Potential Estrogenic Activity Research Articles

The Genomic Response of a Human Uterine Endometrial Adenocarcinoma Cell Line to 17α-Ethynyl Estradiol

We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p </= 0.0001). The annotation available for the genes affected indicates that EE exposure results in changes in multiple molecular pathways affecting various biological processes, particularly associated with development, morphogenesis, organogenesis, cell proliferation, cell organization, and biogenesis. All of these processes are also affected by estrogen exposure in the uterus of the rat. Comparison of the response to EE in both the rat uterus and the Ishikawa cells showed that 71 genes are regulated in a similar manner in vivo as well as in vitro. Further, some of the genes that show a robust response to estrogen exposure in Ishikawa cells are well known to be estrogen responsive, in various in vivo studies, such as PGR, MMP7, IGFBP3, IGFBP5, SOX4, MYC, EGR1, FOS, CKB, and CCND2, among others. These results indicate that transcript profiling can serve as a viable tool to select reliable in vitro systems to evaluate potential estrogenic activities of target chemicals and to identify genes that are relevant for the estrogen response.

Fenvalerate, A Pyrethroid Insecticide, Adversely Affects Sperm Production and Storage in Male Rats

The aim of this study was to investigate the potential estrogenic activity of fenvalerate by examining reproductive and fertility capabilities in Wistar rats. Adult male animals were treated for 30 d with 20 or 40 mg/kg/d fenvalerate or corn oil (vehicle) by oral gavage. Further, a possible estrogenic activity of fenvalerate (0.4, 1, 4, 8, or 40 mg/kg) was tested after a 3-d treatment of immature female rats using the uterotrophic assay. Exposure to the higher dose of fenvalerate was toxic to testis and epididymis as shown by a decrease in the absolute weights and sperm counts in both organs. Although the sperm counts were reduced, the fertility and sexual behavior were similar in control rats and rats treated with 40 mg/kg pesticide. Fenvalerate did not exert estrogenic activity in vivo at the tested doses. Data suggest that fenvalerate treatment in this study failed to compromise fertility, possibly due to enhanced reproductive capacity in rodents compared to humans.

4-Alkylphenols and related chemicals show similar effect on the function of human and rat estrogen receptor α in reporter gene assay

Alkylphenols (APs) are widely used as important industrial materials and have attracted lots of attention because of their potential estrogenic activities. In this study, we developed human estrogen receptor α (hERα) and rat estrogen receptor α (rERα) mediated reporter gene assays and compared the estrogenic activity of APs and related chemicals based on the two ERα. Human breast cancer cell line MCF-7 was co-transfected with Gal4-fused hERα and corresponding reporter plasmid; African green monkey kidney cell line CV-1 was co-transfected with rERα and reporter gene. Both assays showed acceptable response to natural estrogen 17β-estradiol (E2) with EC 50 of 0.16 nM and 4.7 nM. Then the estrogenic activity of 4-APs, 4-phenylphenol and bisphenol-A were evaluated and compared with the effects of E2. The data suggested that test APs and related chemicals possessed weakly estrogenic activity and the activity of test APs increased with the increase of substituent size. This structure–activity relationship helped to infer the activity of chemicals with similar feature. Furthermore, test APs showed similar effect on the function of hERα and rERα. This consistency helped to extrapolate in vivo rodent data to human being when performing risk assessment of endocrine disruptors.

Estrogen- and androgen-sensitive bioassays based on primary cell and tissue slice cultures from three-spined stickleback ( Gasterosteus aculeatus)

Endocrine disrupting compounds are chemicals that may interfere with the endocrine system causing severe effects in organisms. The three-spined stickleback ( Gasterosteus aculeatus L.) offers a potential for the assessment of endocrine disruption caused by a) estrogenic xenobiotics through the estrogen-dependent protein vitellogenin and b) androgenic xenobiotics through the androgen-dependent protein spiggin. The stickleback is presently the only known fish species with a quantifiable androgen and anti-androgen biomarker endpoint. In the current study, hepatocyte and kidney primary cell cultures and liver and kidney tissue slice cultures were prepared and used for detecting estrogenic or androgenic activity in vitro through the action of hormones or municipal sewage water. The results indicate that stickleback male hepatocyte cultures are suitable in detecting estrogenic activity and stickleback female kidney tissue slice cultures in detecting androgenic activity. The tested sewage water showed high estrogenic activity but no significant androgenic activity. Primary cell and tissue slice cultures isolated from the three-spined stickleback will allow simultaneously screening in vitro for potential estrogenic and androgenic activity of complex samples.

