Toxicological Sciences 68, 184-199 (2002)
Copyright © 2002 by the Society of Toxicology
REPRODUCTIVE AND DEVELOPMENTAL TOXICOLOGY |
Gene Expression Profile Induced by 17
-Ethynyl Estradiol, Bisphenol A, and Genistein in the Developing Female Reproductive System of the Rat
The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 538707, No. 805, Cincinnati, OH 45253-8707
Received December 11, 2001; accepted March 6, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure to some compounds with estrogenic activity, during fetal development, has been shown to alter development of reproductive organs, leading to abnormal function and disease either after birth or during adulthood. In order to understand the molecular events associated with the estrogenicity of different chemicals and to determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have used microarray technology to determine the transcriptional program influenced by exposure to this class of compounds during organogenesis and development. Changes in patterns of gene expression were determined in the developing uterus and ovaries of Sprague-Dawley rats on GD 20, exposed to graded dosages (sc) of 17
-ethynyl estradiol (EE), genistein, or bisphenol A (BPA) from GD 11 to GD 20. Dose levels were roughly equipotent in estrogenic activity. We compared the transcript profiles between treatment groups and controls, using oligonucleotide arrays to determine the expression level of approximately 7000 rat genes and over 1000 expressed squence tags (ESTs). At the highest tested doses of EE, BPA, or genistein, we determined that less than 2% of the mRNA detected by the array showed a 2-fold or greater change in their expression level (increase or decrease). A dose-dependent analysis of the transcript profile revealed a common set of genes whose expression is significantly and reproducibly modified in the same way by each of the 3 chemicals tested. Additionally, each compound induces changes in the expression of other transcripts that are not in common with the others, which indicated not all compounds with estrogenic activity act alike. The results of this study demonstrate that transplacental exposure to chemicals with estrogenic activity changes the gene expression profile of estrogen-sensitive tissues, and that the analysis of the transcript profile of these tissues could be a valuable approach to determining the estrogenicity of different compounds.
Key Words: toxicogenomics; gene expression profiling; microarrays; 17-ethynyl estradiol; genistein; bisphenol A.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Significant concern has recently been raised about the potential of environmental chemicals that might disrupt endocrine function. Particular attention has been given to chemicals that are able to alter estrogen functions. There are well-known examples of adverse effects of chemicals with estrogenic activity on humans (diethylstilbestrol, reviewed by Kaufman and Adam, 2002
; Swan, 2000), and wildlife development (DDT: Bowerman et al., 1995; Portelli et al., 1999; Sparling et al., 2001; Vos et al., 2000). Manifestations of abnormal reproductive system development, both structural and functional, may be latent until adolescence or adulthood. This latency has made it difficult to identify rapidly the developmental toxicants that act by an estrogenic mechanism. For this reason, the endocrine-disruptor screening battery proposed by the U.S. Environmental Protection Agency contains no prenatal exposures, despite the fact that development is generally acknowledged as the most sensitive life stage (U.S. EPA, 1998). Thus, the need arises for an accurate, rapid, and cost-effective method for assessing the potential estrogenicity of chemicals during development.
When a biological system is exposed to a toxic insult, it almost invariably modifies its pattern of gene expression as either a direct or an indirect response to the toxicant exposure (Nuwaysir et al., 1999; Steiner and Anderson, 2000). A direct response is likely to be the case for estrogens, since the signal transduction pathway for estrogen receptors (as well as all receptors in the steroid hormone receptor super-family) has been shown to involve binding to DNA and to result in transcriptional regulation of specific genes (reviewed by Katzenellenbogen et al., 2000; Klinge, 2001; Nilsson et al., 2001). We hypothesize that the largely latent developmental effects of estrogens are preceded by immediate changes in gene expression in the embryo and fetus. Therefore, an approach in assessing the potential estrogenic activity of different compounds was to identify those patterns of gene expression elicited in a tissue/organ exposed to these particular classes of chemicals.
Although a variety of long-established methods are available to characterize changes in gene expression in response to toxicants, the utility of determining those changes for hazard identification and risk assessment has not been exploited, primarily because of their lack of high throughput and their labor-intensive requirements. The arrival of new technologies in genomics, such as gene arrays or microarrays, that allow the simultaneous quantitative analysis of thousands of gene-expression changes in a single experiment (The Chipping Forecast, 1999), offers the opportunity to use gene expression profiling as a tool to predict toxic outcomes of exposure to particular chemicals with increased sensitivity and speed compared to traditional approaches. The application of microarray technology in toxicology research has been termed toxicogenomics (Nuwaysir et al., 1999; Pennie et al., 2000; Rodi et al., 1999). The premise of toxicogenomics is that identifying the gene-expression profiles, induced directly or indirectly by different classes of toxicants, should result in recognizable "molecular fingerprints" that are representative of specific toxicities. Once identified, these molecular fingerprints could be used to evaluate new or untested chemicals possessing unidentified toxicities, to improve traditional testing toxicity screens, and to understand mechanisms of action of different toxicants (Farr and Dunn, 1999; Nuwaysir et al., 1999; Pennie et al., 2000, 2001).
The purpose of the present study was to determine whether there is a common set of genes whose expression profile could be altered by exposure to compounds with estrogenic activity during organogenesis and development, and to facilitate the identification of gene transcripts with potentially important roles in estrogen action, many of which may have not been detected thus far by using traditional approaches. If a common set of genes is identified, this could serve as the basis for a screening assay for estrogenic activity. Estrogens have multiple physiological effects, not just in tissues from the reproductive system, but also bone, liver, and brain and from the cardiovascular and the immune systems (Hall et al, 2001; Nilsson et al., 2001). In mammals, the predominant biological effects of estrogens are mediated through 2 distinct intracellular receptors: estrogen receptor (ER)- and ER-ß (Klinge, 2001; Nilsson et al., 2001). There is a considerable variation in the expression levels of the 2 ER isoforms in the different target tissues (Couse et al., 1997). The uterus and ovaries are 2 of the most sensitive tissues to estrogenic regulation; therefore, we have determined the changes in patterns of gene expression in the uterus and ovaries of fetuses of pregnant rats exposed to a potent synthetic estrogen (17 -ethynyl estradiol), a natural phytoestrogen (genistein), and a weakly estrogenic chemical used in the manufacture of polycarbonate plastics (bisphenol A), from day 11 to day 20 of gestation. The estrogenic activity, including dose-response relationships, of this reference set of chemicals has been well established (Ashby and Tinwell, 1988; Branham et al., 1988; Diel et al., 2000; Kwon et al., 2000; McLachlan and Newbold, 1987; Nguyen et al., 1988; Sahlin et al., 2000; Sheehan et al., 1981). We chose fetal uterus and ovaries for the analysis of estrogen-responsive gene expression because of their sensitivity to estrogen effects. Exposure to potent estrogens (DES, EE, estradiol) during various stages of development has been shown to irreversibly modify the morphology and physiology of the uterus (Branham et al., 1988; McLachlan and Newbold, 1987; Newbold, 1995; Newbold et al., 1983; Nguyen et al., 1988; Ozawa et al., 1991; Rothschild et al., 1987; Sheehan et al., 1981). The precursors of the gonads and reproductive tracts are discernible on GD 12. Dosing from GD 11 onwards encompasses the critical period of reproductive development in which most of the organogenesis is occurring and a stage at which the developing fetus is more susceptible to endocrine disruption (Bigsby et al., 1999; Cooper and Kavlock, 1997; McLachlan and Newbold, 1987). The dose levels of 0.5, 1, or 10 µg EE/kg/day; 0.1, 10, or 100 mg genistein/kg/day; and 5, 50 or 400 mg BPA/kg/day were selected, based upon the published estrogenic potency of these compounds (Ashby and Tinwell, 1988; Branham et al., 1988; Diel et al., 2000; Kwon et al., 2000; Nguyen et al., 1988; Sahlin et al., 2000).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Bisphenol A (BPA,
99% purity) was purchased from Aldrich Chemical Company (Milwaukee, WI). 17--Ethynyl estradiol, genistein (4`,5,7-trihydroxyisoflavone), peanut oil, and dimethyl sulphoxide (DMSO) Hybri-Max were obtained from Sigma Chemical Company (St. Louis, MO).
