Research Results: Early Developmental Outcomes
Grantee Research Project Results
Anti-Androgenic Pesticides: Impact on Male Reproduction
Rao Veeramachaneni, Animal Reproduction and Biotechnology Laboratory - Colorado State University, Fort Collins, CO
EPA STAR Grant #R826131
- This research resulted in the development of an animal model (rabbit) for evaluating effects of anti-androgen exposures that is more relevant to human development than previous rodent models.
- Experiments documented and characterized the adverse developmental effects of gestational exposure to DDT, vinclozolin, and dibutyl phthalate (DBP) in male offspring, including impaired spermatogenesis, reduced testis and accessory gland weights, and decreased mating ability. Altered hormonal responses and cryptorchidism were also observed.
Developmental Effects of Dietary Soy Phytoestrogens
Claude L. Hughes, Duke University Medical Center
EPA STAR Grant #R825721
- Developmental exposure of rats to the potent reference estrogen diethylstilbestrol (DES) at low doses and/or the common phytoestrogen genistein at levels comparable to the upper range of human exposure affected some markers of sexual development in both male and female pup.
- Similar exposure of mice to DES or the phytoestrogens genistein or daidzein was found to alter many reproductive outcomes in both males (e.g., testicular weight) and females(e.g., estrogen cycling). Timing of exposure also was an important modifier of the observed effects.
- Neither In utero nor lactational exposure to genistein nor daidzein affected mammary tumor latency or the number of tumors in mice predisposed to development of such tumors.
- In the Los Angeles areas, approximately one in three fetuses are exposed to xenobiotic EACs (e.g., PCBs, the pesticide hexachlorobenzene) and about half of all fetuses are exposed to the phytoestrogens daidzein, genistein, formononetin, biochanin-A, and coumestrol in utero. Amniotic fluid sampling my serve as a useful biomarker of exposure to these agents.
- The results in rodents in conjunction with the detection of EACs in amniotic fluid sustain concerns about potential adverse health effects of developmental exposuresto EACs for the humanfetus/neonate.
Developmental Exposure to Endocrine Disruptors: Fertility and Gene Expression Profiles
Timothy Zacharewski, Michigan State University
EPA STAR Grant #R827402
- Prenatal and lactational exposure to diethylstilbestrol (DES) causes long-term adverse effects on testicular development and sperm function in mice, and these effects are associated with changes in testicular gene expression, even long after the cessation of DES exposure.
- Developmental exposure of male mice to genistein at dietary levels equal to or exceeding that of human populations consuming a soy-rich diet does not result in adverse effects on sperm quality or changes in testicular gene expression similar to DES.
- In utero and lactational exposure to genistein at levels as high, or higher, than human populations consuming a soy-rich diet does not affect the morphology of the mammary gland in pubertal female mice.
- Data demonstrate the potential for developmental exposure to DES, and possibly other estrogenic chemicals, to irreversibly alter testicular growth, sperm function, and testicular gene expression, including genes involved in steroidogenesis, lysosomal function, and testicular development.
- These findings suggest that estrogenic endocrine disruptors may act through multiple mechanisms of action, and estrogenic potency alone may not be an accurate predictor of adverse reproductive effects.
Effects of an Endocrine Disruptor on Prostate Development and Growth
Barry G. Timms - University of South Dakota
EPA STAR Grant # R827403
- Three-dimensional morphometric reconstruction has been successfully used to evaluate developmental patterns of the urogenital complex and prostate of fetal and adult mice under the influence of environmental estrogens.
- The low dose of methoxychlor (MXC) increased the size of the dorsal prostate region and coagulating gland with a similar increase in the tissue volume of the ventral prostate.
- At the high doses, mice treated with MXC or diethylstilbestrol (DES) had a reduced number of prostatic ductal tips, which did not differ between the age groups.
- Morphological changes in the prostate were more apparent in mice at two months compared to one month.
- Anatomical effects on the ducts and the urogenital sinus (UGS) were more readily observed in the 3-D reconstructions compared to measurements of the number of prostatic outgrowths.