Estrogenic properties of traditional Cameroonian medicinal plants

Medicinal plants of tropical and subtropical areas of Cameroon, which are traditionally used to treat female ailments, were investigated towards potential estrogenic activities. In a first series of screening experiments in the yeast estrogen receptor assay on 33 extracts from 18 plants estrogenic activities in the ethyl acetate extracts of the stem bark of Millettia conraui and the stem bark of Millettia drastica, as well as the methanol extracts of the leaves of Bridelia ferruginea, the roots of Pseudarthria hookeri and the roots of Nauclea latifolia could be shown. These results could be verified by alkaline phosphase induction in Ishikawa cells at 10 and 100mg/ml doses, except for the extract of Bridelia ferruginea which was toxic for Ishikawa cells. All stimulatory effects were inhibited by a combined treatment of cells with the pure antiestrogen fulvestrant. In conclusion, the extracts of Millettia conraui, Millettia drastica, Pseudarthria hookeri and Nauclea latifolia may contain secondary metabolites endowed with estrogenic activity. In another experiment, the ethyl acetate extract of the stem bark of Erythrina lysistemon also proved to be estrogenic in the two in vitro assays described above. These results prompted us to investigate the estrogenic activity of this extract in vivo in young ovariectomized female Wistar rats after a 7-day treatment. Preliminary results obtained to date concern the extract of Erythrina lysistemon, which was evaluated through the proliferative status of target sex organs such as uterus and vagina. The oral administration of 200mg/kg BW/d of Erythrina lysistemon extract in comparison to untreated ovariectomized rats significantly increased the vaginal epithelial height by 47% and induced a weak increase of uterine epithelial height around 7%. Both effects were not as pronounced as those elicited in positive control of 100µg/kg BW/d of ethinylestradiol given orally. Overall our results suggest that the extract of Erythrina lysistemon contains secondary metabolites endowed with estrogenic activity.

Environmental Xenoestrogens: Short-term Exposure of Low Doses of Lindane, Dieldrin, Dibutyl Phthalate, and Diethylhexyl Phthalate Increases Uterine Weight in Young Female Mice

An in vivo method was developed to test the potential estrogenic activity of Lindane, Dieldrin, Dibutyl phthalate (DBP), and Diethylhexyl phthalate (DEHP) in 7-8 week old, virgin female mice. The mice were dosed for three consecutive days at the following dosages: a) 3.38 µM kg -1 day -1 for estrogen E2, b) 2.61 µM kg -1 day -1 for lindane, c.) 4.2 µM kg -1 day -1 for dieldrin, d) 6.43 µM kg -1 day -1 for DEHP and at e) 5.77 µM kg -1 day -1 for DBP. The uterine weights without fluids relative to mice weight, which was targeted as the biomarker (response), were significantly affected by the doses administrated. The statistical analyses showed increases in the average relative weights of uteri of 0.0071g for E2, 0.0092 for lindane, 0.0062 for dieldrin, 0.0068g for DEHP and 0.0056g for DBP compared with 0.00225 for the control (n = 5). These results show that all compounds used in this study have estrogenic effects in these mice under these conditions. Lindane was considered to be a strong estrogen while DBP was considered to be a weak estrogen. What are the implications of these findings for human exposure to similar low doses of any of these compounds, singly, sequentally or simultaneously? What are the policy and ethical responsibilities for addressing such potentially adverse effects within humans or wildlife.

