Impact of the phytoestrogen content of laboratory animal feed on the gene expression profile of the reproductive system in the immature female rat.
The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. In fact, soy and alfalfa are commonly used as protein sources in the manufacture of most rodent diets. Some of these ingredients are kmown to contain endocrine modulators, such as the phytoestrogens genistein and daidzein (abundant in soybeans and its products) and their respective glycosides (genistin and daidzin), and coumestrol (found in alfalfa). These phytoestrogens are able to bind to both estrogen receptor (ER) isoforms, ER-[alpha] and ER-[beta], in vitro (Beck et al. 2003; Casanova et al. 1999). They have a higher affinity for ER-[beta] (Boue et al. 2003), but they activate both ER isoforms, although with less potency than estradiol. Both genistein and daidzein have much weaker affinities than does 17[beta]3-estradiol for the rat ERs: genistein binds 3- and 100fold weaker, and daidzein binds 60- and 1,000-fold weaker to rat ER-[beta] and ER-[alpha], respectively (Boue et al. 2003; Casanova et al. 1999). These two phytoestrogens are able to elicit estrogenic responses in vivo (Boettger-Tong et al. 1998; Brown and Setchell 2001; Degen et al. 2002; Jefferson et al. 2002; Levy et al. 1995; Odum et al. 2001; Thigpen et al. 1997). The selective interaction of phytoestrogens with human ER-[alpha] and ER-[beta] is similar in vitro to that described for the rat (Casanova et al. 1999; Kuiper et al. 1998).
Genistein is also known to have other activities, such as inhibition of different enzymes, among them tyrosine kinases (Akiyama et al. 1987), nitric oxide synthase (Duarte et al. 1997), and topoisomerase II (Okura et al. 1988), and decreasing calcium-channel activity in neurons (Potier and Rovira 1999). It also decreases lipid peroxidation (Arora et al. 1998) and diacylglycerol synthesis (Dean et al. 1989). Therefore, the multiple biologic activities of phytoestrogens raise the question of whether they have the potential to influence the outcome and/or interpretation of bioassays used to identify chemicals with estrogenic potential. In particular, questions have been raised about the presence of phytoestrogens in diets fed to animals used in bioassays designed to screen chemicals that may act as weak regulators of ERs and to screen low doses of potent regulators of ERs (Thigpen et al. 1997, 2002). One such bioassay is the uterotrophic and antagonists.
By using a version of the uterotrophic assay in the immature rat, one of the tier I screening assays recommended for detecting the estrogenic properties of endocrine-disrupting chemicals [Organisation for Economic Co-operation and Development (OECD) 2001; U.S. Environmental Protection Agency (U.S. EPA) 1998], we have identified a set of genes from the uterus and ovaries of prepubertal rats for which expression is regulated by estrogen exposure in a dose-dependent manner and which have the potential to be used as biomarkers for estrogen activity (Naciff et al. 2003). Gene expression changes induced by estrogen stimulation are more sensitive than the classical end points (i.e., uterine weight increase) for evaluating estrogenicity (Naciff et al. 2003). Given that components of the rodent diet commonly used in reproductive toxicology studies include chemicals with known estrogenic activity, understanding the influence of diet and dietary components on estrogen response is an important issue, in this study, we used gene expression profiling to evaluate the effect of two diets with different phytoestrogen content on the transcript profile of two organs that are responsive to estrogen stimulation: the uterus and the ovaries of prepubertal rats.
Materials and Methods
Chemicals. 17[alpha]-Ethynyl estradiol (EE) and peanut oil were obtained from Sigma Chemical Company (St. Louis, MO).
Animals and treatments. Fifteen-day-old female Sprague-Dawley rats were obtained (Charles River VAF/Plus; Charles River Laboratories, Raleigh, NC) in groups of 10 pups per surrogate mother. We chose this rat strain because it is commonly used in reproductive and developmental toxicity studies. The rats were acclimated to the local vivarium conditions (24[degrees]C; 12-hr light/12-hr dark cycle) for 5 days and were fed a casein-based diet (soy- and alfalfa-free diet; Purina 5K96, Purina Mills, St. Louis, MO). Starting on postnatal day (PND)20 and during the experimental phase of the protocol, all rats were singly housed in 20 x 32 x 20 cm plastic cages. To test the diet effect, there were two animal groups (n = 20): one group was fed a standard laboratory rodent diet (Purina 5001, Purina Mills), and the other group was maintained on the casein-based diet: The Purina 5001 diet contains phytoestrogens, mostly genistein and daidzein derived from soy and alfalfa, at levels that may have an impact on the gene expression profile (total daidzein + genistein = 0.49 mg/g; Thigpen et al. 1999), particularly in tissues regulated by estrogens such as reproductive tissues. However, those levels are not uterotrophic when evaluated by the traditional end points, uterine weight gain and increase in luminal epithelial cell height. The casein-based diet is essentially phytoestrogen free, consistently containing < 1 ppm aglycone equivalents of genistein, daidzein, and glycitein, and was fed to the four groups of animals from PND16 onward in order to remove any possible effects of the regular rodent diet (Purina 5001) previously fed to the rats by the animal supplier. All the animals were allowed free access to water and specific pelleted commercial diet (Purina 5001 or casein-based 5K96). 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 (Institute of Laboratory Animal Resources 1996).
Starting on PND20, each diet group was divided into two subgroups of 10 animals. One subgroup from each diet subgroup was dosed by subcutaneous injection with 0.1 [micro]g/kg/day EE in peanut oil. This dose is not sufficient to induce a uterotrophic response in juvenile rats (Kanno et al. 2002; Naciff et al. 2003). Animals received 5 mL/kg body weight of dose solution once a day for 4 days. A 4-day dosing regime was selected to optimize detection of any effect of EE exposure at this low dose, both at the histologic level and at the gene expression level. The dose was administered between 0800 and 0900 hr each day. Controls, fed with the appropriate diet, received 5 mL/kg of peanut oil once a day for 4 days. Doses were administered on a microgram per kilogram body weight basis and adjusted daily for weight changes. Body weight (nearest 1.0 g) and the volume of the dose administered (nearest 0.1 mL) were recorded daily. The exact time of the last dose was recorded, to establish a 24-hr waiting period before tissue collection. The animals were sacrificed by C[O.sub.2] asphyxiation 24 hr after the last dosing, on PND24. The body of the uterus, cut just above its junction with the cervix, with the ovaries attached, was carefully dissected free of adhering fat and mesentery and was weighed as a whole. Then, the ovaries were dissected free, and the uterine and ovarian wet weight was recorded. Both the uterus and ovaries were placed into RNAlater (50-100 mg/mL of solution; Ambion, Austin, TX) at room temperature.
