Pregnancy outcomes for mouse preimplantation embryos exposed in vitro to the estrogenic pesticide o,p'-DDT

Pregnancy outcomes were evaluated following uterine transfer of murine preimplantation embryos exposed in vitro to the estrogenic pesticide o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT). Single-cell embryos were incubated 72 h in medium droplets containing 0.1% ethanol (control) or 0.1 microg/ml o,p'-DDT (pesticide). Morula and preblastocyst embryos were transferred in groups of eight to right uterine horns of pseudopregnant mice (n=111) and pups (n=132) were evaluated at Caesarean-section (C-section). In vitro exposure to o,p'-DDT reduced development to morula (P<0.001) and modestly increased blastomere apoptosis (P=0.05). However, treatment differences were not detected for implantation rates (35% versus 39%; P=0.64), pup numbers per dam (2.3 versus 1.9; P=0.36), transfer efficiencies (16% versus 14%; P=0.53), fetal weights (1.56 g versus 1.57 g; P=0.91), skeletal abnormalities (55% versus 66%; P=0.47), or male ratios (54.8% versus 53.8%; P=1.0). In vitro exposure of preimplantation embryos to 0.1 microg/ml o,p'-DDT for 72 h resulted in no measurable effects on subsequent implantation or pup characteristics at C-section.     

Development and characterization of a cell line that stably expresses an estrogen-responsive luciferase reporter for the detection of estrogen receptor agonist and antagonists.

Recently several advisory committees (EDSTAC, ICCVAM) have recommended that stable estrogen-dependent gene expression assays be developed for screening chemicals for estrogenic activity because of the high degree of specificity of the response and potential for use in a high-throughput mode. In this paper we describe a specific, sensitive assay developed for screening chemicals for estrogenic and antiestrogenic activities. T47D human breast cancer cells, which naturally express estrogen receptor (ER) alpha and beta, were stably transfected with a triplet ERE (estrogen-responsive elements)-promoter-luciferase reporter gene construct. The transformed cells were named T47D-KBluc. These cells are sensitive to the potent estrogens, 17beta-estradiol, ethynyl estradiol, and diethylstibesterol, and well-characterized weaker environmental estrogens like genistein, HPTE (an estrogenic pesticide metabolite), and 4-nonylphenol. The EC50 for estradiol was about 0.01 nM, reaching maximal induction at 0.1 nM. The antiestrogen, ICI 182,780, was able to completely inhibit the induction of luciferase expression by 0.1 nM estradiol at 10 nM, with an IC50 of 1 nM. In addition, we were able to replicate, in this in vitro assay, the observation that low concentrations of cadmium were able to induce estrogen-dependent gene expression, an effect that was completely inhibited by the potent antiestrogen ICI 182,780. The potent glucocorticoid receptor agonist, dexamethasone, was without effect as an ER agonist at concentrations up to 10 nM, whereas the potent androgen, dihydrotestosterone (DHT), showed no induction at concentration of 50 microM, but was a partial agonist at high concentrations of 0.2 mM and above. In summary, we have developed a specific, sensitive estrogen-responsive gene expression assay in a stable cell line that could possibly be adapted for high throughput screening of large numbers of chemicals for estrogenic and antiestrogenic activity. In addition, herein we also provide key protocol recommendations necessary to identify and eliminate common problems encountered in in vitro screening for estrogenicity.     

Assessing the estrogenic potential of organochlorine pesticides in primary cultures of male rainbow trout (Oncorhynchus mykiss) hepatocytes using vitellogenin as a biomarker

