Gene Expression Profile Induced by 17alpha-Ethynyl Estradiol in the Prepubertal Female Reproductive System of the Rat

Abstract

The profound effects of 17beta-estradiol on cell growth, differentiation, and general homeostasis of the reproductive and other systems, are mediated mostly by regulation of temporal and cell type-specific expression of different genes. In order to understand better the molecular events associated with the activation of the estrogen receptor (ER), we have used microarray technology to determine the transcriptional program and dose-response characteristics of exposure to a potent synthetic estrogen, 17 alpha-ethynyl estradiol (EE), during prepubertal development. Changes in patterns of gene expression were determined in the immature uterus and ovaries of Sprague-Dawley rats on postnatal day (PND) 24, 24 h after exposure to EE, at 0.001, 0.01, 0.1, 1 and 10 micro g EE/kg/day (sc), for four days (dosing from PND 20 to 23). The transcript profiles were compared between treatment groups and controls using oligonucleotide arrays to determine the expression level of approximately 7000 annotated rat genes and over 1740 expressed sequence tags (ESTs). Quantification of the number of genes whose expression was modified by the treatment, for each of the various doses of EE tested, showed clear evidence of a dose-dependent treatment effect that follows a monotonic response, concordant with the dose-response pattern of uterine wet-weight gain and luminal epithelial cell height. The number of genes whose expression is affected by EE exposure increases according to dose. At the highest dose tested of EE, we determined that the expression level of over 300 genes was modified significantly (p < or = 0.0001). A dose-dependent analysis of the transcript profile revealed a set of 88 genes whose expression is significantly and reproducibly modified (increased or decreased) by EE exposure (p < or = 0.0001). The results of this study demonstrate that, exposure to a potent estrogenic chemical during prepubertal maturation changes the gene expression profile of estrogen-sensitive tissues. Furthermore, the products of the EE-regulated genes identified in these tissues have a physiological role in different intracellular pathways, information that will be valuable to determine the mechanism of action of estrogens. Moreover, those genes could be used as biomarkers to identify chemicals with estrogenic activity.

Phenotypic Anchoring of Gene Expression Changes during Estrogen-Induced Uterine Growth

“…Increasing attention is being paid to the use of gene expression changes in the uterus for the identification of surrogate markers for short-term rodent estrogenicity assays (Naciff et al 2002(Naciff et al , 2003Owens and Ashby 2002;Watanabe et al 2002), and our data reveal a large number of novel candidate marker genes. The insights provided by these data, into how an ER ligand coordinates transcriptional regulatory networks that result in proliferation and differentiation in a complex organ, provide a paradigm for understanding the modes of action of other nuclear receptors.…”

Genomics and developmental risk assessment

“…The juvenile and adult uterus goes through a proliferative (uterotrophic) response after treatment with physiological/ pharmacological levels of estrogens, but this response is transitory and comparable to normal estrous cycling in the adult rat. We identified characteristic transcript profiles for estrogens at both life stages (Naciff et al, 2002(Naciff et al, , 2003. Although literally hundreds of genes are affected by estrogens, we were able to identify a set of approximately 60 genes in the fetus and 120 in the juvenile rat that responded in a robust, doseresponsive manner to a variety of estrogens.…”

“…More importantly, it demonstrated the potential for gene expression analysis to support investigations into biological responses at the low end of the dose response curve and indicate that biologically based dose-response modeling will be feasible. Naciff et al, 2003). Genes are clustered according to the biological process with which they are associated.…”

“…Programs available at NCBI include the Gene Expression Omnibus, which, among other things, clusters genes according to the biological process in which they are involved, or the location at which they are expressed. Figure 1 shows clustering of data from an experiment in which estrogens at various dose levels were given to juvenile rats and gene expression in the uterus analyzed (Naciff et al, 2003). The data are sorted according to gene ontology (GO) terms for biological processes.…”

The Safety Assessment of Food Additives by Reproductive and Developmental Toxicity Studies

“…Parabens are lately reported to act as xenoestrogens which are a class of endocrine disruptors due to the lengths of their alkyl side chains (Okubo et al, 2001). It is known that parabens have an effect on reproductive tissues, induce aberrant estrogenic signaling in cells, cause changes in the expression patterns of multiple genes in rat fetal reproductive system (Naciff et al, 2003). Despite the potential health effects, parabens are approved as food additives by the EU with ADI dose of 0-10 mg/kg bw/day (Ishiwatari et al, 2007) and with NOAEL as 1000 mg/kg bw/day (Boberg et al, 2010).…”