42nd Annual Meeting of the Society of Toxicology. Salt Lake City, UT. March 9-13, 2003.

Temporal expression patterns of hepatic genes of immature, ovariectomized mice treated with ethynyl estradiol.

Boverhof DR, Fertuck KC, Aiyar R, Burgoon LD, Zacharewski TR.

Department of Biochemistry & Molecular Biology, National Food Safety & Toxicology Center, and Institute for Environmental Toxicology, Michigan State University, East Lansing, MI.

Changes in global hepatic gene expression were examined in immature ovariectomized C57BL/6 mice treated with ethynyl estradiol (EE), a synthetic estrogen. Mice were gavaged once or 3x24hr with 0.1mg/kg EE or vehicle and liver tissue was harvested at 0, 2, 4, 8, 12, 24 or 72 hrs. The relative expression levels of 3067 genes was assessed using an independent reference design in which samples from EE treated mice were co-hybridized with samples from time-matched vehicle controls (TMV) on a cDNA/EST array. Significant changes in gene expression were identified using least squares means based on a general linear mixed model of EE samples compared to TMV samples using t-tests. Approximately 3-8% of the genes examined at each time point exhibited significant changes in expression (p<0.01). A gene annotation tool (GAT) that interfaces with our toxicogenomic supportive relational database, dbZach (http://dbzach.fst.msu.edu), was used to associate Gene Ontology functional categories with members of the significant gene set. This identified a large number of second messenger, transcription factor and signal transduction genes (e.g. c-fos, c-jun , insulin-like growth factor 1, transcription factor 12, transglutaminase 2, and laminin B) and various cytochrome P-450 enzymes involved in steroid and cholesterol biosynthesis that were significantly altered in response to EE. The promoters of the responsive genes were retrieved from the UCSC database, screened for the presence of estrogen response elements (EREs) and were found to contain responsive motifs which may suggest regulation by the estrogen receptor. The dramatic alteration of diverse signaling cascades illustrates the complexity of estrogen action in the liver. This, combined with the importance of the liver in metabolism and toxicity, suggests that evaluating hepatic responses will be important in future assessments of estrogen action in the intact organism.