values for both sexes no longer differed from respective controls. Observations at puberty showed that males exposed to High DES and GEN alone exhibited earlier onset of puberty (about 3 days earlier than the controls). The onset of puberty was unaffected by estrogen treatment in all groups of females except low DES, which showed an earlier onset of puberty by approximately 1? days. Observations in the post-pubertal interval showed that the ratios of male reproductive organ weights/body weight (testes, epididymis, seminal vesicle, and prostate) were unaffected by estrogen treatment in all groups except high DES which increased testicle weight and decreased epididymis, seminal vesicle, and prostate weights. Length of the initial vaginal cycles during the first 3 weeks after vaginal opening was 4 days for all groups except for the females exposed to high DES. This group did show cycles, but there were more cycles with additional days of cornifed smears yielding a mean cycle length of 6 days.
This experiment shows that the reference estrogen DES at low doses and the common dietary phytoestrogen GEN at levels comparable to the upper range of human exposure affect some but not all of the markers of sexual development that we assessed in Long-Evans hooded rats.
Second Study. The second study was performed to test our hypothesis that man-made chemicals and phytoestrogens can be quantified in human amniotic fluid during the second trimester. Two groups of amniotic fluid samples obtained at routine amniocentesis were assayed by gas chromatographic/mass spectrometric (GC/MS) analysis. The first group of amniotic fluid samples (n=53) from women (n=51) with a mean (? SEM) age of 36.5 ? 0.5 years and between 15 and 23 weeks of gestation were analyzed for common polychlorinated biphenyl (PCB) congeners, the DDT metabolites p,p'-DDE, and o,p'-DDE as well as the pesticides hexachlorobenzene (HCB) and the three isomers of hexachlorocyclohexane ( -, -, and -HCH). The limit of quantitation (LOQ) for PCBs was 0.01 ng/ml; for the other organochlorines contaminants the LOQ was 0.1 ng/ml. The contaminants -HCH with a mean (? SD) concentration of 0.15 ? 0.06 (ng/ml) and p,p'-DDE with a mean (? SD) concentration of 0.21 ? 0.18 ng/ml were detected in the amniotic fluid. PCB-specific congeners were detected with a much lower frequency and levels were in the range of the LOQ. Overall, one in three amniotic fluid samples tested positive for at least one environmental contaminant. The second group of amniotic fluid samples (n=62) from women (n=56) with a mean (? SD) age of 36.7 ? 5.2 years and between 15 and 23 weeks of gestation were analyzed for the common phytoestrogens daidzein, genistein, formononetin, biochanin-A, and coumestrol. The LOQ for each of these phytoestrogens was 0.50 ng/mL. The mean (? SD) concentrations of daidzein and genistein in amniotic fluid were 1.14 ? 1.04 and 1.37 ? 1.00 ng/ml with maximum levels of 5.52 and 4.86 ng/ml, respectively. Overall, 26 (46.4%) and 34 (60.7%) of the women had quantifiable levels of daidzein and genistein, respectively, in their amniotic fluid. There were no differences in the ethnic background, age, or gestational age of women who had detectable levels of phytoestrogens compared to those who did not.
Conclusions. In this area of Los Angeles, approximately one in three fetuses are exposed to xenobiotic endocrine active environmental chemicals and more than one in two fetuses are exposed to phytoestrogens in utero. Our approach of using amniotic fluid to quantify the presence of hormonally active exogenous factors provides a viable and proximate (to time of reproductive tract differentiation) estimate of fetal exposure to hormonally active compounds. Although the consequences of these exposures for the human fetus remain unknown at this time, our demonstrations: (1) of effects of these EACs in an animal model at levels of exposure that mimic those found in humans in North America during sensitive time frames; and (2) that exposures of the human fetus to pertinent quantities of these same EACs do occur during biologically comparable sensitive time frames, sustain concerns about potential adverse health effects of developmental exposures to EACs for the human fetus/neonate. We conclude that:
- In addition to endogenous steroids, the human fetus is exposed to mixtures of naturally occurring and xenobiotic EACs.
- Exposures to mixtures of EACs occur at times that are known to be critical developmental intervals for the human fetus.
- Although human fetal effects have not yet been assessed, the measured concentrations and receptor-binding affinities of the EACs relative to those of endogenous sex steroids suggest that target tissue effects are likely.
- Developmental exposure of rats to some EACs at levels of exposure that mimic those found in humans in North America produces target tissue effects that indicate changes in cell function are elicited.
