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2004 Progress Report: Determinants of Fetal Male Rat Germ Cell Vulnerability to Phthalate Esters
EPA Grant Number: R830766Title: Determinants of Fetal Male Rat Germ Cell Vulnerability to Phthalate Esters
Investigators: Gaido, Kevin
Institution: The Hamner Institutes
EPA Project Officer: Deener, Kacee
Project Period: February 24, 2003 through February 23, 2006 (Extended to March 31, 2007)
Project Period Covered by this Report: February 24, 2004 through February 23, 2005
Project Amount: $725,736
RFA: Children's Vulnerability to Toxic Substances in the Environment (2002)
Research Category: Children's Health , Health Effects
Description:
Objective:The objective of this research project is to identify key molecular and cellular events associated with germ cell development in the rat testis that are targets for endocrine active chemicals following in utero exposure.
Progress Summary:Exposure of male rats to some phthalate esters during development results in altered expression of genes that may be involved in germ cell proliferation and survival. Our studies revealed that exposure to 500 mg/kg/day di(n-butyl)phthalate (DBP) from gestation day (gd) 12 to 20 significantly increased the number of multinucleated germ cells (gonocytes) in the fetal rat testis. In humans, multinucleated gonocytes (MNG) are believed to be precursors of testicular germ cell cancer, which is the most common cancer in young men. Our immunostaining with markers for proliferation and apoptosis indicated that MNG in the fetal rat testes exposed to DBP were neither proliferating nor undergoing apoptosis on gd 17-21. On gd 21, many gonocytes in DBP-exposed testes were joined by intercellular bridges, which were not observed in control testes. These bridges were detected by only electron microscopy and could not be seen by light microscopy without specific markers. In the normal rats testis, intercellular bridges have been observed occasionally on gd 17 (but not gd 21) and are believed to result from incomplete cytokinesis during gonocyte mitosis, which normally occurs in the rat on gd 16. Gonocyte multinuclearity may result from cytoplasmic confluence of gonocytes joined by intercellular bridges following their enlargement or from plasma membrane fusion. We hypothesize that DBP interferes with division of gonocytes, resulting in persistence of these bridges several days after mitosis on gd16. Persistent bridges and lack of support from Sertoli cells because of altered cell-cell contact between Sertoli cells and gonocytes in DBP-exposed fetal testes may cause an increase in the number of MNG following exposure to DBP.
The effect of DBP exposure on fetal development of gonocytes was most apparent on gd 21. Using morphology of MNG as an endpoint, we obtained a dose-response relationship between the number of MNG per section and several dose levels of DBP including highest estimated human exposure level (0.1 mg/kg/day). Statistical significance of DBP treatment on occurrence of MNG was achieved at the 100 mg/kg/day dose level. At this dose level, the dose-response curve exhibited an effect of threshold. Although MNG develop in the normal fetal rat testis, exposure to DBP in utero increased the number of these genetically altered cells in a dose-dependent manner. Counts of MNG were based on morphological criteria, which is a less sensitive indicator of DBP response as compared to stereological characteristics of the fetal testis, such as the testis volume and total cell number. For these parameters, significance of DBP treatment was reached at 50 and 30 mg/kg/day dose levels, respectively. Persistent bridges in DBP-exposed gonocyes suggest that incomplete cytokinesis contributes to the development of MNG. We will identify molecular markers of incomplete cytokinesis in gonocytes in the rat and use these markers to refine our understanding of the relationship between dose levels of DBP and the number of affected gonocytes. Intercellular bridges between developing male germ cells have been reported in the human fetal testis. Given the high level of conservation of cellular processes underlying testicular development in humans and rats, markers of incomplete cytokinesis will aid in the assessment of MNG occurrence in the developing human testis.
On postnatal days (pnd) 3-5 MNG in DBP-exposed testes became mitotically active but seemed unable to complete mitosis as indicated by abnormal mitotic figures. To gain insights in molecular events regulating response of developing germ cells to DBP, we are establishing a Sertoli-germ cell co-culture using pnd 5 rat testes. To better preserve Sertoli cell structure, which is crucial for germ cell development, and increase cell viability, we are using a mixture of proteins comprising the basal membrane (Matrigel) as a co-culture overlay. After establishing the doubling time and characterizing the Sertoli-germ cell ratio by immunostaining with markers specific for each cell type, co-cultures will be treated to mono-n-butyl phthalate (active metabolite of DBP) and assessed for proliferation and apoptosis. These co-cultures will also be treated with testosterone and stem cell factor to establish the role of these factors on germ cell survival.
Future Activities:The studies for the subsequent reporting period will focus on identification of key molecular pathways that are disrupted by DBP and lead to the development of multinucleated gonocytes. Effects of DBP on mitosis and apoptosis on gd 16-17 will be assessed at low doses of in utero DBP and di(2-ethylhexyl)phthalate exposure. Fate of the genetically abnormal multinucleated gonocytes during postnatal testicular development in the rat will be determined. Sertoli-germ cell co-cultures will be used for establishing whether mono-n-butyl phthalate alters proliferation and apoptosis of these cells. These co-cultures will also be used to assess the role of testosterone and stem cell factor on survival of mono-n-butyl phthalate-exposed germ cells.
Journal Articles on this Report: 1 Displayed | Download in RIS Format
| Other project views: | All 25 publications | 3 publications in selected types | All 2 journal articles |
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Howroyd P, Hoyle-Thaker R, Lyght O, Williams D, Kleymenova E. Morphology of the fetal rat testis preserved in different fixatives. Toxicologic Pathology 2005;33(2):300-304. |
R830766 (2004) R830766 (2005) R830766 (Final) |
not available |
risk assessment, health effects, vulnerability, animal, mammalian, organism, age, chemicals, toxics, biology, pathology, measurement methods, plastics,
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POLLUTANTS/TOXICS, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Endocrine Disruptors - Environmental Exposure & Risk, Biology, Risk Assessments, Health Risk Assessment, Physical Processes, endocrine disruptors, Chemicals, Biochemistry, Environmental Microbiology, Environmental Chemistry, Endocrine Disruptors - Human Health, altered gene expression, fetal exposure, animal models, endocrine disrupting chemicals, molecular mechanisms, EDCs, developmental biology, human health risk, fetal development, in utero exposure, fetal genocyte degeneration, reproductive processes, altered sexual development, phthalates, exposure, gestational exposure, germ cell vulnerability, exposure studies
Progress and Final Reports:
2003 Progress Report
Original Abstract
2005 Progress Report
Final Report