Quantification of organic eluates from polymerized resin-based dental restorative materials by use of GC/MS

Residual monomers, additives and degradation products from resin-based dental restorative materials eluted into the oral cavity may influence the biocompatibility of these materials. Emphasis has been placed on studies addressing cytotoxic, genotoxic and estrogenic potential of these substances. A prerequisite for analyzing the potential of exposure to eluted compounds from dental materials is reliable quantification methods, both real time and accelerated measurements. The purpose of the present study was to quantify nine eluates; 2-hydroxyethyl methacrylate (HEMA), hydroquinone monomethyl ether (MEHQ), camphorquinone (CQ), butylated hydroxytoluene (BHT), ethyl 4-(dimethylamino)benzoate (DMABEE), triethylene glycoldimethacrylate (TEGDMA), trimethylolpropane trimethacrylate (TMPTMA), oxybenzone (HMBP) and drometrizole (TIN P) leaching from specimens of four commonly used resin-based dental materials in ethanol and an aqueous solution. All analyses were performed by use of GC/MS, each component was quantified separately and the results presented in μg mm −2. This study has shown that elution from various materials differs significantly, not only in the types of eluates, but also regarding amounts of total and of single components. A high amount of HMBP, a UV stabilizer with potential estrogenic activity, was detected from one material in both solutions.

In Vitro and In Vivo Evaluation of the Estrogenic, Androgenic, and Progestagenic Potential of Two Cyclic Siloxanes

The purpose of these experiments was to determine the potential estrogenic, androgenic, and progestagenic activity of two cyclic siloxanes, octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5). Receptor-binding experiments and a luciferase reporter gene assay were used to determine if the materials were able to bind and activate either the estrogen receptors (ERs) or progesterone receptors (PRs)-alpha or beta. The rat uterotrophic assay (RUA) for estrogenic activity and the Hershberger assay for androgenic activity were utilized as the in vivo assays. For the ER-binding studies, D4 was shown to bind to ERalpha but not to ERbeta. D5 did not bind to either of the two receptors. D4 activated the reporter gene at 10 microM, while D5 was considered negative in the estrogen reporter gene assay. Neither material was a ligand for the PRs. Both the RUA and Hershberger assays were conducted using whole-body inhalation of the two materials for 16 h/day. D4 resulted in a small but significant increase in both wet and blotted uterine weight as well as increases in both luminal and glandular epithelial cell height in both Sprague Dawley and Fischer 344 rats. D5 was negative in both rat strains, indicating that D5 does not possess estrogenic activity. Neither material possessed any significant antiestrogenic activity. Both materials were negative in the Hershberger assay indicating that neither material possesses any significant androgenic activity. Our studies have shown that D4 exhibits a low affinity for ERalpha in vitro and a weakly estrogenic response in vivo.

Detection of estrogenic activity in herbal teas by in vitro reporter assays

Herbal teas have become popular as alternatives to caffeinated beverages during past two decades. However, toxicological studies of herbal teas have been limited and the safety of herbal teas thus remains unknown. We focused on the estrogenic activities of herbal teas since some of their ingredients are similar to those used in herbal remedies for menopause relief and therefore contain phytoestrogens. To investigate the potential estrogenic activity of extracts prepared from herbal tea mixtures commercially available and to provide useful information for the safety assessment of those products, we initially screened the estrogenic activity in extracts of 15 different herbal teas by an assay using recombinant yeast cells expressing the human estrogen receptor (YES). A distinct estrogenic activity was thus detected in the ethanolic extracts from four herbal tea mixtures. Licorice root was specified as a ingredient responsible for the estrogenic activity in those extracts. In contrast, the aqueous extracts of all herbal tea mixtures we tested exhibited distinct estrogenic activity in YES, thus suggesting the existence of various ingredients that contain estrogenic constituents extractable with water. Among them, the extract of peppermint tea exhibited the highest estrogenic activity. The estrogenic activity in extracts of herbal tea mixtures and specified ingredients were thereafter confirmed by a reporter assay system using transiently transfected HEK293 cells.