Animals and treatments.
Five-month-old male and female Sprague-Dawley rats weighing 300 g were used (Charles River VAF/Plus). We chose this rat strain because it is the most commonly used in reproductive and developmental toxicity studies. The rats were acclimated to the local vivarium conditions (24°C, 12-h light/dark cycle) for 2 weeks. All rats were housed singly in 20 x 32 x 20-cm cages during the experimental phase of the protocol. They were allowed free access to water and to a pelleted commercial diet (Purina 5001; Purina Mills, St. Louis, MO) containing phytoestrogens, mostly genistein and daidzein derived from soy and alfalfa (Thigpen et al., 1999). While we realize that the presence of these compounds may have an impact on the gene expression profile, we chose to use this diet to avoid a potential negative shifting of the baseline data, which would diminish the value of historical comparisons of estrogen-dependent gene-expression data already published. The experimental protocol was carried out according to Procter and Gamble's animal care-approved protocols, and animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Males were used only as breeders, and timed-pregnant females were used for the experiments. Breeding was carried out by co-housing one male and one female overnight. Successful mating was confirmed the following morning by the presence of sperm in the vaginal smear. Sperm positive animals were considered to be at gestation day 0 (GD 0) at that time.
For the genistein study, females were switched to a soy- and alfalfa-free diet, the casein-based diet 5K96 (Purina Mills; St. Louis, MO) 1 day before mating and during the entire experimental phase. This diet has been shown to contain 0.54 µg genistein per gram (Chang et al., 2000) and consistently contains less than 1 ppm aglycone equivalents of genistein, daidzein, and glycitein (Purina Mills). The dams were randomized into 4 groups and housed in individual cages. Each treatment group had a minimum of 7 pregnant females. Starting on GD 11, the dams were dosed by subcutaneous injection with 0, 0.5, 1, or 10 µg/kg/day of 17--ethynyl estradiol in peanut oil; and 0, 0.1, 10, or 100 mg genistein/kg/day; or 0, 5, 50, or 400 mg/kg/day bisphenol A in DMSO. Animals received 1 ml/kg bw of dose solution each day on GD days 11 to 20. The dose was administered between 8 and 9 A.M. each day. Controls received 1 ml/kg of peanut oil or DMSO, respectively. Doses were administered on a µg or mg/kg bw basis and adjusted daily for weight changes. Body weights (nearest 1.0 g) and the volume of the doses administered (nearest 0.1 ml) were recorded daily. The exact time of the last dose was recorded to establish a 2-h waiting period before tissue collection. The animals were sacrificed by CO2 asphyxiation at 2 h after the last dosing on GD 20. The fetuses were harvested and the fetal uterus and ovaries were removed and placed into RNAlater (50–100 mg/ml of solution; Ambion) at room temperature.
Histology.
For the histological examination, the reproductive tissues from 4 fetuses, obtained from different litters within the same dose-treatment group, were fixed in 10% neutral buffered formalin immediately after removal from the fetuses, then dehydrated and embedded in paraffin. Serial 4–5-µm cross sections were made through the ovaries, oviducts, and uterine horns and stained with hematoxylin and eosin. To evaluate the serial sections for abnormalities, we focused on the proliferative state of the endometrial stroma and luminal epithelium along the uterine horns, and on proliferation of the columnar epithelium lining the lumen along the oviduct, by counting the number of mitotic cells per unit area under a light microscope (Nikon Optiphot-2, Nikon).
Expression profiling.
Our goal was to determine the gene expression profile in estrogen-regulated tissues induced by chemicals with estrogenic activity; the uterus and ovaries are 2 of the most sensitive tissues to estrogenic regulation. Therefore, the uterus and ovaries of at least 5 littermates were pooled to yield a representative litter sample for analysis, and total RNA was extracted using TRI-Reagent (Molecular Research Center, Inc., Cincinnati, OH). Total RNA was further purified by an RNeasy kit (QIAGEN, Valencia CA). Ten µg of total RNA from each pool of tissue sample were converted into double-stranded cDNA by using the SuperScript Choice System (GIBCO BRL, Rockville, MD) with an oligo-dT primer containing T7 RNA polymerase promoter. The double-stranded cDNA was purified by phenol/chloroform extraction, and then used for in vitro transcription using ENZO BioArray RNA transcript labeling kit (Affymetrix, Inc. Santa Clara, CA). Biotin-labeled cRNA was purified by the RNeasy kit (QIAGEN), and a total of 20 µg of cRNA were fragmented randomly to 200 bp at 94°C for 35 min (200 mM Tris-acetate, pH 8.2, 500 mM KOAc, 150 mM MgOAc). Labeled cRNA samples were hybridized to the Affymetrix GeneChip Test 2 Array (Affymetrix, Inc. Santa Clara, CA) to assess the overall quality of each sample. After determining the target cRNA quality, samples of pooled uteri-ovaries from five individual dams (replicates) from each treatment group (with high quality cRNA) were selected and hybridized to Affymetrix Rat Genome U34A high-density oligonucleotide microarrays for 16 h. The microarrays were washed and stained on the Affymetrix Fluidics Station 400, using instructions and reagents provided by Affymetrix. Briefly, nonhybridized material is removed, and then the microarray is exposed to streptavidin-phycoerythrin (SAPE) to detect bound cRNA. The signal intensity was amplified by a second staining with biotin-labeled anti-streptavidin antibody and followed by streptavidin-phycoerythrin staining. Fluorescent images were read using the Hewlett-Packard G2500A Gene Array Scanner.
Real-time RT-PCR.
In order to validate the relative change in gene expression induced by estrogenic exposure of the fetal uterus and ovaries of the rat in selected genes identified by the oligonucleotide microarrays, we used a real-time (kinetic) quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) approach. QRT-PCR evaluates product accumulation during the log-linear phase of the reaction, and it is currently the most accurate and reproducible approach for transcript quantification (Morrison et al., 1998; Rajeevan et al., 2001). This approach also allowed us to evaluate the "basal level" of expression of individual genes in samples derived from animals exposed to the 2 different diets used in our study. QRT-PCR was used to compare the transcript level of selected genes in samples derived from animals exposed to Purina 5001, with the levels of the same transcript found in equivalent samples derived from animals exposed to the casein-based diet. The reverse transcription (RT) reaction was carried out with 10, 25, 50, and 100 ng of total RNA, DNAse-I-treated (Ambion; Austin, TX) from control and treated samples using the Access RT-PCR system from Promega (Promega Corp., Madison, WI), according to manufacturer's instructions (45 min at 48°C). Absence of genomic DNA contamination in the total RNA samples was confirmed by performing the same RT reactions, but without reverse transcriptase followed by quantitative PCR. Real-time PCR was performed in the iCycler iQTM Multi-Color Real Time PCR Detection System (Bio-Rad Laboratories; Hercules, CA) to continuously monitor the fluorescence of the high affinity, double-stranded, DNA binding dye SYBR Green I (Bio Whittaker Molecular Applications; Rockland, ME), using an automated detector combined with special software (Bio-Rad). Each QRT-PCR run included a standard curve of 7 points with known amounts of the same purified amplicon being tested (from 5 x 106–10 copies of target), a no-template control, a reverse transcriptase negative control, and the experimental samples being tested, including at least 3 independent samples for each treatment group, run in duplicate and in parallel. Amplification reactions (20 µl) were carried out with the next cycle conditions: one initial step of 4 min at 95°C, followed by 50 cycles of 95°C for 15 s, 55°C for 20 s, and 72°C for 40 s, with a final extension at 72°C for 4 min. The standard curve was generated by plotting the amount of amplicon tested against the corresponding Ct value to calculate the relative expression levels of the different samples, and we interpolated the sample Ct values against the standard curve. To confirm the amplification specificity from each primer pair, the amplified PCR products were size-fractioned by electrophoresis in a 4% agarose gel in Tris borate ethylene diamine tetracetic acid (TBE) buffer and photographed after staining with ethidium bromide. Table 1 shows the nucleotide sequences for the primers used to test the indicated gene products. Preliminary experiments were carried out with each primer pair to determine the overall quality and specificity of the primer design. After RT-PCR, only the expected products at the correct molecular weight were identified.