Many organochlorine pesticides are suspected of impairing natural hormonal function in organisms by mimicking endogenous estrogen. The aim of this study was to assess the estrogenic activity of the organochlorine pesticides o,p'-DDT, dieldrin, aldrin, heptachlor, mirex and DDT in rainbow trout hepatocyte cultures using vitellogenin (Vtg) as the biomarker. A wide range of pesticide concentrations (0.0001-100 microM) was evaluated. Among the pesticides tested, o,p'-DDT was the most potent inducer of Vtg. The lower potency of technical grade DDT relative to o,p'-DDT could be explained by the fact that this pesticide is a mixture of two different pesticides (18% o,p'-DDT and 77% p,p'-DDT). This suggests that o,p'-DDT is a stronger inducer of Vtg than p,p'-DDT. A simple hypothesis could be that pesticides mixed together competed for the same receptor to favor the formation of a complex with reduced activity towards EREs. If these compounds are classified according to the level of Vtg secreted, we observed the following decreasing order: 17beta-estradiol (E(2))>o,p'-DDT>dieldrin>aldrin>DDT. Non-toxic levels of these compounds competed with E(2) for binding to the estrogen receptor. Heptachlor and mirex did not induce Vtg. Since the latter compounds failed to stimulate Vtg production, the possibility that they could interfere with the estrogenic response by inhibiting E(2) action was tested. In the presence of heptachlor, Vtg production triggered by E(2) significantly decreased. The EC50 value for inhibition of ER binding by heptachlor was cytotoxic for hepatocytes in culture, and this could in part explain the lack of Vtg response observed with this compound at the concentrations tested. Our results indicate that organochlorine pesticides can act as positive or negative modulators of estrogenic function in rainbow trout.     

Uncoupling of 5-HT1A receptors in the brain by estrogens: regional variations in antagonism by ICI 182,780

Previously we have shown that 17beta-estradiol (in vivo and in vitro) rapidly decreases the function of serotonin(1A) (5-HT(1A)) receptors, allowing us to hypothesize that 17beta-estradiol accomplished this via activation of a membrane estrogen receptor. Hippocampus and frontal cortex obtained from ovariectomized rats were incubated with 17beta-estradiol or bovine serum albumin (BSA)-estradiol in the presence or absence of the estrogen receptor (ER) antagonist ICI 182,780. Membranes were prepared to measure R(+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding (a measure of 5-HT(1A) receptor coupling and function). In both hippocampus and frontal cortex, 17beta-estradiol and BSA-estradiol (50 nM) decreased R(+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding. ICI 182,780 blocked the effect of both the estrogens in hippocampus, but only the effect of 17beta-estradiol in frontal cortex. Due to the inability of ICI 182,780 to block the effects of BSA-estradiol in frontal cortex, similar experiments were performed using the selective estrogen receptor modulator tamoxifen as the agonist. Tamoxifen (100 nM and 1 microM) decreased R(+)8-OH-DPAT-stimulated [(35)S]GTPgammaS binding. ICI 182,780 (1 microM) blocked the ability of tamoxifen to decrease 5-HT(1A) receptor coupling in the hippocampus, but not in the frontal cortex. Taken together, these data support the existence of a pharmacologically distinct ER in hippocampus vs. frontal cortex that might be responsible for rapid uncoupling of 5-HT(1A) receptors.     

Estradiol and endocrine disrupting compounds adversely affect development of sea urchin embryos at environmentally relevant concentrations

Environmental endocrine disrupting compounds (EDCs) are a wide variety of chemicals that typically exert effects, either directly or indirectly, through receptor-mediated processes, thus mimicking endogenous hormones and/or inhibiting normal hormone activities and metabolism. Little is known about the effects of EDCs on echinoderm physiology, reproduction and development. We exposed developing sea urchin embryos (Strongylocentrotus purpuratus and Lytechinus anamesus) to two known EDCs (4-octylphenol (OCT), bisphenol A (BisA)) and to natural and synthetic reproductive hormones (17beta-estradiol (E2), estrone (E1), estriol (E3), progesterone (P4) and 17alpha-ethynylestradiol (EE2)). In addition, we studied two non-estrogenic EDCs, tributyltin (TBT) and o,p-DDD. Successful development to the pluteus larval stage (96 h post-fertilization) was used to define EDC concentration-response relationships. The order of compound potency based on EC50 values for a reduction in normal development was as follows: TBT(L. anamesus)>OCT>TBT(S. purpuratus)>E2>EE2>DDD>BisA>P4>E1>E3. The effect of TBT was pronounced even at concentrations substantially lower than those commonly reported in heavily contaminated areas, but the response was significantly different in the two model species. Sea urchin embryos were generally more sensitive to estrogenic EDCs and TBT than most other invertebrate larvae. Stage-specific exposure experiments were conducted to determine the most sensitive developmental periods using blastula, gastrula and post-gastrula (pluteus) stages. The stage most sensitive to E2, OCT and TBT was the blastula stage with less overall sensitivity in the gastrula stage, regardless of concentration. Selective estrogen receptor modulators (SERMs) were added to the experiments individually and in combination with estrogenic EDCs to interfere with potential receptor-mediated actions. Tamoxifen, a partial ER agonist, alone inhibited development at concentrations as low as 0.02 ng/ml and was effective at this concentration in decreasing the sensitivities of the embryos to estradiol and estrogenic EDCs. The complete antagonist ICI 182,780 inhibited development at concentrations as low as 0.03 ng/ml but increased embryo sensitivity to estradiol and estrogenic EDCs. Estradiol and estrogenic EDCs all cause developmental toxicity in sea urchins through a TAM-sensitive but an ICI-insensitive mechanism. It remains to be demonstrated whether this mechanism involves an estrogen-responsive nuclear receptor (NR), a membrane receptor (NR or non-NR-related) or a completely different mechanism of toxicity. However, early embryo sensitivity and the differential response to SERM co-incubation further suggests more than one mode of EDC action in the developing sea urchin embryo.     