The Feed Factor: Estrogenic Variability in Lab Animal Diets

Animal studies have long been a cornerstone of biomedical and environmental health research, and scientists need assurance that animals used in these studies are being cared for in ways that will not unknowingly influence experimental outcomes. But a growing number of scientists have voiced concern over the possibility that certain estrogenic compounds present in lab animal feed may skew test results. These compounds are deemed potentially problematic because they can bind to estrogen receptors and induce estrogen-like effects in animals, humans, and cells grown in culture. Some experts have advocated strict standardization of rodent chows and even the removal of dietary phytoestrogens. This emergent controversy was the focus of “DIET II—The Effect of Variability in Estrogenic Activity of Commercial Animal Feeds: Interaction with Manufacturers, NIH Officials, and Scientific Societies to Develop a Solution,” a full-day meeting held 3 August 2006 in Research Triangle Park, North Carolina. The meeting, the second in a series on the topic, was cosponsored by the NIEHS and the NIH Office of Dietary Supplements (ODS). Discussions at the meeting centered on the variation in estrogenic activity between feed batches, the effects of these estrogenic components on endocrine-related end points, and the difficulties inherent in comparing, interpreting, and reproducing these end points over time within and between different laboratories when background levels of diet-related estrogenic activity are not adequately documented. Findings presented at this meeting made it clear that researchers studying estrogen-related end points can not afford to overlook the influence of the test rodent’s diet. “This workshop is an excellent example of the cumulative and self-correcting nature of the scientific process,” said ODS nutritionist Elizabeth Yetley, a conference co-organizer. “That is, through the accumulation of results from a body of experimental evidence, the importance of approaches for better-defined animal diets relative to their potential estrogenic activity have been identified, and measures to improve future research in this area are being undertaken by the scientific community.” Participants at the conference—organized by NIEHS scientists Jerrold Heindel and Julius Thigpen, along with Yetley and University of Missouri–Columbia biologist Frederick vom Saal—included investigators from the endocrine disruptor research community as well as representatives from animal feed companies. This spectrum of representatives reflected the fact that, in Heindel’s words, “researchers and animal care divisions of research institutions are beginning to pay attention to the phytoestrogen issue, and feed manufacturers want to know what the scientific community wants.” Heindel described a sincere interest on the part of both sides to reach a “win–win” solution, one that would yield animal diets of a known estrogenicity that could be used by researchers in all fields of physiology and toxicology, but that also would not unduly burden the feed manufacturers.

Estrogenic properties of isoflavones derived from Millettia griffoniana

In most developing countries, 70-80% of the population still resort to traditional medicine for their primary health care. This medicine utilises medicinal plants which are traditionally taken as concoction and infusion. The root and stem bark of Millettia griffoniana (Leguminosae), has been reported to contain isoflavonoids, alkaloids, and diterpenoids. The possible benefit of some bioactive isoflavones derived from M. griffoniana prompted us to screen them for estrogenic activity. Six isoflavones and coumarin derived from M. griffoniana (bail) namely, compound nos. 1-6 (Fig. 1) were tested for their potential estrogenic activities in three different estrogen receptor alpha (ERalpha)-dependent assays. In a yeast-based ERalpha assay, all test substances and 17beta-estradiol as endogenous agonist, showed a significant induction of beta-galactosidase activity. The test compounds at the concentration of 5 x 10(-6) M could achieve 59-121% of the beta-galactosidase induction obtained with 10(-8) M 17beta-estradiol (100%). In the reporter gene assay based on stably transfected MCF-7 cells (MVLN cells), the estrogen responsive induction of luciferase was also stimulated by the M. griffoniana isoflavones. In Ishikawa cells, all substances exhibited estrogenic activity revealed by the induction of alkaline phosphatase (AlkP) activity. The estrogenic activities of isoflavones from M. griffoniana could be completely suppressed by the pure estrogen antagonist, ICI 182,780, suggesting that the compounds exert their activities through ERalpha. Although all substances showed estrogenic effects, 4'-methoxy-7-O-[(E)-3-methyl-7-hydroxymethyl-2,6-octadienyl]isoflavone (7-O-DHF), Griffonianone C (GRIF-C), and 3',4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO) were found to be the most potent of tested substances. In summary, estrogenic activities of the isoflavones derived from M. griffoniana were described for the first time using reporter gene assays and the estrogen-inducible AlkP Ishikawa model.

Royal jelly has estrogenic effects in vitro and in vivo

Royal jelly (RJ) from honeybees ( Apis mellifera) is traditionally thought to improve menopausal symptoms. The potential estrogenic activities of RJ were investigated using various approaches. RJ competed for binding of 17β-estradiol to the human estrogen receptor α and β but its affinities were weak compared with diethylstilbestrol and phytoestrogens. The reporter gene expression assays suggested that 0.1–1 mg/ml RJ activated estrogen receptors, leading to enhanced transcription of a reporter gene through an estrogen-responsive element. 1 mg/ml RJ stimulated the mRNA expression of estrogen-responsive pS2 and vascular endothelial growth factor (VEGF) by increasing gene transcription in MCF-7 cells. Treatment with RJ at concentrations ranging from 0.5 to 1 mg/ml enhanced MCF-7 cell proliferation, but concomitant treatment with 1 μM tamoxifen blocked this effect. In vivo studies using ovariectomized rats showed that 17β-estradiol (20 mg/kg, s.c.) treatment restored VEGF expression in both uterus and brain, whereas RJ (1 g/kg, s.c.) restored it in uterus but not in brain. These findings provide evidence that RJ has estrogenic activities through interaction with estrogen receptors followed by endogenous gene expressions.