Histology. Reproductive tissues from two animals in each dose group were fixed in 10% neutral buffered formalin immediately after weighing and then dehydrated and embedded in paraffin. Serial 4-5 [micro]m cross sections were made through the ovaries, oviducts, and uterine horns, which were stained with hematoxylin and eosin. The evaluation of the morphologic changes induced by the two different diets with or without EE exposure in the uterus was performed as described previously (Naciff et al. 2003).
Expression profiling. We used 10 [micro]g total RNA, extracted from uterus and ovaries from individual animals (combining only the tissues from the same animal), to prepare biotin-labeled cRNA, as previously described (Naciff et al. 2002, 2003). Labeled cRNA samples were hybridized to the Affymetrix GeneChip Test 3 Array (Affymetrix Inc., Santa Clara, CA) to assess the overall quality of each sample. After determining the target cRNA quality, we selected individual samples of pooled uteri/ovaries from five or six individual females (replicates) from each diet group, from controls, and from EE-treated subgroups (with high quality cRNA) and hybridized them to Affymetrix Rat Genome U34A high-density oligonucleotide microarrays for 16 hr. The microarrays were washed and stained by streptavidin-phycoerythrin to detect bound cRNA. The signal intensity was amplified by 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 (Affymetrix Inc.). Affymetrix image files for the 20 chip hybridizations, and the absolute analysis results of each diet group are available from the authors upon request.
Real-time reverse transcriptase-polymerase chain reaction. In order to corroborate the changes in gene expression identified by the oligonucleotide microarrays, we used a real-time (kinetic) quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) approach, as previously described (Naciff et al. 2002). This approach allowed us to evaluate the "basal level" of expression of individual genes in samples derived from animals exposed to the two different diets used in our study, as well as changes induced by low-dose EE exposure (0.1 [micro]g/kg/day), We compared the transcript level of selected genes in samples derived from animals in all experimental groups. 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 tetraacetic acid 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 done with each primer pair to determine the overall quality and specificity of the primer design. After QRT-PCR, we observed only the expected products at the correct molecular weight.
Data analysis. We addressed potential interindividual variability by using independent samples of each experimental group (n = 5 for each set) for analysis. For the uterine/ovarian weight determination, the luminal epithelial cell height, and the gene expression analysis, we compared the data from the animals fed with the casein-based diet with the data from the animals fed the normal rodent diet (Purina 5001). For gene expression analysis, scanned output files of Affymetrix microarrays were visually inspected for hybridization artifacts and then analyzed using Affymetrix Microarray Suite (version 5.0) and Data Mining Tool (version 3.0) software, as described by the manufacturer (Affymetrix 2002; Lockhart et al. 1996). Arrays were scaled to an average intensity of 1,500 units and analyzed independently. The Affymetrix Rat Genome U34A microarrays used in this study have 8,740 probe sets corresponding to approximately 7,000 annotated rat genes and 1,740 expressed sequence tags (ESTs).
For each transcript in the diet and dose groups, we conducted pairwise comparisons with vehicle controls fed the casein-based diet, using two-sample t-tests: first, we compared the two diet groups, and then we compared each treatment group with its respective diet control. We then conducted analysis of variance (ANOVA) for general diet and treatment effects on the signal value (which serves as a relative indicator of the level of expression of a transcript) and the log of the signal value. General diet effects were evaluated by ANOVA and a nonparametric test for dose-response trend, the Jonkheere-Terpstra test. Genes for which any of the tests had p [less than or equal to] 0.001 was taken as evidence that the expression of those genes was modified by the diet or by EE exposure. For the combined analysis of the two sets (casein-based or Purina 5001 diet), stratified nonparametric tests were conducted that were focused in detecting genes showing a diet response, or where there was a consistent treatment effect versus vehicle for the EE-treated group (0.1 [micro]g kg/day). Here, we used linear models, with terms for both study and treatment effects, on average differences (signal values) and their log transformation, as well as stratified forms of the Wilcoxon-Mann-Whitney nonparametric statistic and a stratified form of the Jonkheere-Terpstra nonparametric statistic for diet response. Fold-change summary values for genes were calculated as a signed ratio of mean signal values (for each diet and EE-treated group compared with the appropriate control). Because fold-change values can become artificially large or undefined when mean signal values approach zero, all the values < 100 were made equal to 100 before calculating the mean signal values that are used in the fold-change calculation. All statistical analyses use the measured signal values, even if they were smaller than 100 units.
Effect of diet on uterine/ovarian and uterine wet weight and uterine luminal epithelial cell height. Both diets, Purina 5001 and casein-based 5K96, were well tolerated by all the animals. We observed no evidence of overt toxicity and no clinical signs of toxicity. No difference was determined in body weights between animals fed either diet (Table 2). We did not detect premature vaginal opening in any of the animals in either diet group or in animals exposed to EE. There were no differences in wet uterine weight or in absolute and relative uterine weight (Table 2) between the two diet groups, even when the animals were exposed to low doses of EE.
The gross anatomy of the uterus and ovaries of animals fed either diet was identical, and no signs of accumulation of fluid in the uterine lumen were noted in any of the animals. We observed no differences in uterine weight gain (wet weight) or uterine epithelial cell height (Figure 1), and we found no change in the number of uterine glands. The classical morphologic changes induced by estrogen stimulation (hypertrophy of luminal epithelial, stromal, and myometrial cells; thickening of stromal layer; and some stromal inflammatory reaction) were not observed in any of the animals exposed to the two different diets, even when exposed to 0.1 [micro]g kg/day EE (Figure 1).
[FIGURE 1 OMITTED]
Effect of diet on gene expression profile of the uterus/ovaries. In order to compare the gene expression profiles induced by the different diets (different phytoestrogen content) and the EE dose tested, we compared the average value of the signal values, a relative indicator of the level of expression of a transcript, between the two groups of independent controls. We then compared the appropriate diet-control group with the respective EE group (0.1 [micro]g/kg/day), for all the 8,740 transcripts represented on the array.