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Data analysis.
Potential inter-individual variability was addressed by pooling the target tissues (uteri and ovaries) from individual fetuses within each litter to yield a representative litter sample for analysis. We analyzed 5 litters per dose group per compound. Scanned output files of Affymetrix microarrays were visually inspected for hybridization artifacts and then analyzed using Affymetrix Microarray Suite (ver. 4.0) and Data Mining Tool (ver. 1.0) software, as described previously (Lockhart et al., 1996
). Arrays were scaled to an average intensity of 1500 and analyzed independently. The Affymetrix Rat Genome U34A microarrays used in this study have 8740 probe sets corresponding to 7000 annotated rat genes and 1740 expressed sequence tags (ESTs). Each gene or EST is represented by 16–20 pairs of 25-mer oligonucleotides that span the coding region. Each probe pair consists of a perfect match sequence that is complementary to the cRNA target and a sequence that is mismatched by a single base change at the middle of the nucleotide, a region critical for target hybridization. The mismatched oligonucleotide serves as a control for nonspecific hybridization. The Microarray Analysis Suite (Affymetrix) was used to generate the data for comparative analysis. Distinct algorithms made an absolute call, presence/absence, for each transcript, and calculated the average difference between perfect match and mismatch probe pairs. The mathematical definitions for each algorithm are described in the Affymetrix Microarray Suite User's Guide, Version 4.0. Transcripts for which an absent call was determined in all the samples, across the dose groups for a given compound, were eliminated from further analysis. For the remaining transcripts, a series of statistical tests was conducted for each transcript separately. For each transcript and chemical, we conducted standard t-tests, comparing each treatment group to its control, and analysis of variance (ANOVA) on the average-difference value (that serves as a relative indicator of the level of expression of a transcript) and the log of average difference. We also conducted a nonparametric test for trend, the Jonkheere-Terpstra test. Genes for which any of the tests had p < 0.001 was taken as evidence that the expression of those genes was modified by the compound being tested. This procedure was done for each treatment vs. control, and for the full set of study results for each individual compound tested (vehicle vs. low, mid, and high dose).
In order to compare the gene expression profiles induced by the 3 chemicals tested, and to address possible issues of diet-induced differences (due to different phytoestrogen content), the average value of the average-difference values, which is a relative indicator of the level of expression of a transcript, was compared among the 3 groups of independent controls, for all the 8740 transcripts represented on the array. In these analyses, we compared the data from animals exposed to Purina 5001 (controls from EE and BPA studies) to those exposed to the casein-based diet (genistein study). Our analysis indicated that approximately 2% of those transcripts showed a significant change on their level of expression that can be correlated to the diet used to feed the dams (data available upon request, JMN). No significant changes were found at the transcript level for selected estrogen-regulated genes by QRT-PCR (Fig. 3, Table 6). Importantly, none of the genes that were identified as part of the fingerprint for chemicals with estrogenic activity was affected by the diet. Data from the 3 chemicals were also pooled for the purposes of identifying genes that are regulated in a similar manner by the 3 compounds. Here, we used linear models, with terms for both study and treatment effects, on average differences and their log transformation, as well as on stratified forms of the Wilcoxon-Mann-Whitney nonparametric statistic and a stratified form of the Jonkheere-Terpstra nonparametric statistic for dose response. In the linear model analysis, study-to-study differences are adjusted for by the presence of a term for study effects in the model and, in the nonparametric statistics, stratification amounts to pooling within-study evidence of treatment effects. Genes regulated differentially among chemicals were identified by the addition of an interaction term to the linear model analyses. In all of these pooled analyses, the expression of a gene was considered affected when any of the relevant tests had p < 0.0001 for that particular gene. Fold-change summary values for genes were calculated as a signed ratio of mean average differences. Because fold-change values can become artificially large or undefined when mean average-difference values approach zero or are negative, all the values <100 were made equal to 100 before calculating the mean average differences that are used in the fold-change calculation. Note that all statistical analyses use the measured average-difference values, except when an average difference is negative; then the log-scale analyses instead use the ranks of the average-difference values.
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Online supplemental materials.
Affymetrix image files for the 60 chip hybridizations, the analysis of the 3 control groups, and the absolute-analysis results of each compound are available upon request (JMN).
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RESULTS |
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Maternal and fetal toxicity/compound and dose selection.
17
-Ethynyl estradiol (EE) at 0.5, 1, and 10 µg/kg/day was used as a reference chemical, with known estrogenic effects at the mid and highest doses tested. For both BPA and genistein, we chose a high dose reported to be uterotrophic, as well as dosages with no effect (5 and 0.1 mg/kg/day for BPA and genistein, respectively). The mid-dosages were selected based upon the published estrogenic potency of these compounds. In this study, treatment of pregnant rats with the indicated doses of EE, genistein, or BPA (see Materials and Methods) was not associated with substantial maternal or fetal toxicity. There were no detectable effects of EE, BPA, or genistein at any of the dosages tested, on either maternal body weights or numbers of live fetuses per litter. Histological examination of fetal ovaries, oviducts, and uteri indicated essentially no changes or gross abnormalities in micromorphology of these organs induced by estrogen exposure at any of the dosages tested (Fig. 1). However, adverse effects were seen with the highest dose of EE and BPA, namely vaginal bleeding and early parturition in 1 of 8 dams given the high dose of EE and 1 of 8 dams given the high dose of BPA. In the fetuses, both EE and BPA induced, at the highest doses tested, prominent nipples/areolas in both female and male fetuses. Even at the highest dose tested, genistein did not have any of these toxic effects in the dams or their fetuses.
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Analysis of the gene expression patterns induced by 17
-ethynyl estradiol, genistein, and bisphenol A by oligonucleotide arrays.Following individual treatment of pregnant rats (from GDal days 11 to 20), RNA from the pooled fetal uteri and ovaries from animals from the same litter were processed for microarray analysis as described. Samples from the pooled tissues (uteri and ovaries) from 5 individual dams, used as 5 independent experimental samples for each treatment group, were analyzed to determine RNA transcript levels. Based on the number of genes expressed in control versus treated samples, as well as on the level of expression of individual genes, the overall gene expression pattern was similar between control (vehicle-treated) and estrogenic compound-treated (EE, BPA or genistein) fetal tissues (Fig. 2
). Although the number of genes whose expression is altered by any chemical tested is not very big, there are many genes whose expression is modified by exposure to each compound.
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Of the 8740 probe sets, corresponding to
7000 annotated rat genes and 1740 ESTs analyzed in this study, there were 366, 397, and 381 genes whose expression level showed some evidence of treatment effect (up- or down-regulated) by EE, BPA, and genistein, respectively, relative to their respective vehicle control and judged by fold change (at least ±1.5 fold, up or down), t-tests or ANOVA analyses (p 
0.001). From those genes, the expression of 26, 35, and 227 was statistically significantly changed by EE, BPA, or genistein exposure in a dose-dependent manner (trend analysis, Jonkheere-Terpstra test, p 0.001), respectively. Global analysis of the data derived from the 3 chemicals tested at the highest dosages indicated that the expression of 66 genes was consistently and significantly regulated in the same direction (p 0.0001), although at a different magnitude, by all 3 estrogenic compounds. These include genes known to be directly regulated by estrogens, such as progesterone receptor, but also other annotated genes and ESTs that have not been previously identified as estrogen-responsive. Table 2 shows the complete list of the 66 genes from our studies that showed a statistically significant (p 0.0001, t-test) change in their expression by estrogenic exposure, along with their accession number and fold change (average calculated by comparing treatment versus control, with n = 5 in each case). Trend analysis of the data from the 3 chemicals tested indicated that 52 of these genes show a significant dose-response relationship (p 0.0001, Jonkheere-Terpstra test). Comparing the relative expression value of each of the genes listed in Table 2 in the 3 control groups (EE vs. genistein, or BPA vs. genistein), no significant differences were found in any of those transcripts (data not shown). Furthermore, no significant changes were found at the transcript level, for selected estrogen-regulated genes, by QRT-PCR (Fig. 3
and Table 6) in any of the control groups. Although there are gene expression changes that potentially could be correlated with the phytoestrogen content of the 2 rodent diets used in the present study (identified by comparing the 3 independent control groups), the presence of a higher phytoestrogen content in one of them (Purina 5001) does not seem to compromise our ability to detect the estrogenic effect of the different chemicals tested, even at the lower doses, on the set of responsive genes of the fetal reproductive tract of the female rat here identified.
