2005 Progress Report: Estrogen Elicited Gene Expression Network Elucidation in the Rat UterusEPA Grant Number: R831847
Title: Estrogen Elicited Gene Expression Network Elucidation in the Rat Uterus
Investigators: Zacharewski, Timothy , Chan, Christina , Gennings, Chris , Harkema, Jack
Current Investigators: Zacharewski, Timothy , Chan, Christina , Harkema, Jack
Institution: Michigan State University
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2004 through August 31, 2007
Project Period Covered by this Report: September 1, 2004 through August 31, 2005
Project Amount: $747,960
RFA: Computational Toxicology and Endocrine Disruptors: Use of Systems Biology in Hazard Identification and Risk Assessment (2004) RFA Text | Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health Effects , Computational Toxicology , Health , Safer Chemicals
The objectives of this research project are to: (1) establish estrogenic endocrine disruptor (EED)-elicited dose- and time-dependant changes in rat uterine gene expression; (2) investigate the role of the estrogen receptor (ER) in mediating changes in gene expression; (3) phenotypically anchor changes in gene expression to histopathological outcomes; and (4) develop a model that describes the EED-elicited uterine gene expression network.
In-life time course and dose response studies for ethynyl estradiol (EE) in immature ovariectomized Sprague Dawley rats have been completed. EE elicited the characteristic uterotrophic effects (up to 7-fold induction in uterine wet weight) in a dose responsive manner, allowing for selection of a dose for the temporal studies that would result in optimal physiological response. The 100 µg/kg dose of EE was thus selected and implemented in a comprehensive time course study (2, 4, 8, 12, 18, 24, and 72, 84, 96, 120, 144, 168 hours posttreatment with one or three daily doses, respectively) wherein a broad spectrum of physiological, histopathological, and molecular endpoints were captured for measurement. This time course design captured the induction and subsequent regression in uterotrophy that occurred at 72 hours. Temporal changes in uterine wet and blotted weights (5- and 7-fold, respectively, peaking at 72 hours) were used to assess gross physiological endpoints including water imbibition, proliferation, and regression. Histopathological samples were processed, mounted, stained (H&E), and evaluated by a veterinary pathologist (Co-Investigator, Dr. Jack Harkema). Subsequent findings of timed stromal edema (Figure 1A), immune cell accumulation, basal lamina thickening, luminal epithelial hypertrophy, proliferation, and apoptosis were used to characterize the morphological changes preceding and overlapping with the uterotrophic response at 72 hours. Immunohistochemical staining for BrdU nuclear incorporation into luminal epithelial cells (Figure 1B) at each time point allowed for morphological quantification of this marker of DNA synthesis and cell cycle progression. Maximal induction in BrdU staining (15-fold) was seen at 24 hours indicating a synchronous progression of these cells through the cell cycle approximately 24 hours after treatment with EE. Luminal epithelial cell height also was significantly increased 1.6- and 2.7-fold at 24 and 72 hours, respectively (Figure 1C).
Figure 1. Rat Uterine Tissue (A) H&E Stained, (B) Immunohistochemically Stained for BrdU, and (C) Showing Luminal Epithelial Cell Height
Custom in-house rat cDNA microarrays were used to characterize the temporal changes in gene expression involved in uterotrophic induction after a single dose of EE as well as monitor the changes at and subsequent to the uterotrophic time point (72 hours) after three doses. Differentially expressed genes were analyzed and functionally annotated using a variety of computational and literature search-based methods. The resulting functional categories represented by the EE expression profiles included transcription factors, water and ion transporters, angiogenic factors, pro- and antiapoptotic genes, redox regulators, xenobiotic metabolism enzymes, heat shock proteins, protein synthesis genes and proteasome family members, DNA replication and cell cycle control genes, and cellular energetics control genes. The expression profiles of several (20) representative genes covering multiple categories of response were verified by quantitative real-time PCR with an average temporal correlation coefficient of 0.83 between the microarray and real-time expression data indicating excellent concordance between the two methods. The temporal regulation of these families of gene responses mapped well to the time points preceding or overlapping with the histopathological and physiological endpoints that were observed in the uterus. Phenotypic linkages between gene responses and cellular or structural endpoints have been established, providing a robust baseline data set for comparison to subsequent studies of estrogenic compounds.
In-life dose response studies of o,p’-DDT have been completed (Figure2) and time course studies are underway to evaluate the estrogenicity of DDTin the rat uterus. The dose response study revealed DDT to behave in amanner less potent and with less efficacy than EE in inducing wet weight; however,induction in blotted weights are more comparable. The difference in inductionin water content suggests a differential effect in water or ion transport. Severalgenes involved in related processes were regulated by EE including Vegf,aquaporins, solute carrier family proteins, and sodium channel proteins. Subsequentexamination of DDT expression profiles will provide evidence to determine ifthese mechanisms are involved in the EE but not DDT induction in water contentamong other endpoints.
Figure 2. Uterotrophy Dose Response Curves
These results demonstrate that EE elicits effects on uterine size, water content, cytoarchitecture, and proliferation. Preliminary investigations indicate organ-, cell-, and gene-level changes are perturbed by EE treatment. The results are consistent with global gene expression results, although more refined studies are need to further elucidate the disrupted pathways, and to determine if these affects are relevant to humans.
No significant change in plans is being proposed for Year 2. Additional treatment and time points will be examined in Sprague Dawley rats. Bioinformatic mapping of putative estrogen response elements to the gene expression changes in addition to probing cis-regulatory elements via chromatin immunoprecipitation will allow for molecular discrimination of estrogen responsiveness.