In comparing the expression profile identified in the uterus/ovaries of animals fed a casein-based diet versus the ones fed a soy/alfalfa-containing diet, we identified the expression of 29 genes that were significantly different (p [less than or equal to] 0.001). A list of those genes, along with their accession numbers, gene symbols, and the average fold changes, is shown in Table 3. The number of genes whose expression is modified by the diet's composition is relatively small, and the average fold change on the expression of these genes affected by the rodent standard diet, compared with the casein-based diet, is relatively low in the uterus and ovaries. Although robust expression differences for specific genes can be attributed to the composition of the diet, this list does not include genes well known to be estrogen regulated, such as progesterone receptor (PgR), intestinal calcium-binding protein (icabp), and complement component 3 (CC3).
One hypothesis is that if the soy/alfalfa-based diet was not estrogenic on its own, perhaps it would have sufficient potency to measurably enhance the effect of a sub-uterotrophic dose of EE. Although the expression of most genes from the prepubertal uterus/ovaries that respond to estrogen exposure is not altered by the diet composition, there are some that show a variable, nonstatistically significant response. For comparison, we calculated the relative fold change induced by diet for genes that showed a dear dose response to 1-10 [micro]g kg/day EE (Naciff et al. 2003). Presumably, those genes have the potential to represent the response to weak or low levels of estrogen stimulation (expected from the dietary phytoestrogens) and are shown in Table 4, The fold change represents the ratio of the relative expression level of each gene in tissues from animals fed Purina 5001 versus those fed the 5K96 diet (as indicated in Table 3). For comparison, in Table 4 the relative expression level of the same transcripts under EE exposure is also shown. Analyzing the effect of EE exposure on the expression of the same set of genes, comparing the relative expression level of each gene in the tissues from animals exposed to EE versus their respective controls that were fed the same diet, and taking into account that lack of statistical significance for those genes listed, the average (n = 5) response to EE exposure is very similar, if not equal, even for those showing a relative large fold change, regardless of the diet fed to the animals. These results suggest that the response to the diet's composition is independent from the EE effect at this dose level of exposure. At higher doses of EE, the contribution of the dietary phytoestrogens is considered negligible because EE is a potent ER agonist able to interact with both isoforms of this receptor and with higher affinity than any of the dietary phytoestrogens (Kuiper et al. 1997). The changes induced by exposure to higher EE doses have been reported (Naciff et al. 2003). Corroboration of the microarray results by QRT-PCR for a selected group of genes is shown in Table 5. With the exception of CC3, which is undetectable by microarray analysis of samples derived from the animals exposed to two different diets (Table 5), the expression levels of the other genes are very similar, determined by either QRT-PCR or microarray analysis.
Dietary phytoestrogens, such as genistein and daidzein (abundant in soybeans and its products) and their respective glycosides (genistin and daidzin), and coumestrol (found in alfalfa), have been found to have estrogenic properties in both in vitro and in vivo (Beck et al. 2003; Boettger-Tong et al. 1998; Boue et al. 2003; Brown and Setchell 2001; Casanova et al. 1999; Degen et al. 2002; Jefferson et al. 2002; Kanno et al. 2002; Levy et al. 1995; Odum et al. 2001; Thigpen et al. 2002). However, the results of the present study showed that phytoestrogens at concentrations present in a given lot of a commercial rodent diet are not able to elicit an estrogenic response in the reproductive system of the immature rat, judged by classical end points and specific gene expression changes characteristic of estrogen exposure in estrogen-responsive target organs (uterus and ovaries). Although a number of gene expression differences were observed with the two rodent diets tested, Purina 5001 and casein-based diet (relatively high vs. low phytoestrogen content, respectively), they cannot be correlated with estrogenic activity. These gene expression changes are more likely to be caused by nutritional differences between the diets, rather than individual dietary components affecting ER pathways. Also, the traditional end points used to assess estrogenic activity, namely, uterine wet weight gain and hypertrophy of luminal epithelial cell layer, were not affected by the phytoestrogen content of the diet. It has to be stressed that we have previously identified gene expression as being far more sensitive than the classical uterotrophic response in assessing estrogenicity (Naciff et al. 2003).
We also tested whether the consumption of phytoestrogen-containing diets was sufficient to render a subuterotrophic dose regimen of EE active. To do this, we evaluated the number and type of genes whose expression is modified in the uterus/ovaries from animals exposed to 0.1 [micro]g/kg/day EE but fed different diets. There is not a statistically different number of genes affected by components of the diet, and those genes affected by EE are the same, regardless of the diet fed to the animals. More important, the different phytoestrogen content of the diets does not modify--by either increasing or decreasing--the response of the estrogen-sensitive genes from the uterus/ovaries to low doses of a potent ER agonist; their expression changes in the same direction and magnitude as a result of EE regardless of whether the rats were fed the phytoestrogen-containing diet. Table 4 shows the transcripts that we have previously identified as being responsive to estrogen exposure, under the uterotrophic assay protocol (Naciff et al. 2003), with their relative expression level calculated by comparing the two diets. This includes genes that have an extremely robust response to estrogen exposure, such as CC3, PgR, and icabp (Heikaus et al. 2002; Krisinger et al. 1992; L'Horset et al. 1990; Li et al. 2002; Naciff et al. 2003). Thus, we are confident that, despite the potential effect of the phytoestrogens in the Purina 5001 diet, the transcript profile determined in the uterus and ovaries is comparable with the one determined in the animals fed the casein-based diet, and truly reflects the lack of estrogenic activity of the soy/alfalfa-based diet.
Our data corroborate the findings of the OECD (Owens et al. 2003), Wade et al. (2003), and Yamasaki et al. (2002) in the uterotrophic assay. As part of the studies conducted by the OECD validation initiative, it has been established that the phytoestrogen contents of the multiple rodent diets employed by the participant laboratories had no important effect on the sensitivity of the uterotrophic assay (Owens et al. 2003). In independent studies, Wade et al. (2003) and Yamasaki et al. (2002) reached the same conclusions by testing the effect of various phytoestrogen-containing diets in the outcome of their immature uterotrophic assays. Our findings also agree with reports on the effects of phytoestrogens on the reproductive system of other species. Foth and Cline (1998) reported that supplementing the diet of postmenopausal macaques with up to 148 mg of phytoestrogen (from soy) per day for 6 months failed to induce any proliferative effects on endometrial histology, a marker for estrogenic stimulation. Anthony et al. (1996) determined that dietary soybean isoflavones improve cardiovascular risk factors (plasma lipids, lipoproteins, and atherosclerosis) without detectable estrogenic effects in the reproductive system of peripubertal rhesus monkeys. The data presented here establish the fact that the phytoestrogens found in a regular Western diet (compared with traditional Asian diets), exemplified here as the standard rodent diet, do not elicit an estrogenic response at the histologic level or at the gene expression level. Thus, the potential benefits for humans derived from consuming a normal diet (not intentionally enriched with phytoestrogens) are not compromised by undesired estrogenic properties.
