Research Grants/Fellowships/SBIR

1998 Progress Report: Developmental Effects of Dietary Soy Phytoestrogens

EPA Grant Number: R825721
Title: Developmental Effects of Dietary Soy Phytoestrogens
Investigators: Hughes, Claude L. , Lewis, Carole , Tyrey, Lee
Current Investigators: Hughes, Claude L. , Davis, Vickie , Tyrey, Lee
Institution: Duke University
Current Institution: Duke University Medical Center
EPA Project Officer: Moore, James C.
Project Period: November 1, 1996 through October 30, 1999
Project Period Covered by this Report: November 1, 1997 through October 30, 1998
Project Amount: $574,747
RFA: Endocrine Disruptors (1996) RFA Text |  Recipients Lists
Research Category: Endocrine Disruptors , Health , Safer Chemicals



Naturally occurring compounds may disrupt or mimic hormone action. For example, dietary intake of soy phytoestrogens has been suggested to provide cardioprotection and aid in breast cancer prevention in post-menopausal women. However, an increase of soy consumption in the North American diet has resulted in increased exposure to dietary estrogen mimics by reproductive-aged women, raising concern regarding possible effects on the fetus and neonate.

Exposure to exogenous sex steroid agonists, antagonists, or modulators of endogenous sex steroid hormone metabolism is known to alter normal sexual dimorphic development and lead to defeminization/ masculinization of females and demasculinization/ feminization of males. Previous studies have shown adverse effects on sexual dimorphic development in rat pups treated parenterally with the phytoestrogens genistein and coumestrol during the prenatal and neonatal intervals. Our preliminary study is focused on the effects of genistein, at levels comparable to the upper range of human exposure on fetal and neonatal development. The effects of genistein (GEN), relative to the reference estrogen diethylstilbestrol (DES), were examined using the developmental markers anogenital distance (AGD), age at onset of puberty and initial estrous cyclicity in Long-Evans hooded rats.

We hypothesize that gestational and neonatal exposure to levels of dietary soy phytoestrogens in the upper range of human intake will alter the normal sexually dimorphic development of the brain and neuroendocrine control of reproduction in the rodent model. Our specifics aims are to determine whether intake of dietary soy phytoestrogens by dams from mid-gestation throughout lactation to weaning will affect progeny by altering normal sexually dimorphic development of the central nervous system, as reflected by changes in:

  1. brain histomorphometry and histochemistry,
  2. neuroendocrine maturation at puberty,
  3. ovarian versus neuroendocrine aging over life span.

Progress Summary:

Fifteen adult dams and four adult sires of the Long-Evans hooded strain were purchased from Harlan-Sprague Dawley (Indianapolis, IN) and maintained under a 12L:12D light schedule with controlled temperature and humidity. Food and water were available ad libitum, with dams and sires started on a purified casein based diet (AIN 93G modified by replacing soybean oil with corn oil; Harlan Teklad, Madison, WI) for three or more weeks prior to mating. Dams, sires and pups were maintained on this diet throughout the study. Four dams were assigned to each of the four treatment and were gavaged from gestation day (GD) 14 through weaning on post-natal day (PND) 21 with either corn oil alone (unexposed controls), GEN (15 mg/kg BW), DES (0.1 µg), or the combination (GEN at 15 mg/kg BW and 0.1 µg DES) in corn oil, with the exception that DES was not administered GD 20 through birth. The day of delivery was designated Day 1 of life (PND 1).

The developmental effect of genistein (GEN), and its interaction with the reference estrogen diethylstilbestrol (DES), was initially examined using the sexual development markers anogenital distance (AGD), age of puberty, and initial cyclicity in Long-Evans hooded rats. On PND 1, litter size, sex, birth anogenital distance (bAGD), and birth body weights (bBW) were recorded and individual pups were tattooed for within litter identification. On PND 21, weaning anogenital distance (wAGD) and weaning body weight (wBW) were documented and pups received ear notches for permanent identification. AGDs were measured in duplicate with Vernier calipers. The age of puberty was determined by inspecting the offspring for the day of vaginal opening (female) or preputial separation (male). Upon vaginal opening, vaginal cytology was assessed daily to monitor initial cyclicity.

Exposure of the pregnant dam to the soy phytoestrogen genistein, alone or in combination with DES, resulted in decreased bBW in both the male and female progeny (p<0.05). DES alone did not cause any effect on bBW. Sex-specific bAGDs did not vary among groups, however, when bAGD was indexed to bBW (bAGD/bBW), effect was evident only in the female pups, showing that GEN increased the bAGD/bBW in comparison to DES and DES + GEN, but showed no effect when compared to control animals. Male pups showed no differences in bAGD/bBW. At weaning (PND 21), there were no differences among sex-specific body weights (wBW). In both male and female pups, the weaning anogenital distance (wAGD) of all estrogen-treated groups was larger than that of the control. Additionally, wAGDs of DES + GEN was larger than DES exposed pups and GEN exposed female pups. GEN exposed males had a larger wAGD than those exposed to DES. When wAGD was indexed to wBW, in both male and female pups the wAGD/wBW of all estrogen-treated groups was larger than those of the control group; and, female pups exposed to the combination DES + GEN had a larger wAGD/wBW than those to DES alone. Male pups within all estrogen-treated groups had an earlier onset of puberty than the control animals. Females exposed to DES, either alone or in combination with GEN, exhibited an earlier onset of puberty than control and GEN. Exposure of the female pups to DES + GEN resulted in a delayed onset of puberty relative to DES. Initial cyclicity appears to be unaffected in all estrogen-treated groups.

Use of AGD or AGD adjusted for body weight, as an endpoint of sexually-dimorphic development has allowed us to distinguish between effects due to in utero and lactational exposure. In this experiment, pups exposed in utero to DES, GEN, or the combination GEN + DES showed no effect on bAGD or bAGD/bBW, whereas in utero + lactational exposure increased wAGD and wAGD/wBW in both sexes in all estrogen-exposed groups compared to control. The sites and mechanisms by which this effect is caused are presently unknown.

Future Activities:

Awaiting histomorphological and histochemical analysis of brains and reproductive organs collected at 2 and 5 months of age. Will collect final subset's organs at 9 months of age. Conduct tests to determine affects of gestational versus lactational exposure and possible dose dependent effects.

Journal Articles:

No journal articles submitted with this report: View all 2 publications for this project

Supplemental Keywords:

Administration, Oral; Animal; Animal Feed; Birth Weight/Drug Effects; Carcinogen; DES/Pharmacology; Dose-Response Relationship/Drug; Drug Effects/Growth and Development; Estrogens, Non-Steroidal; Health Effects; Isoflavones/Administration and Dosage/Metabolism/Toxicity; Rats, Long-Evans; Sex Differentiation; Sex Maturation., RFA, Health, Scientific Discipline, Environmental Chemistry, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Susceptibility/Sensitive Population/Genetic Susceptibility, Children's Health, genetic susceptability, Biology, Endocrine Disruptors - Human Health, adverse outcomes, puberty, sensitive populations, central nervous system, adolescence, childhood development, epidemiology, health risks, females, exposure, exposure studies, gender, children, brain chemistry, animal models, developmental processes, phytochemicals, growth and development, reproductive processes, environmentally caused disease, biological effects, dietary exposure, dietary soy phytoestrogens, pregnancy, female, rodent, toxics, developmental toxicants, environmental hazard exposures

Relevant Websites:

Progress and Final Reports:
Original Abstract
2000 Progress Report
Final Report