Effect of genistein as a selective estrogen receptor beta agonist on the expression of Calbindin-D9k in the uterus of immature rats.

Genistein, a phytoestrogen possessing a high affinity for estrogen receptor beta (ERbeta), is of increasing interest because of its possible influence on the physiology of mammalian reproductive tracts. Although estrogen has been demonstrated to regulate Calbindin-D9k (CaBP-9k) in the rat uterus as with other calcium binding proteins, the role of ERbeta on the modulation of CaBP-9k remains to be elucidated. To elucidate the effect of genistein as a selective ERbeta agonist on uterine expression of CaBP-9k mRNA and protein, immature female rats were injected with genistein daily for three consecutive days in a dose-dependent (0.4, 4, and 40 mg/kg/day) and time-dependent (40 mg/kg/day; 3, 6, 12, 24, 48, and 72 h) manner. Then, the expression of CaBP-9k mRNA and protein was analyzed by Northern hybridization and Western blot, respectively, in the absence or presence of ICI 182,780 (ICI), an estrogen antagonist. In addition, the protein levels of ERalpha and ERbeta and mRNA level of progesterone receptor (PR) were further measured following genistein treatment to elucidate which of ERs is involved in CaBP-9k modulation. In a dose-dependent experiment, the highest dose of genistein (40 mg/kg/day) for 3 days significantly induced uterine CaBP-9k protein as 17beta-estradiol (E2) did. In addition, its maximal mRNA expression was observed at 3 and 6 h, and it returned to control level at 24 h in a time-dependent experiment. In parallel with its mRNA level, the protein level of CaBP-9k was significantly induced by genistein at 3 h and sustained up to 48 h. The pretreatment with ICI, followed by genistein or E2, completely blocked genistein- and E2-induced CaBP-9k protein in the uterus of immature rats. Interestingly, genistein was demonstrated to induce ERalpha protein, but not ERbeta and PR mRNA, an E2-responsive gene, in this tissue. These results imply that genistein, an ERbeta ligand, may regulate CaBP-9k gene through ERalpha pathway. Taken together, the present study demonstrated that genistein enhanced CaBP-9k gene via ERalpha in the uterus of immature rats, suggesting that ERalpha may be a key mediator in uterine CaBP-9k gene induction in immature rats.     

Novel progestogenic activity of environmental endocrine disruptors in the upregulation of calbindin-D9k in an immature mouse model.

Endocrine disruption is a major global health concern in the industrialized world. The induction of uterine calbindin-D9k (CaBP-9k), which belongs to a large family of intracellular calcium binding proteins, was used to assess the exposure of endocrine disruptors (EDs) in an immature mouse model. Sex steroid hormones have been demonstrated to regulate uterine CaBP-9k expression in the uterus of rats and mice. In particular, the mouse CaBP-9k gene was predominantly regulated by progesterone (P4), whereas rat CaBP-9k was mainly induced by 17-beta-estradiol (E2) in the uterus. In the present study, immature (14-day-old) female mice were injected with 4-tert-octylphenol (OP), nonylphenol (NP), bisphenol A (BPA), E2, or P4 to determine their effects on uterine CaBP-9k mRNA and protein expression. In addition, to specify estrogenic or progestogenic activity of EDs in the regulation of CaBP-9k, the mice were co-treated with ICI 182,780, an estrogen receptor (ER) antagonist, or RU486, a progesterone receptor (PR) antagonist,. Treatments with OP, NP, or BPA resulted in an increase in CaBP-9k mRNA and protein in the uterus of immature mice in a dose-dependent and time-dependent manner. The EDs-induced expression of CaBP-9k mRNA and protein was reversed or abolished by pretreatment with RU486 or ICI 182,780, suggesting that these synthetic chemicals may have both progestogenic and estrogenic properties by acting through PR or ER in the induction of uterine CaBP-9k mRNA and protein in this model. These results describe a novel in vivo model for detection of both estrogenic and progestogenic activities of EDs in the induction of CaBP-9k mRNA and protein in the uterus of immature mice.     