Tamoxifen does not reduce the risk of lung cancer in women

7212 Background: Mounting evidence indicates that women have a different susceptibility to lung cancer than men. In particular, female never-smokers are more likely than male never-smokers to develop lung cancer. Moreover, women are more likely to be diagnosed with adenocarcinoma than are men. Coupled with the localization of estrogen receptors alpha and beta to lung cancer cells, gender differences in incidence point to a possible role for circulating estrogens in lung tumorigenesis. We sought to determine if use of the anti-estrogen tamoxifen decreases the risk of lung cancer. Methods: We examined incidence rates of second primary lung cancers by categories of tamoxifen treatment among women enrolled in randomized ECOG adjuvant breast cancer trials. Overall, there were 15,875 women in 14 studies from 1978–2002. The duration of tamoxifen treatment varied by study and was alternately categorized as ≤ 1, ≤2 or ≥5 years. Results: We identified 55 second-primary lung cancers over 97,225 person-years of follow-up. 27 second-primary lung cancers occurred among 6,478 women who did not receive tamoxifen versus 28 lung cancers among the 9,397 women who received tamoxifen of any duration. Compared to women without tamoxifen treatment, those treated for fewer than two years had an age-adjusted relative risk for lung cancer of 1.13 (95% CI 0.52–2.32). Similarly, among women treated with tamoxifen for ≥5 years the relative risk for subsequent lung cancer was 0.80 (95% CI 0.40–1.57). Conclusions: We did not observe a strong association between tamoxifen treatment and the risk of second-primary lung cancer in a sample of women who had received tamoxifen as adjuvant therapy for breast cancer. The results are similar to those previously reported (Fisher, JNCI 1998). However, the potential estrogenic activity of tamoxifen may argue for evaluating adjuvant breast cancer trials of aromatase inhibitors for incident second primary lung cancers. No significant financial relationships to disclose.

Estrogenic Activity in Sediments from European Mountain Lakes

Superficial and bottom sediment samples from 83 European mountain lakes, ranging from Norway to the Pyrenees and East Europe, were tested for estrogenic compounds by the recombinant yeast assay. The results showed widespread potential estrogenic activity arriving at remote lakes. Tatra Mountains (Slovakia) and Scotland Highlands were the regions with the highest prevalence of lakes with high estrogenic values. Comparison of the estrogenic activity in the superficial layer of sediments with pre-industrial age sections showed that estrogenic compounds were predominantly deposited in recent times. Chemical analysis showed that highly estrogenic sediments were significantly enriched in both polycyclic aromatic hydrocarbons (PAH) and organochlorine compounds. For PAH, enrichment ratios in highly estrogenic samples versus nonestrogenic ones were inversely correlated with the vapor pressure value for each compound, indicating a significant relationship between estrogenicity and accumulation of less volatile PAH. Two PAH of predominantly diagenetic origin, retene and perylene, did not show specific enrichment in estrogenic samples. Principal component analysis revealed a strong correlation between estrogenic activity and the presence of contaminants of anthropogenic origin. These data reveal significant amounts of estrogenic compounds in remote lakes, relate them to the overall human activity, and suggest that they may affect organisms inhabiting these ecosystems.