These findings demonstrate that the phytoestrogens present in a regular rodent diet do not affect the biologic response to a potent exogenous ER agonist, at the level of tissue architecture or gene expression, in prepubertal rat uterus and ovaries. From the results of the present study, it is clear that in order to elicit an estrogenic response at the gene expression level, the organism has to be exposed to higher concentrations of phytoestrogens, as has been shown in the developing female rat with pure genistein (Jefferson et al. 2002; Naciff et al. 2002). It must be stressed that the route of administration has an impact on the degree of the response; Ashby (2000) has shown that genistein gives a stronger uterotrophic response in the immature mouse when subcutaneously injected than when given orally at equivalent concentrations.
Some of the gene expression changes attributed to the composition of the diet, determined in the present study, may have an impact on the biologic response of the reproductive system (uterus/ovaries), mostly by influencing various pathways, some of which have an effect on sex hormone axis. However, none of these genes was included in the transcript profile determined for estrogens in the immature rat uterus and ovaries (Naciff et al. 2003). For example, rGrb14, the rat homologue of the human growth factor receptor, bound human Grb14 adaptor protein, a direct inhibitor of the activated insulin receptor (Bereziat et al. 2002; Kasus-Jacobi et al. 1998), whose up-regulation may result in modification of the response of the uterus/ovaries to insulin. Another gene whose expression is modified by the composition of the diet is that of the gonadotropin-releasing hormone receptor, which among other activities regulates gametogenic and hormonal functions of the gonads (Kang et al. 2003). The expression of insulin-like growth factor 1 (IGF-1) is up-regulated in the reproductive tissues of animals fed the diet with a relatively high phytoestrogen content (Table 3). IGF-1 is a critical regulator of uterine growth, and locally produced uterine IGF-1 could mediate the effects of estradiol on growth and cellular proliferation (Sato et al. 2002). The expression of the gene encoding steroid 3-[alpha]-dehydrogenase is also up-regulated by the soy/alfalfa-based diet. This enzyme, a member of the aldo-keto reductase gene superfamily, is an important multifunctional oxidoreductase capable of metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins (Huang and Luu-The 2000). Aquaporin 1 (AQP1) is one of the genes for which expression is down-regulated by a soy/alfalfa-based diet. This gene encodes a protein that is a member of a family of membrane channel proteins which facilitate bulk water transport and possibly other small molecules, the aquaporins. Treatment of adult ovariectomized mice with replacement steroids demonstrates an estrogen-induced shift in AQP1 signals from the myometrium to the uterine stromal vasculature, suggesting a role in uterine fluid inhibition (Richard et al. 2003), one of the physiologic responses of the uterus to estrogen stimulation. However, the relative expression level of AQP1 gene was not determined by Richard et al. (2003). Li et al. (1997) described a stimulatory effect of estradiol at relatively high concentrations (40 [micro]g/kg) in the expression level of an aquaporin gene (AQP-CHIP) in the uterus of immature rats, although this gene was not identified as AQP1. However, the response of AQP1 in the immature uterus of the rat to dietary components is actually a decrease in its expression level, opposite the effect of estrogenic stimulation.
In all, our data indicate that although there is a clear effect of the diet of the gene expression profile of the uterus/ovaries from the immature rats, this effect is subtle and cannot be correlated with the phytoestrogen content of each diet. Most of the gene transcripts represented in the microarray used in this study have an expression level that is very similar in all the animals, regardless of their diet. Further, by analyzing the expression levels of known estrogen-regulated genes (Naciff et al. 2003), we determined that there is not a significant difference in the relative expression level of any of those genes between animals exposed to Purina 5001 or casein-based diets. [n addition, we found no significant changes at the transcript level for selected estrogen-regulated genes by QRT-PCR. Thus, we are confident that--despite the potential effect of the phytoestrogens in the diet of animals used in a bioassay designed to evaluate the potential estrogenic activity of a given chemical--the response to the chemical (which could be the transcript profile induced by exposure) is independent of the diet and has the potential to truly reflect estrogenic activity.