Estrogen receptor subtypes selectively mediate female mouse reproductive abnormalities induced by neonatal exposure to estrogenic chemicals

Perinatal exposure to estrogens such as diethylstilbestrol (DES), and to estrogenic chemicals, induces persistent anovulation caused by alteration of hypothalamic-pituitary-gonadal (HPG) axis, polyovular follicles, uterine abnormalities and persistent vaginal changes in mice. Most activities of estrogenic chemicals are mediated through estrogen receptor alpha (ERalpha) and/or ERbeta. However, little was known about the relative contribution of the individual ER subtypes in induction of abnormalities. We tested the effects of neonatal exposure to ER selective ligands and DES on female mice. Transactivation assays using mouse ERalpha and ERbeta showed that 10(-10)M DES activated both ER subtypes and that the ERalpha agonist (propyl pyrazole triol, PPT) and the ERbeta agonist (diarylpropionitrile, DPN) selectively activated their respective ERs at 10(-9)M. Neonatal female mice were injected subcutaneously with DES, PPT or DPN and the animals were examined at 13 and 15 weeks of age, respectively. Persistent estrous smears and anovulation were induced in all mice by 0.025-2.5 microg DES and 2.5-25 microg PPT, but not by DPN, suggesting that the observed anovulation was primarily mediated through ERalpha. Disorganization of uterine musculature and ovary-independent vaginal epithelial cell proliferation accompanied by persistent expression of EGF-related genes and interleukin-1-related genes were also mediated through ERalpha. In contrast, polyovular follicles were induced by neonatal treatment with both ERalpha and ERbeta ligands, suggesting that ovarian abnormalities are mediated through both ER subtypes.     

Novel Calbindin-D(9k) protein as a useful biomarker for environmental estrogenic compounds in the uterus of immature rats

The compounds that bind the estrogen receptors (ER) and induce or modulate an ER-mediated response can be defined as estrogenic endocrine disruptors (EDs). We demonstrated that environmental estrogens induced the uterine CaBP-9k mRNA in rats using Northern blot assay suggesting that CaBP-9k gene could be a biomarker for the estrogenicity of chemicals in the previous studies. In the present studies, we further collaborated this idea by investigating the regulation and localization of CaBP-9k protein in response to estrogenic compounds. Immature rats were injected with estrogenic chemicals, 4-tert-octylphenol (OP), nonylphenol (NP) and bisphenol A (BPA) or 17beta-estradiol (E2). After treatment with these estrogenic compounds, the effects on the accumulation of CaBP-9k protein and uterine localization were examined by Western blot analysis and immunohistochemical staining (IHC), respectively. A dose- and time-dependent increase in CaBP-9k protein was observed in the uterus of immature rats when treated with OP and NP. In addition, treatment with BPA resulted in a significant increase in CaBP-9k protein at dose of 500mg/kg BW/day. Taken together, CaBP-9k protein is strongly up-regulated by estrogenic compounds (OP, NP and BPA) and E2 itself in the uterus of immature rats. These results indicate that CaBP-9k protein can be a useful biomarker for detection of the estrogenicity of putative estrogenic compounds. Thus, regarding to risk assessment, we propose that CaBP-9k protein assay in the immature rat uterus can be a very sensitive and powerful tool to identify compounds with estrogenic activity when used in combination with classical assays.     

Assessment of estrogenic and androgenic activities of tetramethrin in vitro and in vivo assays