Potential Estrogenic and Antiandrogenic Effects of Permethrin in Rats

Many environmental chemicals including pesticides have been reported to possess hormonal activities, and thus are classified as endocrine disruptors. Permethrin, a synthetic pyrethroid insecticide, is used worldwide, which provides potential environmental exposure. However, relatively few studies have reported on hormonal activities, particularly estrogenic and androgenic activities of permethrin, and the results of these studies are in some respects contradictory. Therefore, this study investigated the potential estrogenic and androgenic activities of permethrin in vitro and in vivo. We conducted an uterine Calbindin-D9k (CaBP-9k) gene expression assay and an uterotrophic assay for estrogenic activity, and a Hershberger assay for androgenic activity. The CaBP-9k gene, one of the intracellular calcium binding proteins, is estrogen-responsive in the uterus. The rat uterotrophic and Hershberger assays are generally used as in vivo short-term screening assays for detecting the estrogenic and androgenic activities of chemicals, although these assays are still being validated by the Organization for Economic Cooperation and Development (OECD). Northern blot analysis showed the induction of uterine CaBP-9k mRNA level in response to permethrin as well as co-administration of permethrin with E2. In the uterotrophic assay using 18-day-old female rats, subcutaneous treatments with permethrin (10 to 800 mg/kg) for three days increased relative uterine wet weights, and E2-induced uterine weights. These effects were statistically significant at 800 and 200 mg/kg, respectively. Moreover, permethrin-induced uterine weights were inhibited by the co-administration of ICI 182,780, an antiestrogen. In the Hershberger assay, the administration of permethrin orally to testosterone propionate-treated castrated male rats led to statistically significant reductions in androgen-dependent sex accessory tissue (ventral prostate, seminal vesicles, levator ani and bulbocavernosus muscles, Cowper's gland and glans penis) weights at all doses tested (10, 50 and 100 mg/kg). These results suggest that permethrin might have estrogen-like effects on female rats, but antiandrogen-like effects on males.

Toxicogenomic Approach to Endocrine Disrupters: Identification of a Transcript Profile Characteristic of Chemicals with Estrogenic Activity

Public concerns have been raised in recent years over the possible adverse effects that may result from exposure to chemicals in the environment that have the potential to interfere with the normal function of the endocrine system in wildlife and humans ("endocrine disrupters"). Regulations have been established that require the testing of pesticides used in food crops and drinking water contaminants, for estrogenicity and other hormonal activities. In the United States, the U.S. EPA proposed the Endocrine Disrupter Screening Program, which consists of a Tier 1 screening battery of tests that is designed to identify chemicals capable of interacting with various hormonal systems, and different Tier 2 testing assays that are designed to verify and broaden the Tier 1 results. We identify 2 main problems with this approach: (1) the fact that the developmental stages that are the most susceptible to endocrine disruption are not represented in the screening tier, mainly because developmental effects tend to be latent, and there is no way to economically screen in developing models; and (2) the expense to screen each chemical to be included in this program. Thus, the need arises for an accurate, rapid, and cost effective method for assessing the potential endocrine activity of multiple chemicals during development. We hypothesize that the largely latent developmental effects of some endocrine disruptors are preceded by immediate changes in gene expression in the embryo and fetus. Therefore, an approach to assess the potential estrogenic (and other steroid hormonal) activity of different compounds is to identify those patterns of gene expression elicited in a tissue/organ exposed to these particular classes of chemicals. In this paper, the potential utility of such an approach for screening and better understanding of mechanism of action for specific chemicals with endocrine disrupter activities is presented, using as an example chemicals with estrogenic activity.

Regulation of gene expression by 8-prenylnaringenin in uterus and liver of Wistar rats.

The potential estrogenic activity of 8-prenylnaringenin has been investigated using several in vitro test systems. 8-Prenylnaringenin is a natural secondary product of the female blossoms of hops. The aim of the present study was to characterize 8-prenylnaringenin for its estrogenic effects in vivo. A three day uterotrophic assay was carried out on ovariectomized young female rats. A single dose of 8-prenylnaringenin (10 mg/day/kg body mass) was administered subcutaneously. 17beta-Estradiol (0.03 mg/day/kg body mass; subcutaneous administration) was used as a positive control. Uterine wet weight, endometrial and vaginal epithelial height were determined by histological methods. Gene expression in uterus and in liver was assessed using realtime RT-PCR. Both estradiol and 8-prenylnaringenin significantly stimulated uterine wet weight accompanied by a proliferative response. The three day treatment resulted in a statistically significant increase of the uterine epithelial height as well as of the vaginal epithelial height, the latter being the more sensitive parameter. In the uterus of ovariectomized animals estrogen receptor-alpha and clusterin gene expression were down regulated following treatment with estradiol, whereas expression of complement C3 was up-regulated. In response to treatment with 8-prenylnaringenin the same gene expression pattern was detectable, but less pronounced. The levels of estrogen receptor-alpha mRNA in rat liver were very low and therefore could not be quantitatively assessed. Like in the uterine tissue, estradiol down regulated clusterin expression. The response to 8-prenylnaringenin was weaker but still significant. Conversely, 8-prenylnaringenin was found to be more potent than estradiol in inducing expression of IGFBP-1. In summary, the multiparametric assessment of the estrogenic activity of 8-prenylnaringenin provides overwhelming evidence that 8-prenylnaringenin has largely to be regarded as a pure estrogen agonist and is therefore a questionable candidate molecule for hormone replacement therapy.