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Tables 3, 4, and 5
show a partial list of genes whose expression is significantly and reproducibly (p 0.001, t test and ANOVAs) modified by 17 -ethynyl estradiol, bisphenol A, and genistein, respectively, in a dose-dependent manner. The 40 genes with the most robust response (p 0.001, t test) and for which the expression is modified by at least 1.8 fold (up or down) by each chemical are shown (Tables 3–5) (complete lists of the genes regulated by the 3 compounds tested are available upon request, J.M.N.). Gene transcripts shown in these partial lists (Tables 3–5) that are also listed in Table 2 are indicated. Transplacental exposure to these 3 compounds with estrogenic activity results in both gene induction (upregulation) and gene repression (downregulation). At the highest dose tested, gene induction appeared to be a more prevalent process in the case of exposure to EE (154 up and 117 down), while BPA (134 up and 168 down) and genistein (60 up and 128 down) exposure resulted in downregulation being more prevalent when criteria for selection is at least a 1.5-fold change. There are genes whose expression is significantly modified only by EE, BPA, or genistein alone, evidence of the independent effects of each chemical on the target tissues (gene transcripts listed in Tables 3–5, in normal font). From those, the expression of 21 genes can be used to distinguish each chemical (linear model analysis, with a p 0.0001). For example, although the 3 compounds induce the expression of the intestinal calcium-binding protein and uterus/ovary-specific putative transmembrane protein (Tables 2–5), in a dose-dependent manner, the response to EE and BPA can be distinguished from the response to genistein by the magnitude of the induction, being dampened in the case of genistein. There are some other genes that only respond to one chemical. For example: the expression of the calponin gene is increased only by EE exposure (Table 3); and the expression of the GTP-binding protein ral B is only decreased by BPA exposure (Table 4); while the expression of the stanniocalcin (rSTC) gene is stimulated only by genistein exposure (Table 5).
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Based upon the number of genes altered and the level of expression of individual genes, the gene expression profile induced by EE and BPA has a higher degree of similarity than either of them with the transcript profile induced by genistein (Tables 2–5
). From the genes with the most robust response to EE or BPA, there are 17 transcripts in common with highly similar expression levels (Table 3 vs. 4), while there are only 5 and 6 transcripts in common between EE and genistein or BPA and genistein, respectively (Table 3 and 4 vs. 5). For example, the expression of the gene coding for SM22 a smooth muscle-marker gene, and the gene represented by the EST AA900769 is only stimulated by EE and BPA, but not by genistein, while only the exposure to this phytoestrogen induces the expression of the serine-threonine kinase pim-3 gene (Tables 3–5). From the genes represented in the oligonucleotide microarray used in these studies (RatU34A), genes previously known to be regulated by estrogens were in fact regulated by estrogen exposure (see Discussion section), including intestinal calcium-binding protein (InCaBP), progesterone receptor (PrgR), 11-ß-hydroxylsteroid dehydrogenase type 2 (11ß-HSD), interleukin 4 receptor, and insulin-like growth factor 1, among others. However, most of the genes responsive to estrogen exposure, identified in the present study, had not been previously identified. The genes showing the most robust response (by fold change and p values in the different statistical test used [see Materials and Methods]) to transplacental exposure to the 3 chemicals include InCaBP, PrgR, 11ß-HSD, dermo-1 protein, FSH-regulated protein, aspartate aminotransferase, phosphodiesterase I, retinal binding protein, interleukin 4 receptor, growth potentiator factor, and multiple ESTs (AA924772, AA848831, AA799421, and AA891949, among others).
The consistency of the gene expression changes from sample to sample within a treatment group was high (individual sample values are available upon request). Analyzing one transcript at a time, comparing control vs. treated samples, in any of the statistical tests used, we consistently found values of p < 0.001, indicating that the expression of this gene was modified by the compound being tested across the samples (n = 5 for every dose group, for each compound tested). Furthermore, the reliability of this approach was independently corroborated by real-time quantitative (kinetic) reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis of selected genes in independent samples used in the microarray experiments. As shown in Figure 3 and Table 6, the expression of InCaBP, PrgR, vascular -actin (VaACTIN), 11ß-HSD, and the EST-AA924772 mRNAs, based on QRT-PCR analysis, followed essentially the same expression profile induced by the different doses of EE, BPA, and genistein as determined by microarray analysis. Similar results were obtained in the expression pattern determined for upregulated (InCaBPA, PrgR, 11ß-HSD) and downregulated (AA924772) genes by QRT-PCR and microarray analysis. No significant changes in the expression of 2 control genes (cylcophilin B and mitochondrial cytochrome oxidase subunits I, II, III, and ATPS subunit-6 gene) were identified by microarray or QRT-PCR analysis (Fig. 3, Table 6). Furthermore, no significant changes were found at the transcript level for those selected genes by QRT-PCR (Fig. 3, Table 6) in any of the control groups. Although there are a number of gene expression changes that can be correlated with the 2 rodent diets used in the present study (identified by comparing the 3 independent control groups), the presence of a higher phytoestrogen content in one of them (Purina 5001) does not seem to compromise our ability to detect the effect of the different chemicals tested, even at the lower doses, on the set of estrogen-responsive genes of the fetal reproductive tract of the female rat here identified.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Multiple studies over the last few years have established that related phenotypes are generally reflected in related patterns of gene expression, implying that the physiological state of a cell/tissue/organ can be characterized and classified by gene-expression patterns. Thus, we have evaluated whether exposure to estrogenic compounds could be identified by transcripts profiling tissue samples from control vs. treated organisms. High density oligonucleotide arrays offer the opportunity to simultaneously evaluate the expression of thousands of genes in multiple samples. In this study we have used this approach to identify the gene-expression profile induced by 3 chemicals with estrogenic activity, EE, BPA, and genistein, in the developing reproductive tissues of the rat. The fetal uterus and ovaries were selected, since they are 2 of the most sensitive tissues to estrogenic regulation, and contain cells with considerable variation in the expression levels of the 2 ER isoforms (Couse et al., 1997
), consequently have the potential to represent gene-expsresion changes induced by activation of any of those isoforms of the ER.
As a result of issues such as cross talk between different cell types, receptors (Muramatsu and Inoue, 2000; Power et al., 1992), protein-protein interactions between estrogen receptors and transcription factors (Rosenfeld and Glass, 2001; Xu et al., 2000), and the identification of key target genes expressed in selected cell types, it appears that the estrogenic response has to be evaluated in an in vivo system capable of fully evaluating the complexity of such a response. The developing reproductive system of the rat used in this study incorporates the complexity necessary to elucidate the molecular mechanisms implicated in the estrogenic response. The results of this study demonstrate that transplacental exposure to estrogens changes the gene expression profile of estrogen-sensitive tissues (uterus and ovaries). Each estrogenic compound induces an identifiable transcript profile (Tables 3–5), reflecting its molecular mechanism of action. Further, we have identified a common set of genes whose expression is consistently and significantly modified (up- or downregulated) in the same way by the 3 chemicals tested. Most of those genes responsive to the 3 chemicals (52 out of 66, 79%) show a significant dose-response relationship (p 0.0001, Jonkheere-Terpstra test). The specificity of these gene-expression changes is distinctive, robust, and reproducible. However, the potential use of this estrogenic molecular "fingerprint" to discriminate estrogenic compounds from different classes of chemicals has to be investigated further.