Tetramethrin, a synthetic pyrethroid insecticide, is used globally for agriculture, and thus potential environmental exposure to tetramethrin is a concern. Environmental chemicals that are hormonally active (particularly estrogen or androgen) may adversely affect the reproductive and endocrine systems. However, little is known about the estrogenic and androgenic activities of tetramethrin. In this study, uterine CaBP-9k gene expression assay and a uterotrophic assay were conducted for estrogenic activity assessment of tetramethrin, and a Hershberger assay was conducted for androgenic activity. Estrogen receptor (ERalpha and ERbeta) protein levels were also measured in tetramethrin-treated rat uteri. Northern blot analysis showed reduction in uterine CaBP-9k mRNA levels in response to tetramethrin, as well as when rats were given both tetramethrin and 17beta-estradiol (E2). In the uterotrophic assay using 18-d-old female Sprague-Dawley rats, subcutaneous treatment with tetramethrin (5 to 800 mg/kg/day) for 3 d led to a statistically significant decrease in absolute and relative uterine wet weights at all doses tested. Moreover, tetramethrin blocked the effect of E2 on uterine weights. In addition, tetramethrin reduced absolute and relative vaginal wet weights, and also inhibited the increases of vaginal weights produced by E2. Tetramethrin showed no androgenic on antiandrogenic activities in the Hershberger assay. These results suggest that tetramethrin might exert endocrine-disrupting effects on female rats through antiestrogenic action.     

Induction of uterine calbindin-D9k through an estrogen receptor-dependent pathway following single injection with xenobiotic agents in immature rats

Various environmental chemicals, both natural and synthetic, are believed to act as endocrine disruptors (EDs) in mammals. In this study, a new in vivo model of immature rats was used to explore the induction of calbindin-D9k (CaBP-9k) following a single injection of EDs. In a time-dependent experiment, immature rats at postnatal day 16 were treated with high doses (600 mg/kg body weight [BW]) of 4-tert-octyphenol (OP), p-nonylphenol (NP), or bisphenol A (BPA), and euthanized at different time points (3, 6, 12, 24, or 48 h). For a dose-dependent study, immature rats were given different doses (200, 400, or 600 mg/kg BW) and euthanized at 24 h after injection. After treatment with these EDs, the effects on CaBP-9k mRNA and protein were examined by Northern and Western blot analyses, respectively. An anti-estrogen, ICI 182,780, was employed to examine the potential involvement of estrogen receptor (ER) in the induction of estrogen receptor-mediated physiologic responses in vivo. A single treatment with each of the chemicals, at 600 mg/kg BW, resulted in a significant increase in the expression of CaBP-9k mRNA and protein 24 h after injection. In addition, treatment with OP, NP, or BPA resulted in a positive uterotrophic response. Cotreatment with the ER antagonist ICI 182,780 completely prevented the ED-induced uterine weight gain. Taken together, these results demonstrate that a single injection of OP, NP, or BPA results in an increase of CaBP-9k mRNA and protein via an ER-dependent pathway in the uterus of immature rats. This new model may be important to elucidate the mechanism of action of xenoestrogens on estrogen-sensitive tissue.     

ERbeta-selective estrogen receptor modulators produce antianxiety behavior when administered systemically to ovariectomized rats.

17beta-Estradiol (E2) may influence anxiety behavior; however, its effects and mechanisms are not well understood. To determine whether E2's effects on anxiety behavior may involve actions at intracellular estrogen receptor (ER) alpha or beta isoforms, selective ER modulators (SERMs) were administered (10 microg; s.c.) to ovariectomized rats 48 h before testing for anxiety behavior. Rats received sesame oil vehicle, 17beta-E2, which has a high affinity for ERalpha and ERbeta, or SERMs that vary in their activity at ERalpha and beta. ERalpha-selective SERMs were propyl pyrazole triol (PPT), which has more selective effects at ERalpha, than does the other ERalpha SERM utilized, 17alpha-E2, which also binds ERbeta. ERbeta-selective SERMs were diarylpropionitrile (DPN) and 7,12-dihydrocoumestan (coumestrol). DPN is more selective at ERbeta than coumestrol, which also binds ERalpha. 17beta-E2 and ERbeta-selective SERMs (DPN, coumestrol) produced clear antianxiety behavior in the open field, elevated plus maze, emergence, light-dark transition, defensive freezing, and Vogel punished drinking tasks. Anxiety behavior of rats administered ERalpha-selective SERMs (PPT, 17alpha-E2) was not different from vehicle; however, PPT and 17alpha-E2 enhanced sexual receptivity in a manner similar to 17beta-E2. Coadministration of tamoxifen (10 mg/kg) blocked the antianxiety behavior produced by 17beta-E2, DPN, or coumestrol. Together, these data suggest that actions at ERbeta may underlie some of E2's antianxiety effects.     