Estrogenic activities of Ginkgo biloba extracts

Ginkgo biloba extracts (GBE) are extracted from the leaves of Ginkgo biloba tree. GBE contains 24% of phytoestrogens, which is kaempferol, quercetin, and isorhamnetin. It has been reported that phytoestrogens could be a part of SERMs (Selective estrogen receptor modulators) and possibly the alternative HRT (Hormone replacement therapy) for postmenopausal women. The goal of this study was to investigate the potencies of GBE and its major components (quercetin, kaempferol, isorhamnetin) for estrogenic effect, which confirms the capacity as an alternative HRP. It was found that GBE and its major components exerted a dual action on ER-α and ER-β in competitive binding assay.The binding affinity of these chemicals to ER-β was higher than to ER-α. In the E-screen assay, these chemicals induced cell proliferation in ER-positive MCF-7 cell, but not in ER-negative MDA-MB-231 cells. The cell proliferation induced by these chemicals was blocked by tamoxifen. Also, GBE and its major components induced pS2 and PR (progesterone receptor) transcription in MCF-7 cells. Therefore these results indicated that GBE and its major components had the weak estrogenic activities through the estrogen response pathway by an interaction with the ER. In conclusion, we provided the evidence of potential estrogenic activities of GBE, which could be useful as an alternative HRP. However, further studies are required to assess the physiological significance of GBE in animals and humans.

Estrogenic effects of ethanol and ether extracts of propolis

Propolis obtained from honeybee hives has been used in Oriental folk medicine as an anti-inflammatory, anti-carcinogenic, or immunomodulatory agent. The potential estrogenic activity of propolis was investigated in vitro using the MCF-7 human breast cancer cell proliferation, human estrogen receptor (hER) binding and yeast-based steroid receptor transcription, and in vivo using the immature rat uterotrophic effect. Treatments with ethanol extract of propolis (EEP) and ether extract of propolis (REP) enhanced MCF-7 cell proliferation in concentrations ranging from 0.8 to 4 μg/ml. Both EEP and REP competed for binding of [ 3H]17β-estradiol to the hER with IC 50 values of 9.14 and 9.72 μg/ml, respectively. In yeast estrogen receptor transcription assay, both EEP and REP were found to be estrogenic with EC 50 values of 9.48, and 8.55 μg/ml, respectively. Animals treated with EEP or REP for 4 days (500–1000 mg/kg per day, s.c.) exhibited significant dose-dependent increases in uterine wet weight. However, in the yeast androgen and progesterone receptor transcription assays, either EEP or REP was found not to be active. The results suggest that propolis produces estrogenic effects through activation of estrogen receptors.

Modulation of Prolactin Expression by Xenoestrogens

Xenoestrogens are widely used environmental chemicals that have recently been under scrutiny because of their possible role as endocrine disrupters. Among them are endosulfan and chlordane, two persistent insecticides suspected to act as estrogens in living organisms. To test and better understand the potential estrogenic activity of these chemicals, we used a pituitary cell line (GH3) known to respond to estrogens by increasing its secretion of prolactin (PRL), a hormone that is well known for its many physiological functions, especially in fetal growth, development, and reproduction. We measured the levels of PRL secretion and PRL mRNA transcription using immunometric tests, Northern blots, and relative quantitative RT-PCR. We also employed the XTT proliferation assay to compare the growth of GH3 cells stimulated with 17-β estradiol and endosulfan or chlordane. Our results show that endosulfan and chlordane are able to induce a substantial increase of PRL expression while these two chemicals do not increase cell growth. Together, our results suggest that endosulfan and chlordane could indeed modulate an estrogen-inducible gene such as PRL, possibly acting via second messenger-mediated cellular mechanisms instead of solely competing with estrogens for the nuclear estrogen receptor sites.