CONCLUSIONS  TAM shows its partial agonist activity in less efficacious uterotrophy as well as overall lower induction of gene expression relative to EE while activating few if any unique targets.  DDT is less potent and efficacious than EE, but also exhibits an expression pattern almost identical to that of EE in the immature, OVX rodent uterotrophic response.  Conserved and divergent gene expression profiles are being further analyzed for unique ligand- ER activity.  Further analysis of cross tissue and cross species comparisons will lend valuable insight into TAM and DDT’s conserved and divergent mechanisms. ABSTRACT Changes in uterine physiology, cell morphology and gene expression were evaluated in a comprehensive time course study after oral administration of 100 µg/kg bw ethynylestradiol (EE), 100 µg/kg tamoxifen (TAM) or 300 mg/kg o,p’-DDT (DDT) to immature, ovariectomized Sprague-Dawley rats. Animals were dosed once or once daily for 3 consecutive days and uteri were harvested 2, 4, 8, 12, 18, 24, or 72 hrs after treatment. EE, TAM, and DDT elicited uterotrophic EC50s of 16.2 µg/kg, 16.5 µg/kg and >64.6 mg/kg, respectively, in the dose response studies. TAM and DDT only induced a 3.3-fold increase in wet weight with a marked decrease in water imbibition compared to the 7-fold induction with EE. DDT treated histological samples were characterized by marked apoptosis in the luminal epithelia and stroma at 72 hr compared to EE while TAM treatment exhibited significantly higher induction of luminal epithelial cell height at 72hr. Temporal gene expression profiles were measured using custom rat cDNA microarrays consisting of 8,567 features representing 4,342 genes and identified conserved and divergently regulated genes. Of the 1984 genes commonly regulated by all three compounds, 1574 demonstrated highly correlated temporal profiles between all three datasets indicating a large degree of similarity in the expression profiles. However 10 and 183 genes exhibiting expression profiles unique from EE for TAM and DDT, respectively, suggest additional non-estrogen receptor mediated pathways being regulated by DDT but not TAM. Supported by: US EPA RD , R21 GM075838, ES MICROARRAY DATA: COACTIVITY ANALYSIS ETHYNYLESTRADIOL, TAMOXIFEN AND O,P’-DDT INDUCE CONSERVED AND DIVERGENT GENE EXPRESSION PROFILES DURING UTEROTROPHY IN THE RAT UTERUS JC Kwekel 1,3, KJ Williams 2 TR Zacharewski 1,3 1 Departments of Biochemistry & Molecular Biology, 2 Pathobiology & Diagnostic Investigation, 3 Center for Integrative Toxicology and National Food Safety & Toxicology Center, Michigan State University, East Lansing, MI INTRODUCTION AND OBJECTIVES Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM); prescribed as an adjuvant therapy for ER- positive, breast cancer treatment or prophylactically for patients with increased breast cancer risk or family history. It acts by binding the ER and inhibiting estradiol’s proliferative effects in tumor progression and growth. Tamoxifen’s activity as a SERM has been well documented clinically, exhibiting antagonistic properties in the breast but partial agonistic activity in the reproductive tract as evidenced by an increased incidence of uterine tumors in patients taking the drug. DDT is an environmentally persistent organochlorine widely used until the early 1970’s as a common pesticide. Its wide spread production and use was in large part due to its role in vector control for malaria (mosquitoes). The ortho, para’ enantiomer of DDT has been shown to bind the estrogen receptor and disrupt normal reproductive health and development. Despite this knowledge, the subsequent transcriptional activities of the TAM- or DDT-ER complex and the full spectrum of target genes has not been fully delineated. This project has set out to characterize the gene expression changes associated with TAM and DDT activity in the rodent uterus in conjunction with its physiological and morphological effects. The rodent uterotrophic assay has been used for decades to assess the estrogenicity of compounds by their ability to stimulate an increase in uterine wet weight. This assessment, while being a gold standard for screening estrogenic compounds, provides limited insight into the pathways involved. We therefore have employed and extended the enhanced uterotrophic assay adapted from Diel, et al in which in vivo estrogenicity is evaluated in a three tiered approach while incorporating a comprehensive time course study. This approach combines assessments of the uterotrophic, morphological and transcriptional responses of the rodent uterus into an integrated evaluation, wherein:  TAM and DDT treatments will be compared to the prototypical oral estrogen: ethynylestradiol in the rat uterus.  Classic estrogenic endpoints of uterotrophy and luminal epithelial cell height will be quantified and compared.  