Antidepressant effects of ERbeta-selective estrogen receptor modulators in the forced swim test

Estradiol (E2) may influence depressive symptomology of women and decrease depressive behavior among rodents. The mechanism(s) for E2's antidepressant effects are not well understood. To determine whether antidepressant effects of E2 may involve actions at intracellular estrogen receptor (ER) alpha or beta isoforms, selective ER modulators (SERMs) were administered (10 microg sc) to ovariectomized rats 48 h before testing in the forced swim test, an animal model of depression, and the horizontal crossing task. Rats received sesame oil vehicle, 17beta-E2, which has a high affinity for ERalpha and ERbeta, SERMs that vary in their activity at ERalpha and beta, or a tricyclic antidepressant (desipramine; 30 mg/kg ip), as a positive control. ERalpha-selective SERMs were propyl pyrazole triol (PPT) and 17alpha-E2. PPT has more selective effects at ERalpha than does 17alpha-E2, which also binds ERbeta. ERbeta-selective SERMs were diarylpropionitrile (DPN) and 7,12-dihydrocoumestan (coumestrol). DPN is more selective at ERbeta than coumestrol, which also binds ERalpha. 17beta-E2, ERbeta-selective SERMs (DPN, coumestrol), and desipramine administration produced antidepressive behavior (decreased immobility, increased struggling and swimming). ERalpha-selective SERMs (PPT, 17alpha-E2) were not different from vehicle. There were no differences among groups in the number of beam breaks made in the horizontal crossing task. These data suggest that E2's antidepressive effects may involve actions at ERbeta.     

Tetrabromodiphenyl ether (BDE 47) evokes estrogenicity and calbindin-D9k expression through an estrogen receptor-mediated pathway in the uterus of immature rats.

Polybrominated diphenyl ethers (PBDEs), a class of organic brominated flame retardants, have been increasing in the environment and in the tissues and milk of animals, including humans. To date, 209 PBDE congeners have been reported. Among these, 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) is the dominant congener found in humans and animals. A number of studies have suggested that BDE 47 possesses the potential to disrupt the endocrine system, as well as reproductive functions. This suggests that BDE 47 may act as a developmental neurotoxin and endocrine disruptor. In this study, we employed immature rats as a developmental model to examine the potential involvement of BDE 47 in the induction of calbindin-D9k (CaBP-9k), which is a novel biomarker for screening estrogenic compounds. Beginning on postnatal day 16, BDE 47 was administered to immature rats in a dose- and time-dependent manner for 3 days. The biological effects of BDE 47 on the induction of CaBP-9k mRNA and protein were examined by semiquantitative RT-PCR and western blotting, respectively. In addition, the physiological role of the estrogen receptor (ER) in BDE 47-induced CaBP-9k expression was examined in vivo. Treatment with a high dose of BDE 47 (200 mg/kg body weight [BW]/day) resulted in a significant increase in CaBP-9k mRNA and protein 24 h after injection, whereas a modest increase was observed with low and medium doses (50 and 100 mg/kg BW/day). Additionally, treatment with the high dose of BDE 47 induced a clear uterotrophic response. Cotreatment with ICI 182,780, an ER antagonist, completely reversed the BDE 47-induced increases in uterine wet weight and CaBP-9k mRNA and protein. Taken together, these results demonstrate that BDE 47 exposure results in increases in CaBP-9k mRNA and protein in the uteri of immature rats. The biochemical pathway for BDE 47-induced activity may involve the ER-mediated signaling pathway. These results provide new insights into the estrogenic effects of BDE 47 at a critical developmental stage of the female reproductive system.     