Table 1. Primers used to verify the array-based gene expression changes induced by the two different diets, by QRT-PCR. GenBank accession Gene name no. (a) Forward primer Complement component 3 (CC3) M29866 5'-CGTGAGCAGCACAGAAGAGA-3' Progesterone receptor (PgR) L16922 5'-CATGTCAGTGGACAGATGCT-3' Intestinal calcium-binding protein (icabp) K00994 5'-ATCCAAACCAGCTGTCCAAG-3' 11-[beta]-Hydroxylsteroid dehydrogenase type 2 (11[beta]HSD) U22424 5'-ATGGCATTGCCTGACCTTAG-3' Vascular [alpha]-actin (VaACTIN) X06801 5'-GACACCAGGGAGTGATGGTT-3' Cyclophilin B AF071225 5'-CAAGCCACTGAAGGATGTCA-3' Cytochrome P450 subfamily XVII M21208 5'-AAGTGGATCCTGGCTTTCCT-3' (Cyp17) AA924771 EST Rattus norvegicus AA924772 5'-TTTGCTGTGCATGGGATTTA-3' Amplicon Gene name Reverse primer size (bp) Complement component 3 (CC3) 5'-CCAGGTGGTGATGGAATCTT-3' 204 Progesterone receptor (PgR) 5'-ACTTCAGACATCATTTCCGG-3' 428 Intestinal calcium-binding protein (icabp) 5'-TGTCGGAGCTCCTTCTTCTG-3' 196 11-[beta]-Hydroxylsteroid dehydrogenase type 2 (11[beta]HSD) 5'-CTCAGTGCTCGGGGTAGAAG-3' 194 Vascular [alpha]-actin (VaACTIN) 5'-GTTAGCAAGGTCGGATGCTC-3' 202 Cyclophilin B 5'-AAAATCAGGCCTGTGGAATG-3' 239 Cytochrome P450 subfamily XVII 5'-CAATGCTGGAGTCGACGTTA-3' 211 (Cyp17) AA924771 EST Rattus norvegicus 5'-CCCTGCAGGATGTGAGAAGT-3' 202 (a) From GenBank (2004). Table 2. Diet effect on body, uterine, and ovarian weight and luminal epithelial cell height of the juvenile (PND24) rat. Casein-based diet (5K96) Body Ovarian weight weight (g) (mg) Peanut oil Mean [+ or -] SD (absolute) 68.1 [+ or -] 4.8 32.0 [+ or -] 2.6 Mean [+ or -] SD (relative) (a) 0.50 [+ or -] 0.04 p-Value (b) 0.1 EE ([micro]g/kg/day) Mean [+ or -] SD (absolute) 68.5 [+ or -] 5.4 36.2 [+ or -] 2.1 Mean [+ or -] SD (relative) (a) 0+52 [+ or -] 0.1 p-Value (b) Casein-based diet (5K96) Uterine Epithelial cell weight height (mg) ([micro]m) Peanut oil Mean [+ or -] SD (absolute) 56.1 [+ or -] 8.2 13.3 [+ or -] 1.3 Mean [+ or -] SD (relative) (a) 0.58 [+ or -] 0.04 p-Value (b) 0.1 EE ([micro]g/kg/day) Mean [+ or -] SD (absolute) 61.9 [+ or -] 11.2 13.1 [+ or -] 1.6 Mean [+ or -] SD (relative) (a) 0.93 [+ or -] 0.1 p-Value (b) Purina 5001 diet Body Ovarian weight weight (g) (mg) Peanut oil Mean [+ or -] SD (absolute) 70.1 [+ or -] 4.9 34.8 [+ or -] 3.3 Mean [+ or -] SD (relative) (a) 0.49 [+ or -] 0.07 p-Value (b) 0.05 0.18 0.1 EE ([micro]g/kg/day) Mean [+ or -] SD (absolute) 71.1 [+ or -] 5.8 37.1 [+ or -] 1.8 Mean [+ or -] SD (relative) (a) 0.52 [+ or -] 0.1 p-Value (b) 0.05 0.14 Purina 5001 diet Uterine Epithelial cell weight height (mg) ([micro]m) Peanut oil Mean [+ or -] SD (absolute) 59.6 [+ or -] 10.8 14.0 [+ or -] 2.2 Mean [+ or -] SD (relative) (a) 0.56 [+ or -] 0.08 p-Value (b) 0.41 0.26 0.1 EE ([micro]g/kg/day) Mean [+ or -] SD (absolute) 66.4 [+ or -] 13.2 14.5 [+ or -] 1.3 Mean [+ or -] SD (relative) (a) 0.93 [+ or -] 0.2 p-Value (b) 0.36 0.18 During the experimental phase, PND20 female rats were fed with a standard laboratory rodent diet (Purina 5001) or with a soy- and alfalfa-free diet (casein-based diet, 5K96) for 5 days (from PND20 to PND24). Epithelial cell height values were obtained from tissue sections from the midregion of each uterine horn, at equivalent areas, and with clear representation of the epithelium lining the lumen along the uterus (as shown in Figure 1). Epithelial cell height was determined by obtaining five measurements from five areas from two animals for each group. These values were used to determine the mean cell height SD for each treatment group, and the corresponding p-value. (a) Relative weight (mg/g body weight). (b) Two-tailed t-test comparing 5K96 with Purina 5001, in control or treated animals; n = 15 for each diet group (controls) and n = 10 for EE-treated groups. Table 3. Genes whose expression is modified by exposure to diet in the uterus/ovaries of the immature rat. GenBank accession Gene no. (a) Gene name symbol X67948 Aquaporin 1 (aquaporin channel forming integral protein) AQP1 U56839 Purinergic receptor P2Y, G-protein coupled 2 P2ry2 AF017756 GSK-3beta interacting protein rAxin Axin AA859529 Diacylglycerol acyltransferase Dgat L06096 Inositol 1,4,5-triphosphate receptor 3 Itpr3 U90887 Arginase type II Arg2 U78977 ATPase, class II, type 9A Atp9a AA892562 EST196365, high homology to nucleolar protein NAP57 and dyskeratosis congenita 1, dyskerin Dkc1 AI639534 ESTs, similar to properdin (factor P) AI231213 ESTs, high homology to kangai 1 (suppression of tumorigenicity 6), prostate Kai1 D10874 Vacuolar H(+)-transporting ATPase, X56133 Mitochondrial H+-ATP synthase alpha subunit Atp5a1 D13417 Transcription factor HES-1 homolog of hairy and enhancer of split 1, (Drosophila) Hes1 Z71925 Polymerase (RNA) II (DNA directed) polypeptide G Polr2g AA818487 ESTs, high homology to cyclophilin B Ppib AI112237 ESTs, moderately similar to JE0384 NADH dehydrogenase AA818858 Peptidylprolyl isomerase A (cyclophilin A) Ppia AA686579 ESTs, similar to ubiquitin-like protein SMT3C precursor U64705 Protein synthesis initiation factor 4All gene and E3 small nucleolar RNA gene S69316 GRP94/endoplasmin (5 and 3 regions) M15481 Insulin-like growth factor 