2006 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, 2005 through August 31, 2006
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 the 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.
Studies and Results
Time course and dose response studies for ortho,para'-dichlorodiphenyltrichloroethane (DDT) in immature ovariectomized Sprague Dawley rats have been completed. DDT elicited a characteristic uterotrophic effect (up to 3.5-fold induction in uterine wet weight) in wet and blotted weight which was blocked by co-treatment with pure ER antagonist ICI 182780 (Figure 1). DDT induced uterotrophy in a dose responsive manner after treatment with 1, 3, 10, 30, 100 or 300 mg/kg body weight (b.w.), allowing for selection of a dose for the temporal studies that would result in optimal physiological response. EE and DDT elicited uterotrophic EC50s of 16.2 μg/kg and > 64.6 mg/kg, respectively, in dose response studies. DDT only induced a 3.3-fold increase in wet weight with minimal water imbibition compared to the 7-fold induction observed with EE. The 300 mg/kg dose of DDT was used in a comprehensive time course study (2, 4, 8, 12, 18, 24, and 72 hours post treatment with one or three daily doses, respectively) wherein a broad spectrum of physiological, histopathological, and molecular endpoints were assessed. Temporal changes in uterine wet and blotted weights (approximately 3.5-fold for both at 72 hours) were used to assess gross physiological endpoints including water imbibition and proliferation. Histopathological samples were processed and stained (hematoxylin and eosin [H&E]) and evaluated by a veterinary pathologist (Co-Investigator, Dr. Kurt Williams). Subsequent findings of stromal and epithelial apoptosis, immune cell accumulation, luminal epithelial hypertrophy, and proliferation characterized the morphological changes during the uterotrophic response at 72 hours. Morphological measurements of luminal epithelial cell height revealed statistically lower induction in cell height when compared to ethynylestradiol positive control treated rats (Figure 2).
Custom in-house rat cDNA microarrays were used to characterize the dose-responsive uterotrophic changes in gene expression after a single dose of DDT as well as monitor the changes at the uterotrophic time point (72 hours) after three daily 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 DDT expression profiles included water and ion transporters, angiogenic factors, pro- and anti-apoptotic genes, redox regulators, xenobiotic metabolism enzymes, cell cycle control genes, and cellular energetics control genes. The expression profiles of several representative genes covering multiple categories of response were verified by quantitative real-time polymerase chain reaction (QRT-PCR) and showed excellent concordance between the two methods.
A number of genes regulating cell death and water imbibition (Pycard and MIP/Aqp0) demonstrated a particularly large response, whereas classically estrogen regulated genes (C3 and Calb3) exhibited equivalent or less efficacious responses in response to DDT (Figure 3). These results suggest that DDT and EE elicit selective ER modulating activities that may be responsible for differences in uterotrophic and pathological effects.
The temporal gene expression studies are currently being analyzed and complete interpretation of both the dose response and time course data will be integrated into a comprehensive assessment along with the histomorphological and physiological endpoints. Several genes involved in related processes regulated by EE, including Vegf, aquaporins, solute carrier family proteins, and sodium channel proteins, will be examined for their role in the differential water imbibition between the two compounds. Subsequent examination of DDT expression profiles will provide evidence to determine if these mechanisms are involved in the EE but not DDT induction in water content among other endpoints.
These results demonstrate that DDT elicits effects that are dramatically different in effect and potency on uterine size, water content, cytoarchitecture and apoptosis while exhibiting conserved overall proliferative effects. Preliminary investigations indicate organ-, cell- and gene-level changes are perturbed by DDT treatment in the rat uterus. The results are consistent with global gene expression results, although more comparative studies are needed to further elucidate the disrupted pathways and to determine if these effects are relevant to humans.
No significant change in plans is being proposed. 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. Comparative analysis to other ER ligands will be conducted as well as cross-species effects observed in comparable studies to assess the rat gene expression relative to the mouse, which has more mature genomic annotation.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
|Other project views:||All 89 publications||14 publications in selected types||All 13 journal articles|
||Kwekel JC, Burgoon LD, Burt JW, Harkema JR, Zacharewski TR. A cross-species analysis of the rodent uterotrophic program: elucidation of conserved responses and targets of estrogen signaling. Physiological Genomics 2005;23(3):327-342.||