Anti-progestogenic effect of flutamide on uterine expression of calbindin-D9k mRNA and protein in immature mice

A calcium binding protein, calbindin-D_9k (CaBP-9k), is a cytosolic protein and regulated by steroid hormones in the reproductive tissues. Mouse CaBP-9k gene was predominantly regulated by progesterone (P4), whereas rat CaBP-9k was mainly regulated by 17beta-estradiol (E2) in the uterus. The induction of CaBP-9k can be employed as a biomarker for steroidal substrates as endocrine disruptors (EDs). Flutamide (FLU) is a non-steroidal anti-androgen or pro-drug that is rapidly metabolized to hydroxyflutamide, which may have both an anti-androgenic and anti-progestogenic activities. Thus, in the present study, we employed immature mice (14-day-old) subcutaneously injected with P4 (20 mg/kg/day) and/or FLU (5 mg/kg/day) for 3 consecutive days in the presence or absence of RU486, a pure PR antagonist (30 mg/kg/day), to analyze uterine CaBP-9k expression in this model. When immature mice were treated with P4, the expression levels of CaBP-9k mRNA and protein were significantly increased by P4. P4-induced expression levels of CaBP-9k mRNA and protein were abolished by FLU, in part, suggesting that FLU is a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mice. In addition, P4-induced CaBP-9k expression was completely reversed by RU486. Increased expression levels of CaBP-9k mRNA and protein were maintained for 24 h after final injection with P4 in a time-dependent manner. However, CaBP-9k mRNA rapidly disappeared after 48 h and its protein level is similar with its mRNA. Treatment with FLU suppressed partially P4-induced CaBP-9k mRNA and protein until 24 h. Taken together, these results indicate that FLU has an anti-progestogenic activity and plays a role as a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mouse model.     

Effects of estrogen receptor subtype-selective agonists on immune functions in ovariectomized mice

Estrogens have multiple influences on immune functions. Estrogen receptors (ERs) have two distinct subtypes - alpha and beta. To explore the specific roles of each ER subtype in estrogen-mediated immunomodulation, we investigated the effects of ER subtype-selective agonists on immune functions in ovariectomized Balb/c mice. Treatment with ERalpha-selective agonist propyl pyrazole triol (PPT) caused thymic atrophy and significant changes in thymic CD4/CD8 phenotypic profile. In contrast, ERbeta-selective agonist diarylpropionitrile (DPN) alone had no effect on thymic weight, cellularity or CD4/CD8 phenotype expression. When coadministered with PPT, DPN partially antagonized PPT-evoked decrease in thymic cellularity and also partially attenuated PPT-induced shifts in thymic T-cell phenotype. These results indicate that ERalpha plays a predominant role in estrogen-induced thymic atrophy and ERbeta activation may partially down-regulate ERalpha-mediated effects on thymic cellularity and T-cell phenotype expression. In addition, PPT administration induced a reduction in the percentage of mature B cells in the spleen, and enhanced IFN-gamma production but suppressed IL-6 production from in vitro Con A-stimulated splenocytes as estradiol (E(2)) did, whereas DPN treatment had no effects either alone or with PPT, suggesting ERalpha mediates these estrogen actions. Treatment with PPT or DPN did not augment anti-DNP antibody production after DNP-KLH immunization as E(2) did, implying that not merely one ER signaling pathway is involved in mediating estrogen's effects on specific humoral immune responses. Our study further indicates ER subtype-selective agonists provide a novel approach to explore each ER subtype-mediated immunomodulation.     