Although there is a common set of genes whose expression is similarly regulated by the 3 estrogenic compounds we have tested, the gene-expression profiles induced by EE and BPA have a higher degree of similarity to each other than either of them do to the profile induced by genistein (Tables 2–5). These results may reflect the differences in biological activity among these compounds. While EE and BPA may behave as "pure" estrogens, genistein clearly has other activities, such as inhibition of different enzymes, among them tyrosine kinases (Akiyama et al., 1987), nitric oxide synthase (Duarte et al., 1987), topoisomerase-II activity (Okura et al., 1988), and decreasing calcium-channel activity in neurons (Potier and Rovira, 1999). This isoflavone also decreases lipid peroxidation (Arora et al., 1998) and diacylglycerol synthesis (Dean et al., 1989). Although we did not identify all transcripts whose products are directly implicated in these activities (Table 5), these various functions of genistein could be reflected directly or indirectly on the transcript profile determined for genistein in the present study. For example, MAP-kinase is inhibited by genistein, and we have determined that genistein exposure (at high doses, 100 mg/kg) enhances the expression of the corresponding gene (1.4-fold). The expression of topisomerase II is also induced by genistein (1.2-fold), while the expression of phospholipase A2 is downregulated by this phytoestrogen (1.8-fold). The expression of these genes is not affected by EE or BPA. Although the elucidation of the mechanism of action of genistein, or any of the other 2 chemicals tested in our study, is beyond the scope of this manuscript, our findings clearly support the use of gene-expression profiling to understand mechanism of action of these compounds. Since the comparison of the relative expression value of each of the genes listed in Table 2 (estrogenic molecular "fingerprint") in the 3 control groups (EE vs. genistein, or BPA vs. genistein), indicated no significant differences in any of those transcripts among the three control groups exposed to Purina 5001 (EE and BPA case) or casein-based diets (genistein case) (data not shown), it is clear that the presence of phytoestrogen in the diet (Purina 5001) does not obliterate the effect of the different chemicals tested, even at the lower doses, on the set of responsive genes identified. However, the impact of the laboratory rodent diets, containing various amounts of phytoestrogens, on the gene expression profile of any tissue has to be further addressed.
BPA has been classified as a weak estrogen. It has been demonstrated that this chemical is uterotrophic at very high doses (400–800 mg/kg) in the immature AP rat model (Ashby and Tinwell, 1998); however, pre- and postnatal exposure of rats to lower dosages of BPA (3.2, 32, or 320 mg/kg) did not induce any apparent adverse effects on female rat pubertal development or reproductive functions (Kwon et al., 2000). Our results clearly indicate that BPA has estrogen-like actions at the gene expression level, but only at the medium- to high-dose ranges (50 to 400 mg/kg; Tables 2 vs. 4).
Contrary to expectations, there were a few gene products, notably the immediate early genes, c-fos and c-jun, that were not upregulated by estrogen exposure in our studies. This may be the result of our treatment regimen, which involves daily dosing for 10 days. Those early genes (c-fos, c-jun, and others) respond to estrogen stimulation within minutes to hours (Bigsby and Li, 1994; Hyder et al., 1999). Some other genes, known to be estrogen-responsive, are not represented in the oligonucleotide microarray used in the present studies, such as lactoferrin (Shigeta et al., 1996; Teng et al., 1986; Ward et al., 1999). Alternatively, these and other genes may not be affected by the treatment at this developmental stage. We tried to address the issue of not seeing the induction of immediate early genes such as c-fos and c-jun by evaluating the gene expression profile induced by EE exposure, from GD 19 to GD 20 only, and obtaining fetal tissues 2 h after the final dose (data not shown). We could not detect changes in the expression of those early genes. The 2 most compelling explanations are that (1) the genes are fully expressed at this developmental stage, since both c-fos and c-jun are equally expressed in control and treated samples, and/or their transcript levels have reached a steady-state level by the time we isolate the RNA; or (2) the genes simply are not estrogen-sensitive in the prenatal uterus. Whatever the reason, the lack of response of c-fos and c-jun, or other genes to treatment, does not diminish our ability to distinguish a unique signature for estrogenic compounds under our experimental conditions.
The gene expression profile induced by estrogen exposure identified in the present study by no means should be considered a complete list of genes whose expression can be regulated by estrogenic compounds, given that not all gene transcripts of the rat genome are represented in the microarray and the stringent criteria (p < 0.0001 in the statistical test used for analysis) used in the selection of a transcript profile. Nevertheless, the transcripts identified in this study may be used to recognize a subset of marker genes to develop a reliable screening assay to investigate the estrogenicity of different chemicals. Among the genes regulated by the 3 estrogenic compounds are genes whose products are involved in cell growth (ILGF-1, GHR, GPF, bFGF, NTAK or neural- and thymus-derived activator for the ErbB kinase, etc.), differentiation (PrgR, retinol-binding protein, NRP-tyrosine kinase, dermo 1, etc.), stress response (GADD45, or growth arrest and DNA damage inducible gene 45, glutathione S-transferase M5, non-neuronal enolase, etc.), and apoptosis (ILGF, ILGF-binding protein, FGF, FSH-regulated protein, IL4 receptor, etc.). Thus, this approach also offers the possibility to identify the molecular mechanisms involved in the action of natural and synthetic estrogenic compounds, providing information on interrelationships among the responsive genes. At the same time, key molecules and pathways involved in estrogenic response are identified.
Some of the gene-expression changes induced by estrogens identified in these studies were validated by real-time QRT-PCR. The expression of InCaBP, PrgR, vascular -actin (VaACTIN), 11ß-HSD, and the EST-AA924772 mRNAs, based on QRT-PCR analysis, followed essentially the same expression profile induced by the different doses of EE, BPA, and genistein as determined by microarray analysis. Those changes and others are consistent with reported observations described in the literature. These include the induction of intestinal calcium-binding protein, also known as vitamin D-dependent calcium-binding protein (L`Horset et al, 1990; Krisinger et al., 1992); progesterone receptor (Diel et al, 2000; Kraus et al., 1993); 11ß-HSD (Eyre et al., 2001); interleukin-4 receptor (Rivera-Gonzalez et al, 1998); and insulin-like growth factor I (Sahlin and Eriksson, 1996; Sahlin et al., 2000); and the repression of retinol-binding protein (Bucco et al., 1996; Eberhardt et al., 1999; Funkenstein, 2001) and glutathione S-transferase (Waters et al, 2001). The upregulation of the uterus/ovary-specific putative transmembrane protein mRNA can be correlated with estrogen exposure, since the original clone was derived from estrogen-induced rat uterus (Huynh, T. H. and Fotouhi-Ardakani, N., unpublished), has a very high homology to the novel estrogen-regulated gene (ERG1; Chen et al., 1999), and was consistently upregulated by the 3 estrogens tested in the present study. Although the influence of estrogen on the uterine expression of the VLDL receptor gene has not been reported, the expression of this gene in the heart is stimulated by estradiol (Masuzaki et al., 1994). In agreement with our data, Williams and Boots (1980) determined that, in the rat uterus, the aspartate aminotransferase activity per mg of tissue and total activity of this enzyme increases by ethynyl estradiol exposure. Although the identity of the ESTs responsive to estrogens is unknown, AA859581 has high homology to a glucocorticoid-inducible gene (Kaplan et al., 1999). The estrogen influence on the expression of FSH-regulated protein has not been studied. However, the sequence of this transcript has a very high homology to a group of transcription factors termed Kruppel proteins. One of these proteins, the erythroid Kruppel-like factor (EKLF), a transcription factor of the C2H2 zinc-finger class that is essential for definitive erythropoiesis, is induced by estrogens (Coghill et al., 2001). It is possible that the FSH-regulated protein gene encodes a member of the Kruppel family of transcription factors expressed in the reproductive tissues; however, this speculation has to be tested.
We conclude that prenatal estrogenic exposure alters the fetal gene-expression pattern of the rat reproductive system (uterus/ovaries), resulting in a characteristic molecular fingerprint. These findings suggest that the evaluation of the transcript profile of these tissues could be a valuable approach to determining the estrogenicity of different compounds.
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ACKNOWLEDGMENTS |
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The authors wish to thank Drs. Marilyn Aardema, Frank Gerberick, and Robert LeBoeuf for their helpful discussions.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (513) 627-0323. E-mail: naciff.jm{at}pg.com.
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REFERENCES |
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Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987). Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262, 5592–5595.