Design, synthesis, and estrogenic activity of a novel estrogen receptor modulator--a hybrid structure of 17beta-estradiol and vitamin E in hippocampal neurons

We recently discovered that ICI 182,780 (1), an antagonist of estrogen receptor (ER)-dependent proliferation in reproductive tissues, functions as an estrogenic agonist in primary neurons. The present study investigated whether the agonist properties of 1 in neurons could be translated into structural analogs. 7alpha-[(4R,8R)-4,8,12-trimethyltridecyl]estra-1,3,5-trien-3,17beta-diol (2), a hybrid structure of 17beta-estradiol and vitamin E, was synthesized and found to bind to both ERalpha and ERbeta. In vitro analyses demonstrated that 2 was neuroprotective and effective in activating molecular mechanisms associated with estrogenic agonist activity in rat primary hippocampal neurons. Collectively, the data support an estrogenic agonist profile of 2 action comparable to 1 in primary neurons, confirming that estrogenic activity of 1 in neurons is not a unique phenomenon. These results provide support for the development of a brain-selective ER modulator, with potential as an efficacious and safe estrogen alternative to prevent Alzheimer's disease and cognitive decline in postmenopausal women.     

Estrogenic activity of DDT: estrogen-receptor profiles and the responses of individual uterine cell types following o,p'-DDT administration

The administration of o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT) to immature rats stimulates DNA synthesis and cell division in the uterine luminal epithelium (LE), stroma (S), and myometrium (M). The time course of DNA synthesis/cell division in the S and M is similar following administration of o,p'-DDT or 17 beta-estradiol, but the maximum response following pesticide treatment is only 70% of that produced by the hormone. In the LE both compounds yield the same maximum response, but the time course of DNA synthesis/cell division is delayed following o,p'-DDT administration relative to 17 beta-estradiol treatment. The patterns of estrogen receptor retention in uterine nuclei following o,p'-DDT administration are prolonged relative to those observed after 17 beta-estradiol treatment. o,p'-DDT thus produces the uterine hyperplasia characteristic of estrogens, but the magnitude and timing of the response is dependent on the specific cell type observed and is different from that produced by estradiol.     

Methoxychlor and its metabolite HPTE inhibit cAMP production and expression of estrogen receptors α and β in the rat granulosa cell in vitro

The major metabolite of the estrogenic pesticide methoxychlor (MXC) HPTE is a stronger ESR1 agonist than MXC and acts also as an ESR2 antagonist. In granulosa cells (GCs), FSH stimulates estradiol via the second messenger cAMP. HPTE inhibits estradiol biosynthesis, and this effect is greater in FSH-treated GCs than in cAMP-treated GCs. Therefore; we examined the effect of MXC/HPTE on FSH-stimulated cAMP production in cultured GCs. To test involvement of ESR-signaling, we used the ESR1 and ESR2 antagonist ICI 182,780, ESR2 selective antagonist PHTPP, and ESR2 selective agonist DPN. ESR1 and ESR2 mRNA and protein levels were quantified. Both HPTE and MXC inhibited the FSH-induced cAMP production. ICI 182,780 and PHTPP mimicked the inhibitory action of HPTE. MXC/HPTE reduced FSH-stimulated Esr2 mRNA and protein to basal levels. MXC/HPTE also inhibited FSH-stimulated Esr1. The greater inhibition on FSH-stimulated GCs is likely due to reduced cAMP level that involves ESR-signaling, through ESR2.     