Anti-progestogenic effect of flutamide on uterine expression of calbindin-D9k mRNA and protein in immature mice

A calcium binding protein, calbindin-D_9k (CaBP-9k), is a cytosolic protein and regulated by steroid hormones in the reproductive tissues. Mouse CaBP-9k gene was predominantly regulated by progesterone (P4), whereas rat CaBP-9k was mainly regulated by 17beta-estradiol (E2) in the uterus. The induction of CaBP-9k can be employed as a biomarker for steroidal substrates as endocrine disruptors (EDs). Flutamide (FLU) is a non-steroidal anti-androgen or pro-drug that is rapidly metabolized to hydroxyflutamide, which may have both an anti-androgenic and anti-progestogenic activities. Thus, in the present study, we employed immature mice (14-day-old) subcutaneously injected with P4 (20 mg/kg/day) and/or FLU (5 mg/kg/day) for 3 consecutive days in the presence or absence of RU486, a pure PR antagonist (30 mg/kg/day), to analyze uterine CaBP-9k expression in this model. When immature mice were treated with P4, the expression levels of CaBP-9k mRNA and protein were significantly increased by P4. P4-induced expression levels of CaBP-9k mRNA and protein were abolished by FLU, in part, suggesting that FLU is a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mice. In addition, P4-induced CaBP-9k expression was completely reversed by RU486. Increased expression levels of CaBP-9k mRNA and protein were maintained for 24 h after final injection with P4 in a time-dependent manner. However, CaBP-9k mRNA rapidly disappeared after 48 h and its protein level is similar with its mRNA. Treatment with FLU suppressed partially P4-induced CaBP-9k mRNA and protein until 24 h. Taken together, these results indicate that FLU has an anti-progestogenic activity and plays a role as a partial antagonist of P4 in the regulation of uterine CaBP-9k in immature mouse model.     

Functional characterization of estrogen receptor subtypes, ERalpha and ERbeta, mediating vitellogenin production in the liver of rainbow trout

The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ERalpha and ERbeta, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ERalpha selective agonist, methyl-piperidino-pyrazole (MPP) an ERalpha selective antagonist, and diarylpropionitrile (DPN) an ERbeta selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbow trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [(3)H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ERalpha could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ERbeta. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ERalpha. On the other hand, once blocked ERalpha with MPP, the only manifestation of agonist activity of estradiol would be achieved via ERbeta. In conclusion, the present results indicate that vitellogenin production is mainly mediated through ERbeta, implying, furthermore that compounds which only exhibit ERalpha selectivity are not detected by vitellogenin bioassay.     

Potential estrogenic activity of triclosan in the uterus of immature rats and rat pituitary GH3 cells

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol; TCS) is used as an antimicrobial agent in personal care, pharmaceutical, industrial, and household products. In this study, we established an in vivo model for screening estrogenic activity of TCS in the uteri of immature rats. In addition, we employed temporarily transfected cells with plasmids containing estrogen response element (ERE) and progesterone (P4) response element (PRE) sequences. We found that uterine weight was significantly increased by 17α-ethinylestradiol (EE) as a positive control and TCS at doses of 7.5, 37.4, and 187.5 mg/kg. In addition, the expressions of calbindin-D_9k (CaBP-9k) and complement C3 (C3) were significantly induced by EE and TCS in the uteri of immature rats, indicating that TCS can induce their expression mediated by estrogenic activity. Co-treatment with steroid antagonists ICI 182,780 (ICI) and RU 486 in conjunction with TCS (37.5 mg/kg) reversed TCS-induced uterine weight and CaBP-9k mRNA and protein expression increases in immature rats. Moreover, ERE and PRE luciferase activity was evaluated in GH3 cells following treatment with TCS. Concentrations of TCS at increasing doses (10⁻⁹, 10⁻⁷, and 10⁻⁵ M) resulted in a significant increase in ERE luciferase activity compared to control; however, no difference was observed in PRE luciferase activity following TCS treatment. To confirm that ER signaling is involved in TCS-induced CaBP-9k expression, we treated GH3 cells with the anti-estrogen ICI, which can block TCS-induced up-regulation of CaBP-9k in these cells. Taken together, these results indicate that TCS has an estrogen-like property, which may be mediated through an ER-involved signaling pathway in both in vivo and in vitro models.     