Arora, A., Nair, M. G., and Strasburg, G. M. (1998). Antioxidant activities of isoflavones and their biological metabolites in liposomal system. Arch. Biochem. Biophys. 356, 133–141.[Web of Science][Medline]
Ashby, J., and Tinwell, H. (1998). Uterotrophic activity of bisphenol A in the immature rat. Environ. Health Perspect. 106, 719–720.[Web of Science][Medline]
Bigsby, R, Chapin, R. E., Daston, G. P., Davis, B. J., Gorski, J., Gray, L. E., Howdeshell, K. L., Zoeller, R. T., and vom Saal, F. S. (1999). Evaluating the effects of endocrine disruptors on endocrine function during development. Environ. Health Perspect. 107(Suppl. 4), 613–618.
Bigsby, R. M., and Li, A. (1994). Differentially regulated immediate early genes in the rat uterus. Endocrinology 134, 1820–1826.
Bowerman, W. W., Giesy, J. P., Best, D. A., and Kramer, V. J. (1995). A review of factors affecting productivity of bald eagles in the Great Lakes region: Implications for recovery. Environ. Health Perspect. 103(Supp. 4), 51–59.
Branham, W. S., Zehr, D. R., Chen, J. J., and Sheehan, D. M. (1988). Alterations in developing rat uterine cell populations after neonatal exposure to estrogens and antiestrogens. Teratology 38, 271–279.[Web of Science][Medline]
Bucco, R. A., Zheng, W. L., Wardlaw, S. A., Davis, J. T., Sierra-Rivera, E., Osteen, K. G., Melner, M. H., Kakkad, B. P., and Ong, D. E. (1996). Regulation and localization of cellular retinol-binding protein, retinol-binding protein, cellular retinoic acid-binding protein (CRABP), and CRABP II in the uterus of the pseudopregnant rat. Endocrinology 137, 3111–3122.[Abstract]
Chang, H. C., Churchwell, M. I., Delclos, K. B., Newbold, R. R., and Doerge, D. R. (2000). Mass spectrometric determination of genistein tissue distribution in diet-exposed Sprague-Dawley rats. J. Nutr. 130, 1963–1970.
Chen, D., Xu, X., Zhu, L. J., Angervo, M., Li, Q., Bagchi, M. K., and Bagchi, I. C. (1999). Cloning and uterus/oviduct-specific expression of a novel estrogen-regulated gene (ERG1). J. Biol. Chem. 274, 32215–32224.
The Chipping Forecast (1999). (Review). Nature Genetics 21(Suppl. 1), 1–60.
Coghill, E., Eccleston, S., Fox, V., Cerruti, L., Brown, C., Cunningham, J., Jane, S., and Perkins, A. (2001). Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice. Blood 97, 1861–1868.
Cooper, R. L., and Kavlock, R. J. (1997). Endocrine disruptors and reproductive development: A weight-of-evidence overview. J. Endocrinol 152, 159–166.
Couse, J. F., Lindzey, J., Grandien, K., Gustafsson, J. A., and Korach, K. S. (1997). Tissue distribution and quantitative analysis of estrogen receptor-alpha (ER-) and estrogen receptor-beta (ER-ß) messenger ribonucleic acid in the wild type and ER- knockout mouse. Endocrinology 138, 4613–4621.
Dean, D. M., Kanemitsu, M., and Boynton, A. L. (1989). Effects of the tyrosine-kinase inhibitor genistein on DNA synthesis and phospholipid-derived second messenger generation in mouse 10T1/2 fibroblasts and rat liver T51B cells. Biochem. Biophys. Res. Commun. 165, 795–801.[Web of Science][Medline]
Diel, P., Schulz, T., Smolnikar, K., Strunck, E., Vollmer, G., and Michna, H. (2000). Ability of xeno- and phytoestrogens to modulate expression of estrogen-sensitive genes in rat uterus: Estrogenicity profiles and uterotropic activity. J. Steroid. Biochem. Mol. Biol. 73, 1–10.[Web of Science][Medline]
Duarte, J., Ocete, M. A., Perez-Vizcaino, F., Zarazuelo, A., and Tamargo, J. (1987). Effect of tyrosine kinase and tyrosine phosphatase inhibitors on aortic contraction and induction of nitric oxide synthase. Eur. J. Pharmacol. 338, 25–33.
Eberhardt, D. M., Jacobs, W. G., and Godkin, J. D. (1999). Steroid regulation of retinol-binding protein in the ovine oviduct. Biol. Reprod. 60, 714–720.
Eyre, L. J, Rabbitt, E. H., Bland, R., Hughes, S. V., Cooper, M. S., Sheppard, M. C., Stewart, P. M., and Hewison, M. (2001). Expression of 11ß-hydroxysteroid dehydrogenase in rat osteoblastic cells: Pre-receptor regulation of glucocorticoid responses in bone. J. Cell. Biochem. 81, 453–462.[Web of Science][Medline]
Farr, F., and Dunn, R. T., II (1999). Concise review: Gene expression applied to toxicology. Toxicol. Sci. 50, 1–9.
Funkenstein, B. (2001). Developmental expression, tissue distribution, and hormonal regulation of fish (Sparus aurata) serum retinol-binding protein. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 129, 613–622.[Medline]
Hall, J. M., Couse, J. F., and Korach, K. S. (2001). The multifaceted mechanisms of estradiol and estrogen receptor signaling. J. Biol. Chem. 276, 36869–36872.
Kaplan, F., Ledoux, P., Kassamali, F. Q., Gagnon, S., Post, M., Koehler, D., Deimling, J., and Sweezey, N. B. (1999). A novel developmentally regulated gene in lung mesenchyme: Homology to a tumor-derived trypsin inhibitor. Am. J. Physiol. 276, L1027–L1036.
Katzenellenbogen, B. S., Choi, I., Delage-Mourroux, R., Ediger, T. R., Martini, P. G., Montano, M., Sun, J., Weis, K., and Katzenellenbogen, J. A. (2000). Molecular mechanisms of estrogen action: Selective ligands and receptor pharmacology. J. Steroid Biochem. Mol. Biol. 74, 279–285.[Web of Science][Medline]
Kaufman, R. H., and Adam, E. (2002). Findings in female offspring of women exposed in utero to diethylstilbestrol. Obstet. Gynecol. 99, 197–200.[Web of Science][Medline]
Klinge, C. M. (2001). Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res. 29, 2905–2919.
Kraus, W. L., Montano, M. M., and Katzenellenbogen, B. S. (1993). Cloning of the rat progesterone receptor gene 5`-region and identification of two functionally distinct promoters. Mol. Endocrinol. 7, 1603–1616.
Krisinger, J., Dann, J. L., Currie, W. D., Jeung, E. B., Leung, P. C. (1992). Calbindin-D9k mRNA is tightly regulated during the estrous cycle in the rat uterus. Mol. Cell. Endocrinol. 86, 119–123.[Web of Science][Medline]
Kwon, S., Stedman, D. B., Elswick, B. A., Cattley, R. C., and Welsch, F. (2000). Pubertal development and reproductive functions of Crl:CD BR Sprague-Dawley rats exposed to bisphenol A during prenatal and postnatal development. Toxicol. Sci. 55, 399–406.
L'Horset, F., Perret, C., Brehier, A., and Thomasset, M. (1990). 17 ß-Estradiol stimulates the calbindin-D9k (CaBP9k) gene expression at the transcriptional and posttranscriptional levels in the rat uterus. Endocrinology 127, 2891–2897.
Lockhart, D. J., Dong, H., Byrne, M. C., Follettie, M. T., Gallo, M. V., Chee, M. S., Mittmann, M., Wang, C., Kobayashi, M., Horton, H., and Brown, E. L. (1996). Expression monitoring by hybrydization to high-density oligonucleotide arrays. Nat. Biotech. 14, 1675–1680.[Web of Science][Medline]
Masuzaki, H., Jingami, H., Yamamoto, T., and Nakao, K. (1994). Effects of estradiol on very low-density lipoprotein receptor mRNA levels in rabbit heart. FEBS Lett. 347, 211–214.[Web of Science][Medline]
McLachlan, J. A., and Newbold, R. R. (1987). Estrogens and development. Environ. Health Perspect. 75, 25–27.[Web of Science][Medline]
Morrison, T. B., Weis, J. J., and Wittwer, C. T. (1998). Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24, 954–962.[Web of Science][Medline]
Muramatsu, M, and Inoue, S. (2000). Estrogen receptors: How do they control reproductive and nonreproductive functions? Biochem. Biophys. Res. Commun. 270, 1–10.[Web of Science][Medline]
Newbold, R. R. (1995). Cellular and molecular effects of developmental exposure to diethylstilbestrol: Implications for other environmental estrogens. Environ. Health Perspect. 103(Suppl. 7), 83–87.