- Estrogenic endocrine disruption in fetal mice results in a permanent change in growth parameters that are specific to certain regions of the UGS and associated structures.
Effects of Early Exposure to Xenoestrogens on the Prostate Gland
Gail S. Prins - University of Illinois at Chicago
EPA STAR Grant # R826299
- Neonatal exposure to low doses of nonylphenol (NP) may advance puberty in male rats, and appeared to increase the expression of liver enzymes known to be under androgenic control in SD rats, but these results could not be duplicated.
- Dose-response study of estradiol benzoate (EB) showed that neonatal exposure to estrogens results in an inverted U-shaped dose-response curve, with low doses transiently increasing prostate weights at day 35 and high doses decreasing weights compared to controls. At 90 days, increased organ weights are seen at lower doses than at 35 days.
- Hepatic 2α- and 16 α-testosterone hydroxylase activities were significantly increased in low-dosed EB animals, suggesting that increased prostate sizes in the low-dose groups were due to advancement of puberty.
- Fisher 344 rats appear to be more sensitive to estrogen treatment than the traditionally used Sprague-Dawley strain, suggesting that the former strain may be the better model for testing environmental endocrine disruption.
Endocrine Disruption in Adolescence
Mari S. Golub, University of California - Davis, Davis CA
EPA STAR Grant #R827404
- Prepubertal exposure to methoxychlor (MXC) and diethylstilbestrol (DES) adversely affected reproductive development in rhesus monkeys, with effects including growth retardation, premature emergence of secondary sex traits and increased incidence of ovarian cysts/masses.
- DES exposure resulted in broad-ranging effects on a number of hematological and immunological parameters. MXC exposure during development affected fewer immunohistological parameters than DES.
- Exposure to MXC during development resulted in more severe behavioral deficits than exposure to DES, even though DES is the more potent estrogen. Differential effects of the two agents at the estrogen receptor subtypes (ER" and ER$) may be relevant to the differential behavioral outcomes.
- The effectiveness of using rhesus monkeys has been demonstrated as a model for potential endocrine disrupting effects in humans.
- Disruption in hematological and immunological systems during puberty could alter adolescent risk for anemia and infectious disease and subsequent adult risk for diseases such as osteoporosis, heart disease, and autoimmune disease.
Endocrine Disruptors and Testis Development
Michael K. Skinner - Center for Reproductive Biology, Washington State University, Pullman, WA
EPA STAR Grant #R827405
- An embryonic testis organ culture system was developed to assess the effects of endocrine disruptors on organ morphogenesis and tissue function. Methods also were developed to assess transforming growth factor (TGF) and neurotropin (NT) gene expression and gene product levels.
- Endocrine disruptors (e.g., methoxychlor [MXC] and vinclozolin) were found to alter early embryonic testis development when maternal exposures occurred at the time of fetal sex determination and testis morpho-genesis.
- In vivo exposure of gestating rats to MXC demonstrated a heritable reduction in sperm function and increased spermatogenic cell apoptosis affecting males in generations F1 through F4. Studies show that this heritable effect may be associated with alteration of the DNA methylation patterns in male germ cell precursors.
Examination of the Estrogen Response Pathways in a New Vertebrate Model
Elwood Linney - Duke University Medical Center, NC
EPA STAR Grant #R825297
- Transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos.
- Technologies were developed to help in the isolation, characterization, and study of a zebrafish estrogen receptor beta (ER-β).
- Researchers developed a YFP reporter that minimized autofluorescence while providing a bright reporter gene product
- Computerized time-lapse fluorescent images of developing zebrafish covering several days of growth demonstrated the progression of developmental gene expression. Analysis of the images allowed the researchers to visualize gene in relations to specific embryonic events, which could be done with mammalian embryonic models.
- Developmental expression studies found indicates maternal loading with ER-β, followed by degradation and transcription between 24 and 48 hours postfertilization, coinciding with aromatase expression.
- Sequence similarity of other species indicate that ER-β has been highly conserved during evolution and is likely used during later embryogenesis in zebrafish.