Transfer of maternally injected endocrine disruptors through breast milk during lactation induces neonatal Calbindin-D9k in the rat model

The uterus is a highly estrogen-responsive tissue, which can be measured through changes in CaBP-9k expression. In this study, we investigated the potential for estrogenic compounds 4-tert-octylphenol (OP), nonylphenol (NP), bisphenol A (BPA), diethylstilbestrol (DES) and 17beta-estradiol (E2) to be transferred through breast milk from dam to neonate during lactation using the induction of CaBP-9k in uterine tissue as a biomarker. Dams were treated with OP, NP and BPA, dissolved in corn oil, at doses of 200, 400 and 600 mg/kg body weight per day l for 5 days after delivery. Dams and neonates were euthanized after 24h. Treatment with these estrogenic compounds increased the expression of CaBP-9k mRNA in the maternal uterus, in a dose-dependent manner. All doses of estrogenic compounds resulted in an increase in CaBP-9k protein levels. These compounds have an estrogenic effect on the maternal uterus during the lactation period as shown by the induction of both CaBP-9k mRNA and protein. In the neonatal uterus, the expression of CaBP-9k mRNA and protein significantly increased with DES exposure. There was a significant increase in CaBP-9k mRNA in neonatal uterus when the dams were treated with high doses of estrogenic compounds, but protein levels of CaBP-9k were undetectable. Taken together, these findings suggest that maternally injected estrogenic compounds may be transferred to neonates through breast milk and thus affecting uterine function, as shown by the induction of CaBP-9k gene expression in the neonatal uterus.     

Effects of the pesticides prochloraz and methiocarb on human estrogen receptor alpha and beta mRNA levels analyzed by on-line RT-PCR.

Exposure to endocrine disrupters such as dioxins, PCBs and certain pesticides are suspected to affect human reproductive health. We have analyzed the effect of the currently used pesticides prochloraz and methiocarb on the estrogen receptor (ER)alpha and beta mRNA levels in parallel with the natural ligand, 17beta-estradiol (E2). Using the highly sensitive on-line RT-PCR technique we were able to quantify the ERalpha and ERbeta mRNA levels in the human breast cancer cell line, MCF7-BUS. Upon exposure with E2 or prochloraz a down regulation of ERalpha and ERbeta mRNAs was observed after 48 h of treatment. Co-treatment with the ER antagonist ICI 182,780 abolished these mRNA down regulations. Western blot analyses elicited a decreased ER protein level after 3 h of exposure with prochloraz but after 24 h the ERalpha protein level had recovered to basal level. Methiocarb exposure had no effect on the ERalpha mRNA level, whereas an increase in the ERbeta mRNA level was observed after 3 h of exposure. Our study demonstrates that like E2, prochloraz had the potential to down regulate the expression of ERalpha and ERbeta mRNAs as well as the ERalpha protein level in MCF7-BUS cells.     

ERbeta mediates the estradiol increase of D2 receptors in rat striatum and nucleus accumbens

Estradiol was previously reported to increase striatal D(2) receptor density. The following experiments investigated the contribution of each estrogen receptor in estradiol modulation of D(2) receptors. Ovariectomized Sprague-Dawley rats were treated for 2 weeks with an agonist for ERalpha, 4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), an agonist for ERbeta, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and compared to estradiol treatment. Ovariectomy decreased D(2) agonist and antagonist striatal binding sites, specific binding was measured using [(3)H]quinpirole and [(3)H]spiperone. Estradiol prevented this decrease, while DPN but not PPT mimicked the estradiol increase of D(2) receptor specific binding. In the nucleus accumbens, ovariectomy decreased [(3)H]quinpirole specific binding in the core and left the shell unchanged. Similarly, estradiol and DPN but not PPT prevented this decrease. Neither ovariectomy nor treatments affected [(3)H]spiperone specific binding in this area. In the olfactory tubercle, neither ovariectomy nor treatments changed D(2) receptor binding. Finally, both ovariectomy and treatments did not affect D(2L), D(2S) mRNA and D(2L)/D(2S) ratios measured by semi-quantitative RT-PCR. The present results show, for the first time, that an ERbeta agonist treatment modulates D(2) receptors and suggest that ERbeta is involved in the estradiol modulation of D(2) receptors.