Newbold, R. R., Tyrey, S., Haney, A. F., and McLachlan, J. A. (1983). Developmentally arrested oviduct: A structural and functional defect in mice following prenatal exposure to diethylstilbestrol. Teratology 27, 417–426.[Web of Science][Medline]
Nguyen, B. L., Hatier, R., Jeanvoine, G., Roux, M., Grignon, G., and Pasqualini, J. R. (1988). Effect of estradiol on the progesterone receptor and on morphological ultrastructures in the fetal and newborn uterus and ovary of the rat. Acta Endocrinol. (Copenhagen) 117, 249–259.
Nilsson, S., Makela, S., Treuter, E., Tujague, M., Thomsen, J., Andersson, G., Enmark, E., Pettersson, K., Warner, M., and Gustafsson, J. A. (2001). Mechanisms of estrogen action. Physiol. Rev. 81, 1535–1565.
Nuwaysir, E. F., Bittner, M., Trent, J., Barrett, J. C., and Afshari, C. A. (1999). Microarrays and toxicology: The advent of toxicogenomics. Mol. Carcinog. 24, 153–159.[Web of Science][Medline]
Okura, A., Arakawa, H., Oka, H., Yoshinari, T., and Monden, Y. (1988). Effect of genistein on topoisomerase activity and on the growth of [Val 12]Ha-ras-transformed NIH 3T3 cells. Biochem. Biophys. Res. Commun. 157, 183–189.[Web of Science][Medline]
Ozawa, S., Iguchi, T., Sawada, K., Ohta, Y., Takasugi, N., and Bern, H. A. (1991). Postnatal vaginal nodules induced by prenatal diethylstilbestrol treatment correlate with later development of ovary-independent vaginal and uterine changes in mice. Cancer Lett. 58, 167–175.[Web of Science][Medline]
Pennie, W. D., Tugwood, J. D., Oliver, G. J., and Kimber, I. (2000). The principles and practice of toxicogenomics: Applications and opportunities. Toxicol. Sci. 54, 277–283.
Pennie, W. D., Woodyatt, N. J., Aldridge, T. C., and Orphanides, G. (2001). Application of genomics to the definition of the molecular basis for toxicity. Toxicol. Lett. 120, 353–358.[Web of Science][Medline]
Portelli, M. J., de Solla, S. R., Brooks, R. J., and Bishop, C. A. (1999). Effect of dichlorodiphenyltrichloroethane on sex determination of the common snapping turtle (Chelydra serpentina serpentina). Ecotoxicol. Environ. Saf. 43, 284–291.[Web of Science][Medline]
Potier, B., and Rovira, C. (1999). Protein tyrosine kinase inhibitors reduce high-voltage activating calcium currents in CA1 pyramidal neurons from rat hippocampal slices. Brain Res. 23, 587–597.
Power, R. F., Conneely, O. M., and O'Malley, B. W. (1992). New insights into activation of the steroid hormone receptor superfamily. Trends Pharmacol. Sci. 13, 318–323.[Medline]
Rajeevan, M. S., Vernon, S. D., Taysavang, N., and Unger, E. R. (2001). Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR. J. Mol. Diagn. 3, 26–31.
Rivera-Gonzalez, R., Petersen, D. N., Tkalcevic, G., Thompson, D. D., and Brown, T. A. (1998). Estrogen-induced genes in the uterus of ovariectomized rats and their regulation by droloxifene and tamoxifen. J. Steroid. Biochem. Mol. Biol. 64, 13–24.[Web of Science][Medline]
Rodi, C. P., Bunch, R. T., Curtiss, S. W., Kier, L. D., Cabonce, M. A., Davila, J. C., Mitchell, M. D., Alden, C. L., and Morris, D. L. (1999). Revolution through genomics in investigative and discovery toxicology. Toxicol. Pathol. 27, 107–110.
Rosenfeld, M. G., and Glass, C. K. (2001). Coregulator codes of transcriptional regulation by nuclear receptors. J. Biol. Chem. 276, 36865–36868.
Rothschild, T. C., Calhoon, R. E., and Boylan, E. S. (1987). Genital tract abnormalities in female rats exposed to diethylstilbestrol in utero. Reprod. Toxicol. 1, 193–202.[Medline]
Sahlin, L., Elger, W., Akerberg, S., Masironi, B., Reddersen, G., Schneider, B., Schwarz, S., Freyschuss, B., and Eriksson, H. (2000). Effects of estradiol and estradiol sulfamate on the uterus of ovariectomized or ovariectomized and hypophysectomized rats. J. Steroid. Biochem. Mol. Biol. 74, 99–107.[Web of Science][Medline]
Sahlin, L., and Eriksson, H. (1996). Influence of blockers for the estrogen receptor (ER) and type 1 IGF-receptor on the levels of ER, ER mRNA, and IGF-I mRNA in the rat uterus. J. Steroid. Biochem. Mol. Biol. 58, 359–365.[Web of Science][Medline]
Sheehan, D. M., Branham, W. S., Medlock, K. L., Olson, M. E., and Zehr, D. R. (1981). Uterine responses to estradiol in the neonatal rat. Endocrinology 109, 76–82.
Shigeta, H., Newbold, R. R., McLachlan, J. A., and Teng, C. (1996). Estrogenic effect on the expression of estrogen receptor, COUP-TF, and lactoferrin mRNA in developing mouse tissues. Mol. Reprod. Dev. 45, 21–30.[Web of Science][Medline]
Sparling, D. W., Fellers, G. M., and McConnell, L. L. (2001). Pesticides and amphibian population declines in California, USA. Environ. Toxicol. Chem. 20, 1591–1595.[Web of Science][Medline]
Steiner, S., and Anderson, N. L. (2000). Expression profiling in toxicology—potentials and limitations. Toxicol. Lett. 112–113, 467–471.
Swan, S. H. (2000). Intrauterine exposure to diethylstilbestrol: Long-term effects in humans. APMIS 108, 793–804.[Web of Science][Medline]
Teng, C. T., Walker, M. P., Bhattacharyya, S. N., Klapper, D. G., DiAugustine, R. P., and McLachlan, J. A. (1986). Purification and properties of an oestrogen-stimulated mouse uterine glycoprotein (approx. 70 kDa). Biochem. J. 240, 413–422.[Web of Science][Medline]
Thigpen, J. E., Setchell, K. D., Ahlmark, K. B., Locklear, J., Spahr, T., Caviness, G. F., Goelz, M. F., Haseman, J. K., Newbold, R. R., and Forsythe, D. B. (1999). Phytoestrogen content of purified, open- and closed-formula laboratory animal diets. Lab. Anim. Sci. 49, 530–536.[Web of Science][Medline]
U.S. EPA (1998). U.S. Environmental Protection Agency, Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), Final Report. Accessible at http://www.epa.gov/scipoly/oscpendo/history/finalrpt.htm. Accessed August, 1998.
Vos, J. G., Dybing, E., Greim, H. A., Ladefoged, O., Lambre, C., Tarazona, J. V., Brandt, I., and Vethaak, A. D. (2000). Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation. Crit. Rev. Toxicol. 30, 71–133.[Web of Science][Medline]
Ward, P. P., Mendoza-Meneses, M., Mulac-Jericevic, B., Cunningham, G. A., Saucedo-Cardenas, O., Teng, C. T., and Conneely, O. M. (1999). Restricted spatiotemporal expression of lactoferrin during murine embryonic development. Endocrinology 140, 1852–1860.
Waters, K. M., Safe, S., and Gaido, K. W. (2001). Differential gene expression in response to methoxychlor and estradiol through ER, ERß, and AR in reproductive tissues of female mice. Toxicol. Sci. 63, 47–56.