The major emphasis of our ongoing work is now focused on developmental effects of dietary soy phytoestrogens in target tissues in mice. This study is primarily under the supervision of Dr. Vicki Davis, who has particular expertise in the use of transgenic mice in assessing the actions of hormonal agents. Reproductive effects of estrogens on development have been studied extensively in mouse models, especially with DES. In addition, many transgenic mouse models have been developed that allow us to investigate the mechanisms involved in the induced effects. For example, in this study we will use the MMTV-neu transgenic mouse to induce mammary cancer that is relevant to human breast cancer. In addition, extensive data are available on estrogen effects on the mammary gland of the mouse that is a focus of this study. Plus, future studies will be designed to use KO models for the estrogen receptor. Therefore, the mouse model allows us to pursue lines of research that would not be available with other models.
Soy isoflavones are chemicals in soy that can act similar to the female hormone, estrogen. Isoflavones in soy appear to have beneficial effects on the health of menopausal women including reduction in heart disease and breast cancer. As the trend toward increased use of soy in the U.S. diet continues, younger women who may be pregnant or lactating will consume isoflavones in their diets. Development of the human fetus and newborn can be altered in adverse ways by exposure to other estrogens, such as DES. Because isoflavones also have estrogenic properties, it is important to determine whether these compounds can have similar developmental effects. In the proposed experiments, we will investigate the effects of feeding pregnant and lactating female mice the two prominent soy isoflavones, genistein and daidzein, on the fetal and neonatal development of their offspring. The mothers will ingest the chemicals either late in pregnancy, during lactation, or both to expose the pups. We will examine the effects on reproductive markers, such as anogenital distance (an indicator of maleness or femaleness) and onset of puberty, as well as effects on mammary gland development. In addition, we will examine whether exposure to these compounds has beneficial (as seen in adult females) or adverse effects on mammary tumor development using a transgenic mouse model that spontaneously develops tumors.
Female FVB/N mice will be rotated into the males' cages and checked for seminal plugs to determine if mating has occurred. When plugs are found, these females will be group housed to monitor for pregnancy (up to five/cage and watched for fighting) and fed a semi- purified diet prepared by Harlan-Teklad. Pregnant females will be individually housed. The pregnant and/or lactating mothers will be treated as follows: dams will be treated daily by gavage (1) from gestational day 14 (gd14) until birth (gd19), (2) from gd14 until weaning (d20), or (3) birth until weaning with 40 or 4 mg/kg/day of genistein, 40 mg/kg/day daidzein, 0.2 µg/day DES, or control (corn oil). The dams that receive treatments starting on day of delivery will begin the gavage procedures (without treatment) around d14 of pregnancy to allow adjustment to the procedure and prevent the possible event of the mother not taking care of the pups due to the stress associated with initiating gavage treatments. All treatments will occur in the dams and not in the pups. All pups will be weaned on day 19-21 and multiple housed by sex. The pups will be ear punched for identification before weaning. After weaning, the dams will be euthanized by CO2 inhalation.
The birth weight and anogenital distance of the pups will be determined at birth. Body weight and anogenital distance will be measured again at weaning. Because we need to compare the mice at birth and weaning, the newborn pups will be marked with tatoo ink intradermally in the tail and/or ankle/wrist region for identification. The pups will receive ear punches for further identification before or at weaning. Body weight will be measured again at the onset of puberty. Onset of puberty (vaginal opening in females, preputial separation in males) also will be assessed. Cycling will be checked daily in female pups from the time of vaginal opening until necropsy using vaginal smears. The pups will be euthanized by CO2 inhalation at weaning or post-puberty (around 2 months of age).
One group of dams will be mated with transgenic male mice that spontaneously develop mammary cancer (MMTV-neu). Treatments are as indicated above to provide either in utero (daily gavage during gd14-gd18) or lactational exposure (birth-wean) for the genistein, daidzein, and control. The pups will be weaned and assessed as indicated above. However, the females will be allowed to age and monitored for mammary tumor onset by weekly palpations beginning at 4-5 months of age until necropsy. After tumors are found, the size will be measured weekly using a caliper to assess growth. The mice will be euthanized at 12-14 months of age unless the tumors reach 2 cm in size or when mice bearing tumors show signs of distress (i.e., difficulty breathing, problems with mobility, hunched posture, scruffy fur). Male pups will be euthanized by CO2 inhalation at weaning.