The pathway-selective estrogen receptor ligand WAY-169916 reduces infarct size after myocardial ischemia and reperfusion by an estrogen receptor dependent mechanism

Previous studies have shown that estrogen treatment protects the heart from reperfusion injury. The adverse effects of long-term estrogen treatment limit its clinical use and emphasize the need for the development of specific pharmacological interventions such as pathway-selective estrogen receptor (ER) ligands. Pathway-selective ER ligands are compounds that retain estrogen's anti-inflammatory ability, but they are devoid of conventional estrogenic action. In the present study, the pathway-selective ER ligand WAY-169916 was assessed for its cardioprotective potential in an in vivo model of ischemia-reperfusion injury. Anesthetized, ovariectomized rabbits were administered WAY-169916 (1 mg/kg), 17beta-estradiol (E2; 20 microg/rabbit), or vehicle intravenously 30 minutes before a 30-minute occlusion and 4 hours of reperfusion. Acute treatment with either WAY-169916 or E2 resulted in a decrease in infarct size, expressed as a percent of area at risk (WAY-169916, 21.2 +/- 3.3; P < 0.001 and E2, 18.8 +/- 1.7; P < 0.001) compared with vehicle 59.4 +/- 5.4). Pretreatment with estrogen receptor antagonist ICI 182,780 significantly limited the infarct size sparing effect of both WAY-169916 and E2 when expressed as a percent of the risk region (WAY 169916, 47.4 +/- 4.4; E2, 53.01 +/- 5.0). The results demonstrate that WAY-169916 protects the heart against ischemia-reperfusion injury through an ER-dependent mechanism.     

Effect of methoxychlor and estradiol on cytochrome p450 enzymes in the mouse ovarian surface epithelium.

Although the ovarian surface epithelium (OSE) is responsive to hormones and endocrine-disrupting chemicals, little information is available on the metabolizing capabilities of the OSE. Thus, we tested the hypothesis that the OSE is capable of expressing genes regulating phase I metabolism of estrogen and the estrogenic endocrine disruptor methoxychlor (MXC). To test this hypothesis, we isolated mouse OSE cells and cultured them with vehicle (dimethylsulfoxide; DMSO), 3 microM MXC, or 0.1 microM 17beta-estradiol (E2) +/- the anti-estrogen ICI 182,780 (1 microM) for 14 days. After culture, the cells were subjected to quantitative real-time polymerase chain reaction for cytochrome P450s (CYPs) 1A1, 1B1, 2C29, and 1A2, and estrogen receptor alpha (ERalpha). Our results indicate that E2 and MXC did not alter the expression of CYP1A1 or CYP1A2. In contrast, E2 significantly increased expression of CYP1B1 compared to controls (DMSO = 0.93 +/- 0.1, E2 = 3.12 +/- 0.64 genomic equivalents (GE), n = 4, p < or = 0.01). The E2-induced increase in CYP1B1 was abolished by co-treatment with ICI 182,780 (0.41 +/- 0.17 GE). MXC treatment did not affect CYP1B1 expression. Both MXC and E2 increased expression of CYP2C29 (DMSO = 0.02 +/- 0.003; MXC = 0.04 +/- 0.008; E2 = 0.46 +/- 0.03 GE, n = 4, p < or = 0.05). MXC- and E2-induced elevations in CYP2C29 were abolished by co-treatment with ICI 182,780 (0.02 +/- 0.005; 0.02 +/- 0.07 GE). In addition, E2 increased ERalpha expression 15-fold compared to controls (DMSO = 1.10 +/- 0.09, E2 = 15.0 +/- 3.60 GE, n = 3, p < or = 0.05), and ICI 182,780 abolished the E2-induced increase in ERalpha expression (1.85 +/- 1.09 GE). MXC treatment did not affect ERalpha expression. These data indicate that the OSE expresses enzymes known to metabolize native and xenoestrogens and that MXC and E2 modulate expression of some of them through ER-linked mechanisms.     