Temporal gene expression profiles of EE, TAM and DDT will be examined by cDNA microarray analysis and comparative analysis of the expression profiles will determine conserved or divergent pathways. UTERINE WEIGHT: DOSE RESPONSE AND TIME COURSE Figure 3 Dose Response experiments were performed which indicate comparable potency of TAM but lower efficacy of TAM and DDT in uterine wet weight induction and water imbibition. A.A. MICROARRAY DATA: COMPARATIVE ANALYSIS Figure 8 Genes shown to be active in response to at least one ligand were compiled into a common list of genes and hierarchically clustered agglomer- atively (GeneSpring) by gene for visual representation of compar- able levels of gene induction and repression between all three ligands. This cluster plainly shows the highly conserved and correlated nature of the expression profiles of EE, TAM and DDT in the rat uterus. QRT-PCR VERIFICATION OF MICROARRAY DATA EXPERIMENTAL DESIGN Figure 2 The Dose Response study consisted of three daily oral doses, of EE, TAM and DDT (6 dose groups) or sesame oil vehicle to immature, ovariectomized Sprague Dawley rats followed by sacrifice and tissue harvest at 72 hr. The Time Course study consisted of the same dosing schedule for 100 ug/kg bw of EE, 100ug/kg TAM, or 300 mg/kg bw DDT followed by sacrifice at times indicated. Tissues for gene expression were snap frozen in liquid nitrogen while histological samples were fixed for 24 hours in 10% neutral buffer formalin. Each treatment group consisted of five animals each for both studies. Figure 1 The enhanced uterotrophic assay consists of three daily doses of estrogen, followed by physiological, morphological and biochemical assessments 72 hrs after initial treatment (adapted from Diel, et al. 2002, ). Uterine wet weight and water content; histopathological and morphometric assessments; and gene expression studies through microarray analysis and QRT- PCR verification of active genes were assessed. The current study extended the Diel protocol by incorporating a comprehensive time course throughout the first 24 hrs at which the above endpoints were assessed. Uterotrophic Response Morphological Response Transcriptional Response EEDC Compounds Ethynylestradiol Tamoxifen O-p-DDT Bisphenol A Genistein Rodent Models Rat Mouse Target Tissues Uterus Liver Mammary Kidney Bone Dosing Times (h) Sacrifice Time (h) TIME COURSE EE 100 ug/kg bw TAM 100 ug/kg bw DDT 300 mg/kg bw Sesame Oil Vehicle EE (ug/kg bw) 0.01, 0.1, 1, 10, 100, 300 TAM (ug/kg bw) 0.1, 1, 10, 100, 300, 1000 DDT (mg/kg bw) 1, 3, 10, 30, 100, 300 Sesame Oil Vehicle DOSE RESPONSE Sacrifice Dosing Times (h) h MORPHOLOGICAL ASSESSMENTS OF ESTROGENIC RESPONSE Figure 6 Morphometric analysis of EE, TAM, and DDT induced changes in luminal epithelial cell height. Measurement of LEH was repeated with a smaller cohort of animals to confirm TAM’s increased induction in LEH relative to EE. DDT exhibited a lower induction in LE cell height at 72hr. Mean+/-SEM, n=5, p<0.05, student’s t- test, Tukey’s HSD test, a compared to vehicle, b compared to EE. CONSERVED AND DIVERGENT GENES Rat Array 8,567 spots (4,717 genes) Differential Expression P1t ≥ |fold change| ≥ 1.5 Figure 9 Quantitative real time PCR confirmed the temporal expression patterns of several known estrogen responsive genes including Fos and C3. Bars and lines represent fold change relative to time matched vehicle control for QRT- PCR and microarray data, respectively. Error bars represent the SEM for the average fold change in QRT-PCR data. N=5, *p < 0.05, student’s t-test. Figure 4 Time Course experiments monitored uterine wet weight (A) and water content (B), which was calculated as the difference between wet and blotted weights. TAM and DDT exhibited early water imbibition (4hr-12hr), but not at 72hr. Error bars represent the SEM for the average. N=5, *p < 0.05, student’s t-test. B.B. Figure 5 Histological sections at the uterotrophic time point (72hr) exhibit classic changes in uterine cell morphology including increased luminal epithelial cell height (LEH), thickened basal lamina and prolifer- ation as evidenced by increased luminal diameter. TAM treated samples elicited comparable induc- tion in stromal area and increased induction of LE cell height compared to EE. DDT treated samples showed increased incid- ence of apoptosis thickened myometrium and immune cell accumulation in comp- arison to EE treated samples. Figure 7 Microarray data for all three ligands were filtered by a statistical cutoff (P1t ≥ 0.999) and fold change cutoff (± 1.5) relative to vehicle controls at any time point and were thus designated as differentially regulated or “active”. Number of active genes per time point (A) were calculated for each compound. EE and DDT exhibit similar kinetics in time of gene response while TAM lagged by hr due to TAM’s need for bioactivation to the 4-OH form. Numbers of active genes were comparable at 72 hr. Poland Sucks! EE (2,409) TAM (2,087) DDT (2,458) 1, EE TAM DDT TAM 72hr CoActivity Analysis CAS 1565 CAD 18 DAS 362 DAD 39 Correlation of EE-DDT Correlation of TAM-DDT Correlation of EE-TAM α-Ethynyl- Estradiol Tamoxifen o,p’-DDT Figure 10 Genes meeting activity criteria for at least one ligand were compared in a Venn analysis (A) to find overlapping active genes. Genes active in all three data sets were then further processed for coactivity whereby genes exhibiting similarities or differences in time of expression and direction of change were subsequently designated as coactive-similar, CAS; coactive-divergent, CAD; displaced active-similar, DAS; or displaced active-divergent, DAD using CAIT (CoActivity Index Tool). The temporal correlations of EE-TAM, TAM-DDT and DDT-EE were plotted (B) for each gene along with coactivity designations to visualize conserved responses. A. B.