1 IGF-1 S69315 GRP94/endoplasmin (5 and 3 regions) D17310 3-alpha-Hydroxysteroid dehydrogenase(3-alpha-HSD) X67859 Autoantigen or Sjogren syndrome antigen B Ssb AA685903 ESTs, similar to glucose regulated protein, 94 kDa GRP94 S68578 Gonadotropin-releasing hormone receptor Grhr AI009141 EST203592, Rattus norvegicus AF076619 Growth factor receptor bound protein 14 or molecular adapter rGrb14 (Grb14), an inhibitor of insulin actions Grb14 Average fold change p-Values Gene name (b) (c) Aquaporin 1 (aquaporin channel forming integral protein) 1.6 0.000159 Purinergic receptor P2Y, G-protein coupled 2 1.4 0.000448 GSK-3beta interacting protein rAxin 1.4 0.000130 Diacylglycerol acyltransferase 1.3 0.000470 Inositol 1,4,5-triphosphate receptor 3 1.3 0.000420 Arginase type II 1.3 0.000728 ATPase, class II, type 9A 1.3 0.000022 EST196365, high homology to nucleolar protein NAP57 and dyskeratosis congenita 1, dyskerin 1.3 0.000747 ESTs, similar to properdin (factor P) 1.3 0.000446 ESTs, high homology to kangai 1 (suppression of tumorigenicity 6), prostate 1.2 0.000561 Vacuolar H(+)-transporting ATPase, 1.2 0.000865 Mitochondrial H+-ATP synthase alpha subunit -1.1 0.000854 Transcription factor HES-1 homolog of hairy and enhancer of split 1, (Drosophila) -1.2 0.000045 Polymerase (RNA) II (DNA directed) polypeptide G -1.2 0.000379 ESTs, high homology to cyclophilin B -1.2 0.000253 ESTs, moderately similar to JE0384 NADH dehydrogenase -1.2 0.000192 Peptidylprolyl isomerase A (cyclophilin A) -1.3 0.000943 ESTs, similar to ubiquitin-like protein SMT3C precursor -1.3 0.000954 Protein synthesis initiation factor 4All gene and E3 small nucleolar RNA gene -1.3 0.000405 GRP94/endoplasmin (5 and 3 regions) -1.3 0.000120 Insulin-like growth factor 1 -1.3 0.000068 GRP94/endoplasmin (5 and 3 regions) -1.4 0.000174 3-alpha-Hydroxysteroid dehydrogenase(3-alpha-HSD) -1.4 0.000397 Autoantigen or Sjogren syndrome antigen B -1.4 0.000103 ESTs, similar to glucose regulated protein, 94 kDa -1.5 0.000878 Gonadotropin-releasing hormone receptor -1.5 0.000322 EST203592, Rattus norvegicus -1.8 0.000468 Growth factor receptor bound protein 14 or molecular adapter rGrb14 (Grb14), an inhibitor of insulin actions -2.1 0.000158 (a) From GenBank (2004). (b) The average fold change was determined by comparing the average signal value of the indicated transcripts obtained from the uterus/ovaries from five females fed with the casein-based diet (5K96) versus the average signal value obtained from the same tissues from five females fed the standard rodent diet (Purina 5001). (c) Transcripts listed are those showing a robust response to the different diet (p < 0.001) using the stratified form of the Jonkheere-Terpstra nonparametric statistic to identify the diet response. Table 4. Diet effect on genes whose expression is modified by exposure to of 0.1 [micro]g/kg EE in the uterus/ovaries of the immature rat. GenBank accession Gene no. (a) Gene name symbol M29866 Complement component 3 CC3 Y08358 Eotaxin or small inducible cytokine A11 Scya11 AI013389 ESTs, similar to calcium-binding protein, intestinal, vitamin D-dependent Calb3 K00994 Intestinal calcium-binding protein icabp U49062 CD24 antigen Cd24 L14004 Polymeric immunoglobulin receptor pigr AA859661 ESTs, similar to glutaminyl-peptide cyclotransferase precursor M57718 Cytochrome P450 IV A1 CYP4A1 U22424 Hydroxysteroid dehydrogenase, 11-[beta] type 2 Hsd11b2 L07114 Apolipoprotein B editing protein Apobec1 S79730 Opioid receptor-like ORL1 receptor Oprl1 M88469 f-Spondin Sponf X66845 Dynein, cytoplasmic, intermediate chain 1 Dncic1 L46593 Small proline-rich protein gene Sprr L00191 Fibronectin, encoding three mRNAs, exons 1, 2, 3 fn M22323 Gamma-enteric smooth muscle actin Actg2 D15069 Adrenomedullin Adm AA893870 EST197673 Rattus norvegicus AI232078 Transforming growth factor-[beta] (TGF-[beta]) masking protein Ltbp1 U82612 Fibronectin (fn-1) gene fn-1 X05834 Fibronectin (fn-3) gene fn-3 L00382 Skeletal muscle [beta]-tropomyosin and fibroblast tropomyosin 1 tpm1 AA800908 EST190405 Rattus norvegicus M25758 Phosphatidylinositol transfer protein Pitpn AA799773 ESTs, Rattus norvegicus AA892829 EST, similar to mouse bifunctional 3'-phosphoadenosine (PPS1) PPS1 AB010963 Potassium large conductance calcium-activated channel Kcnmb1 AF083269 Actin-related protein complex 1b Arpc1b AA891760 EST195563 Rattus norvegicus AJ005394 Collagen [alpha] 1 type V Col5a1 L11930 Cyclase-associated protein homologue Cap1 X07467 Glucose-6-phosphate dehydrogenase G6pd U26310 Tensin Tns AA891542 EST195345 Rattus norvegicus, similar to mouse heat shock protein hsp40-3 Dnajb5 U44948 Cysteine-rich protein 2 or smooth muscle cell LIM protein (SmLIM) Csrp2 S61868 Ryudocan or heparan sulfate proteoglycan core protein or syndecan-4 SDC4 L41254 Corticosteroid-induced protein or FXYD domain-containing ion transport regulator 4 Fxyd4 AF023087 Nerve growth factor induced factor A, or early growth response 1 Egr1 U07181 Lactate dehydrogenase B Ldhb X89225 Solute carrier family 3, member 2 Slc3a2 AF054826 Vesicle-associated membrane protein 5 Vamp5 X75253 Phosphatidylethanolamine binding protein Pbp AA924772 