Williams, S. A., and Boots, L. R. (1980). Uterine and kidney aspartate aminotransferase activity in ethinylestradiol- and norgestrel-treated rats. Contraception 21, 659–663.[Web of Science][Medline]
Xu, J., Liao, L., Ning, G, Yoshida-Komiya, H., Deng, C., and O'Malley, B. W. (2000). The steroid receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is required for normal growth, puberty, female reproductive function, and mammary gland development. Proc. Natl. Acad. Sci. U.S.A. 97, 6379–6384.
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 R. Dip, S. Lenz, J.-P. Antignac, B. Le Bizec, H. Gmuender, and H. Naegeli Global gene expression profiles induced by phytoestrogens in human breast cancer cells Endocr. Relat. Cancer, March 1, 2008; 15(1): 161 - 173. [Abstract] [Full Text] [PDF]
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J. M. Naciff, G. J. Overmann, S. M. Torontali, G. J. Carr, Z. S. Khambatta, J. P. Tiesman, B. D. Richardson, and G. P. Daston Uterine Temporal Response to Acute Exposure to 17{alpha}-Ethinyl Estradiol in the Immature Rat Toxicol. Sci., June 1, 2007; 97(2): 467 - 490. [Abstract] [Full Text] [PDF]
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 J. W. Erdman Jr., D. Balentine, L. Arab, G. Beecher, J. T. Dwyer, J. Folts, J. Harnly, P. Hollman, C. L. Keen, G. Mazza, et al. Flavonoids and Heart Health: Proceedings of the ILSI North America Flavonoids Workshop, May 31-June 1, 2005, Washington, DC J. Nutr., March 1, 2007; 137(3): 718S - 737S. [Abstract] [Full Text] [PDF]
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 G. E. Akbas, X. Fei, and H. S. Taylor Regulation of HOXA10 expression by phytoestrogens Am J Physiol Endocrinol Metab, February 1, 2007; 292(2): E435 - E442. [Abstract] [Full Text] [PDF]
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A. D. Benninghoff Toxicoproteomics--The Next Step in the Evolution of Environmental Biomarkers? Toxicol. Sci., January 1, 2007; 95(1): 1 - 4. [Full Text] [PDF]
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 J.A. Horcajadas, A. Pellicer, and C. Simon Wide genomic analysis of human endometrial receptivity: new times, new opportunities Hum. Reprod. Update, January 1, 2007; 13(1): 77 - 86. [Abstract] [Full Text] [PDF]
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L. N. Moens, K. van der Ven, P. Van Remortel, J. Del-Favero, and W. M. De Coen Expression Profiling of Endocrine-Disrupting Compounds Using a Customized Cyprinus carpio cDNA Microarray Toxicol. Sci., October 1, 2006; 93(2): 298 - 310. [Abstract] [Full Text] [PDF]
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 M. Messina, W. McCaskill-Stevens, and J. W. Lampe Addressing the soy and breast cancer relationship: review, commentary, and workshop proceedings. J Natl Cancer Inst, September 20, 2006; 98(18): 1275 - 1284. [Abstract] [Full Text] [PDF]
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 T. Buterin, C. Koch, and H. Naegeli Convergent transcriptional profiles induced by endogenous estrogen and distinct xenoestrogens in breast cancer cells Carcinogenesis, August 1, 2006; 27(8): 1567 - 1578. [Abstract] [Full Text] [PDF]
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J. M. Naciff, K. A. Hess, G. J. Overmann, S. M. Torontali, G. J. Carr, J. P. Tiesman, L. M. Foertsch, B. D. Richardson, J. E. Martinez, and G. P. Daston Gene Expression Changes Induced in the Testis by Transplacental Exposure to High and Low Doses of 17{alpha}-Ethynyl Estradiol, Genistein, or Bisphenol A Toxicol. Sci., August 1, 2005; 86(2): 396 - 416. [Abstract] [Full Text] [PDF]
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 C. A White and L. A Salamonsen A guide to issues in microarray analysis: application to endometrial biology Reproduction, July 1, 2005; 130(1): 1 - 13. [Abstract] [Full Text] [PDF]
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G.-S. Lee, H.-J. Kim, Y.-W. Jung, K.-C. Choi, and E.-B. Jeung Estrogen Receptor {alpha} Pathway Is Involved in the Regulation of Calbindin-D9k in the Uterus of Immature Rats Toxicol. Sci., April 1, 2005; 84(2): 270 - 277. [Abstract] [Full Text] [PDF]
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G.-S. Lee, K.-C. Choi, H.-J. Kim, and E.-B. Jeung Effect of Genistein As a Selective Estrogen Receptor Beta Agonist on the Expression of Calbindin-D9k in the Uterus of Immature Rats Toxicol. Sci., December 1, 2004; 82(2): 451 - 457. [Abstract] [Full Text] [PDF]
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J. Ashby and J. Odum Gene Expression Changes in the Immature Rat Uterus: Effects of Uterotrophic and Sub-Uterotrophic Doses of Bisphenol A Toxicol. Sci., December 1, 2004; 82(2): 458 - 467. [Abstract] [Full Text] [PDF]
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M. Suzuki, H. Kobayashi, Y. Tanaka, N. Kanayama, and T. Terao Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) - ITI target genes in mouse ovary identified by microarray analysis J. Endocrinol., October 1, 2004; 183(1): 29 - 38. [Abstract] [Full Text] [PDF]
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S. K. Dey, H. Lim, S. K. Das, J. Reese, B. C. Paria, T. Daikoku, and H. Wang Molecular Cues to Implantation Endocr. Rev., June 1, 2004; 25(3): 341 - 373. [Abstract] [Full Text] [PDF]
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S. Barnes Soy Isoflavones--Phytoestrogens and What Else? J. Nutr., May 1, 2004; 134(5): 1225S - 1228S. [Abstract] [Full Text] [PDF]
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 J. M. Naciff and G. P. Daston Toxicogenomic Approach to Endocrine Disrupters: Identification of a Transcript Profile Characteristic of Chemicals with Estrogenic Activity Toxicol Pathol, February 1, 2004; 32(2_suppl): 59 - 70. [Abstract] [PDF]
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H. Ellinger-Ziegelbauer, B. Stuart, B. Wahle, W. Bomann, and H.-J. Ahr Characteristic Expression Profiles Induced by Genotoxic Carcinogens in Rat Liver Toxicol. Sci., January 1, 2004; 77(1): 19 - 34. [Abstract] [Full Text] [PDF]
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 K. C. Fertuck, J. E. Eckel, C. Gennings, and T. R. Zacharewski Identification of temporal patterns of gene expression in the uteri of immature, ovariectomized mice following exposure to ethynylestradiol Physiol Genomics, October 17, 2003; 15(2): 127 - 141. [Abstract] [Full Text] [PDF]
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 S. C. Hewitt, B. J. Deroo, K. Hansen, J. Collins, S. Grissom, C. A. Afshari, and K. S. Korach Estrogen Receptor-Dependent Genomic Responses in the Uterus Mirror the Biphasic Physiological Response to Estrogen Mol. Endocrinol., October 1, 2003; 17(10): 2070 - 2083. [Abstract] [Full Text] [PDF]
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 X. Wu, S.-T. Pang, L. Sahlin, A. Blanck, G. Norstedt, and A. Flores-Morales Gene Expression Profiling of the Effects of Castration and Estrogen Treatment in the Rat Uterus Biol Reprod, October 1, 2003; 69(4): 1308 - 1317. [Abstract] [Full Text] [PDF]
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G. P. Daston, J. C. Cook, and R. J. Kavlock Uncertainties for Endocrine Disrupters: Our View on Progress Toxicol. Sci., August 1, 2003; 74(2): 245 - 252. [Abstract] [Full Text] [PDF]
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M. Belenky, J. Prasain, H. Kim, and S. Barnes DING, a Genistein Target in Human Breast Cancer: A Protein Without a Gene J. Nutr., July 1, 2003; 133(7): 2497S - 2501. [Abstract] [Full Text] [PDF]
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J. M. Naciff, G. J. Overmann, S. M. Torontali, G. J. Carr, J. P. Tiesman, B. D. Richardson, and G. P. Daston Gene Expression Profile Induced by 17{alpha}-Ethynyl Estradiol in the Prepubertal Female Reproductive System of the Rat Toxicol. Sci., April 1, 2003; 72(2): 314 - 330. [Abstract] [Full Text] [PDF]
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