ESTs, similar to metallothionein 3 Mt3 AA894027 EST197830 Rattus norvegicus AA894030 EST197833 Rattus norvegicus AA946532 ESTs, similar to ATP-binding cassette, sub-family D (ALD), member 3 Abcd3 M32754 Inhibin [alpha]-subunit Inha AA874794 ESTs, similar to nerve growth factor receptor (TNFRSF16) associated protein 1 Ngfrap1 M21060 Superoxide dismutase 1, soluble Sod1 X08056 Guanidinoacetate methyltransferase GAMT D00729 [delta]3, [delta]2-enoyl-CoA isomerase U90829 APP-binding protein 1 Appbp1 AI170613 ESTs, similar to heat shock 10 kDa protein 1 Hspe1 D63761 Adrenodoxin reductase Fdxr D78303 Splicing factor YT521-B YT521 L48060 Prolactin receptor Prlr AA849036 ESTs, similar to guanylate cyclase Gucy1a3 1, soluble, [alpha]-3 M33648 Mitochondrial 3-hydroxy-3- methylglutaryl-CoA synthase HMGCS2 E05646 Phosphatidylethanolamine binding protein Pbp AA858520 ESTs, similar to follistatin Fst L02842 Follicle-stimulating hormone receptor FSHR X04229 Glutathione-S-transferase, [mu] type 1 (Yb1) Gstm1 L23148 Inhibitor of DNA binding 1, helix-loop-helix protein Id1 D63761 Adrenodoxin reductase Fdxr AF076619 Growth factor receptor bound protein 14 Grb14 M33648 Mitochondrial 3-hydroxy-3- methylglutaryl-CoA synthase AA858520 Follistatin Fst X62660 Glutathione transferase subunit 8 AI175776 FST219344 Rattus norvegicus J03914 Glutathione-S-transferase, [mu] type 2 (Yb2) Gstm2 J02592 Glutathione-S-transferase, [mu] type 2 (Yb2) Gstm2 S59525 Gonadotropin-releasing hormone receptor grhr M36453 Inhibin [alpha] Inha X54793 Heat shock protein 60 (liver) Hsp60 AA858640 ESTs L19998 Minoxidil sulfotransferase PST-1 X78848 Glutathione-S-transferase, [alpha] type (Ya) Gsta1 AF001898 Aldehyde dehydrogenase 1, subfamily A1 Aldh1a1 X97754 Hydroxysteroid dehydrogenase 17[beta] type 1 Hsd17b1 AF000942 Inhibitor of DNA binding 3, dominant negative helix-loop-helix Id3 AI171268 EST217223 Rattus norvegicus, identical to inhibitor of DNA binding 3, dominant negative helix-loop-helix Id3 D84336 Delta-like homolog (Drosophila), a novel member of the epidermal growth factor (EGF)-like family of proteins Dlk1 S63167 3 [beta]-Hydroxysteroid dehydrogenase isomerase type II HSD3R2 M12492 Type II cAMP-dependent protein kinase regulatory subunit prkar2a S72505 Glutathione S-transferase Yc1 subunit AA874919 Mismatch repair protein Msh2 M14656 Sialoprotein (osteopontin) Spp1 X01115 SVS-protein F, or seminal vesicle secretion 5 Svs5 M21208 Cytochrome P450, subfamily XVII Cyp17 Average told change (b) EE vs. EE vs. control, Purina control, Purina Gene name 5001/5K96 5K96 5001 Complement component 3 A 14.7 7.0 Eotaxin or small inducible cytokine A11 A 2.7 2.9 ESTs, similar to calcium-binding protein, intestinal, vitamin D-dependent 2.0 2.4 1.9 Intestinal calcium-binding protein 1.1 5.2 2.1 CD24 antigen 1.1 1.3 1.2 Polymeric immunoglobulin receptor 1.5 1.4 1.1 ESTs, similar to glutaminyl-peptide cyclotransferase precursor A 2.1 1.7 Cytochrome P450 IV A1 A A A Hydroxysteroid dehydrogenase, 11-[beta] type 2 1.0 2.2 1.6 Apolipoprotein B editing protein A A A Opioid receptor-like ORL1 receptor 1.2 1.3 1.4 f-Spondin 1.7 1.7 1.1 Dynein, cytoplasmic, intermediate chain 1 A A A Small proline-rich protein gene 2.4 2.5 -1.0 Fibronectin, encoding three mRNAs, exons 1, 2, 3 -1.2 1.6 1.2 Gamma-enteric smooth muscle actin 1.4 1.5 1.3 Adrenomedullin 1.9 2.4 1.2 EST197673 Rattus norvegicus A 1.7 2.0 Transforming growth factor-[beta] (TGF-[beta]) masking protein -1.1 1.2 1.1 Fibronectin (fn-1) gene 1.5 1.6 1.1 Fibronectin (fn-3) gene 1.0 1.4 1.2 Skeletal muscle [beta]-tropomyosin and fibroblast tropomyosin 1 1.2 1.7 1.3 EST190405 Rattus norvegicus 1.2 1.6 1.4 Phosphatidylinositol transfer protein 1.1 1.3 1.3 ESTs, Rattus norvegicus 1.3 1.3 1.2 EST, similar to mouse bifunctional 3'-phosphoadenosine (PPS1) 1.2 1.3 1.1 Potassium large conductance calcium-activated channel 1.2 1.4 1.2 Actin-related protein complex 1b 1.0 1.3 -1.0 EST195563 Rattus norvegicus A A A Collagen [alpha] 1 type V -1.1 1.6 1.2 Cyclase-associated protein homologue 1.1 1.3 1.2 Glucose-6-phosphate dehydrogenase 1.2 1.2 -1.1 Tensin 1.2 1.3 1.1 EST195345 Rattus norvegicus, similar to mouse heat shock protein hsp40-3 1.3 1.3 1.1 Cysteine-rich protein 2 or smooth muscle cell LIM protein (SmLIM) -1.1 -1.6 -1.4 Ryudocan or heparan sulfate proteoglycan core protein or syndecan-4 -1.2 -1.5 -1.1 Corticosteroid-induced protein or FXYD domain-containing ion transport regulator 4 -1.1 -1.4 -1.1 Nerve growth factor induced factor A, or early growth response 1 -1.5 -1.7 1.1 Lactate dehydrogenase B -1.3 -1.2 -1.1 Solute carrier family 3, member 2 -1.3 -1.5 -1.3 Vesicle-associated membrane protein 5 -1.2 -1.8 -1.2 Phosphatidylethanolamine binding protein -1.2 -1.4 -1.1 ESTs, similar to metallothionein 3 A A A EST197830 Rattus norvegicus A A A EST197833 Rattus norvegicus A A A ESTs, similar to ATP-binding cassette, sub-family D (ALD), member 3 -1.3 -1.4 -1.3 Inhibin [alpha]-subunit -1.3 -1.6 1.0 ESTs, similar to nerve growth factor receptor (TNFRSF16) associated protein 1 1.0 -1.4 -1.2 Superoxide dismutase 1, soluble -1.2 -1.2 -1.2 Guanidinoacetate methyltransferase -1.2 -1.3 -1.0 [delta]3, [delta]2-enoyl-CoA isomerase -1.2 -1.2 -1.3 APP-binding protein 1 -1.2 -1.6 -1.1 ESTs, similar to heat shock 10 kDa protein 1 -1.2 -1.3 -1.6 Adrenodoxin reductase -1.1 -1.5 -1.1 Splicing factor YT521-B -1.1 -1.2 -1.2 Prolactin receptor 1.0 -1.3 -1.2 ESTs, similar to guanylate cyclase -1.2 -1.3 -1.1 1, soluble, [alpha]-3 Mitochondrial 3-hydroxy-3- methylglutaryl-CoA synthase -1.2 -1.6 -1.3 Phosphatidylethanolamine binding protein -1.2 -1.3 -1.2 ESTs, similar to follistatin -1.3 -1.5 -1.1 Follicle-stimulating hormone receptor A A A Glutathione-S-transferase, [mu] type 1 (Yb1) -1.2 -1.4 -1.2 Inhibitor of DNA binding 1, helix-loop-helix protein 1.0 -1.1 1.0 Adrenodoxin reductase 1.0 -1.4 -1.2 Growth factor receptor bound protein 14 -1.5 -1.8 -1.3 Mitochondrial 3-hydroxy-3- methylglutaryl-CoA synthase -1.3 -2.0 -1.6 Follistatin -1.3 -1.4 -1.3 Glutathione transferase subunit 8 -1.2 -1.5 -1.3 FST219344 Rattus norvegicus -1.2 -1.7 -1.4 Glutathione-S-transferase, [mu] type 2 (Yb2) -1.2 -1.2 -1.1 Glutathione-S-transferase, [mu] type 2 (Yb2) -1.1 -1.3 -1.2 Gonadotropin-releasing hormone receptor -1.1 -1.8 -1.4 Inhibin [alpha] -1.2 -1.6 -1.6 Heat shock protein 60 (liver) -1.3 -1.3 -1.6 ESTs -1.2 -1.4 -1.4 Minoxidil sulfotransferase -1.2 -1.4 -1.4 Glutathione-S-transferase, [alpha] type (Ya) -1.2 -1.5 -1.4 Aldehyde dehydrogenase 1, subfamily A1 1.1 -1.5 -1.5 Hydroxysteroid dehydrogenase 17[beta] type 1 -1.5 -2.3 -1.6 Inhibitor of DNA binding 3, dominant negative helix-loop-helix -1.2 -1.3 -1.1 EST217223 Rattus norvegicus, identical to inhibitor of DNA binding 3, dominant negative helix-loop-helix -1.4 -1.4 -1.1 Delta-like homolog (Drosophila), a novel member of the epidermal growth factor (EGF)-like family of proteins A A A 3 [beta]-Hydroxysteroid dehydrogenase isomerase type II -1.1 -1.4 -1.6 Type II cAMP-dependent protein kinase regulatory subunit -1.3 -1.9 -1.8 Glutathione S-transferase Yc1 subunit -1.2 -1.5 -1.6 Mismatch repair protein -1.1 -1.5 -1.1 Sialoprotein (osteopontin) 1.0 -3.5 -2.4 SVS-protein F, or seminal vesicle secretion 5 -1.4 -3.9 1.1 Cytochrome P450, subfamily XVII 1.1 -2.4 -2.2 A, absent (undetectable by Microarray Suite 5.0; Affymetrix). Transcripts listed are those previously reported to show a robust response to graded doses of EE in the uterotrophic assay (p < 0.001) (Naciff et al. 2003); n = 5 per group. (a) From GenBank (2004). (b) The average fold change is the ratio of the relative expression level of each gene in uterus/ovaries from animals fed Purina 5001 versus those fed 5K96 diet (n = 5 per group). Table 5. Selected gene expression changes verified by QRT-PCR. CC3 icabp Treatment Q M Q M Vehicle in Purina 5001 vs. 5K96 1.0 A 1.2 1.1 0.1 EE [micro]g vs. control in 5K96 18.7 14.7 3.7 5.2 0.1 FF [micro]g vs. control in Purina 5001 21.2 7.0 3.3 2.1 11[beta] HSD PgR Treatment Q M Q M Vehicle in Purina 5001 vs. 5K96 1.2 1.0 1.1 1.1 0.1 EE [micro]g vs. control in 5K96 2.7 2.2 1.8 1.6 0.1 FF [micro]g vs. control in Purina 5001 2.9 1.6 2.2 1.5 EST AA924772 VaACTIN Treatment Q M Q M Vehicle in Purina 5001 vs. 5K96 -1.1 A 1.2 1.1 0.1 EE [micro]g vs. control in 5K96 -1.5 A 1.1 1.0 0.1 FF [micro]g vs. control in Purina 5001 -1.4 A 1.1 1.1 Cyp17 Cyclo B Treatment Q M Q M Vehicle in Purina 5001 vs. 5K96 -1.2 1.1 1.1 1.0 0.1 EE [micro]g vs. control in 5K96 -3.5 -2.4 1.0 1.1 0.1 FF [micro]g vs. control in Purina 5001 -3.4 -2.2 1.1 1.0 Abbreviations: 11[beta]HSD, 11-[beta]-hydroxylsteroid dehydrogenase type 2 gene; A, absent (undetectable by Microarray Suite 5.0; Affymetrix); cyclo B, cyclophilin B gene; Cyp17, cytochrome P450 subfamily XVII gene; M, microarray-derived fold change; Q, QRT-PCR-derived fold change; VaACTIN, vascular [alpha]-actin gene. The relative fold change is the ratio of the relative expression level of each gene in uterus/ovaries from animals fed Purina 5001 versus those fed 5K96 diet. The microarray-derived fold change and the QRT-PCR-derived fold change were determined as described in "Materials and Methods," using the same amount of total RNA derived from three independent animals, in duplicate. These genes were chosen on the basis of their response to estrogenic stimulation in the uterotrophic assay (Naciff et al. 2003); we also included two control genes, cycle B and VaACTIN.
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Jorge M. Naciff, Gary J. Overmann, Suzanne M. Torontali, Gregory J. Carr, Jay P. Tiesman, and George P. Daston Miami Valley Laboratories, The Procter and Gamble Company, Cincinnati, Ohio, USA
Address correspondence to J.M. Naciff, The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 538707 #805, Cincinnati, OH 45253-8707 USA. Telephone: (513) 627-1761. Fax: (513) 627-0323. E-mail: firstname.lastname@example.org
We thank C. Ryan and W. Owens for their helpful discussions.
The authors are employed by the Procter and Gamble Company.
Received 10 November 2003; accepted 16 August 2004.
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|Title Annotation:||Research / Article|
|Author:||Daston, George P.|
|Publication:||Environmental Health Perspectives|
|Date:||Nov 1, 2004|
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