Analysis of the effects of oestrogen receptor alpha (ERalpha)- and ERbeta-selective ligands given in combination to ovariectomized rats

BACKGROUND AND PURPOSE: Studies with oestrogen receptoralpha (ERalpha)- and ERbeta-selective compounds have already shown that the effects of 17beta-estradiol (E2) on body weight, movement drive and bone-protection are mediated via ERalpha. This study was based on the hypothesis that activation of ERbeta may antagonize ERalpha-mediated effects and designed to investigate potential effects of ERalpha/ERbeta heterodimers. EXPERIMENTAL APPROACH: Ovariectomized (OVX) female Wistar rats were treated with combinations of the ERalpha-specific agonist 16alpha-LE2 (ALPHA; 1 and 10 microg kg(-1) d(-1)), the ERbeta-specific agonist 8beta-VE2 (BETA; 100 microg kg(-1) d(-1)), the phytoestrogen, genistein (10 mg kg(-1) d(-1)) and with the anti-oestrogen compound, ICI 182,780 (3 mg kg(-1) d(-1)) for three weeks. The combined effects of the substances on body weight increase, tibial bone mineral density (BMD) and the influence on running wheel activity (RWA) were investigated. KEY RESULTS: OVX-induced body weight increase was reduced by co-administration of genistein and BETA. Co-application of BETA or genistein with ALPHA had no effect on ALPHA-mediated bone-protection. The RWA of OVX animals was significantly reduced by treatment with genistein but stimulated by application of ALPHA. The stimulatory effect of ALPHA on RWA could be antagonized by co-treatment with the pure antioestrogen ICI 182,780 but also by co-administration of genistein or BETA. CONCLUSIONS AND IMPLICATIONS: Our results indicate that activation of ERbeta may modulate ERalpha-mediated physiological effects in vivo. The observation that substances with selective affinity for ERbeta are able to antagonize distinct physiological functions, like RWA, may be of great relevance to the pharmaceutical use of such drugs.     

Developmental alterations in murine embryos exposed in vitro to an estrogenic pesticide, o,p'-DDT

Culturing pronuclear embryos from CD-1 mice with o,p′-DDT and p,p′-DDT was examined as a means for directly evaluating toxicant risk and for increasing the speed of screening developmental toxicants. Pronuclear (2PN) embryos from CD-1 mice were cultured 96 h in modified Earle’s balanced salt solution containing 0.1% (v/v) ethanol (control) or 10-fold dilutions of 17β-estradiol, o,p′-DDT, or p,p′-DDT. Compared to control treatment, 96 h incubation of 2PN embryos with 0.1 μg/mL o,p′-DDT significantly reduced embryo development to blastocyst and mean cell number, and increased the percentage of cells undergoing apoptosis. The effects of o,p′-DDT on developmental parameters were dose-responsive. Embryo sexing by multiplex polymerase chain reaction indicated that both sexes were susceptible to toxicant injury with comparable reduction in development to blastocyst (27% and 24%, respectively) in the presence of o,p′-DDT. Results of this study suggest that in vitro exposure of preimplantation embryos to xenobiotics may provide a useful tool for rapidly screening developmental toxicants.     

Methoxychlor metabolites may cause ovarian toxicity through estrogen-regulated pathways.

The pesticide methoxychlor (MXC) is a reproductive toxicant that targets antral follicles of the mammalian ovary. Cytochrome P450 enzymes metabolize MXC to mono-OH MXC (1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane [mono-OH]) and bis-OH MXC (1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane [HPTE]), two compounds that are proposed to be more toxic than the parent compound, can interact with the estrogen receptor (ER), and are proposed to be responsible for ovarian toxicity. Thus, this work tested the hypothesis that MXC metabolites may be responsible for inducing antral follicle-specific toxicities in the ovary and that this toxicity may be mediated through ER-regulated pathways. Mouse antral follicles were isolated and exposed to mono-OH (0.01-10 microg/ml), HPTE (0.01-10 microg/ml), or MXC (100 microg/ml) alone or in combination with ICI 182,780 (ICI; 1 microM) or 17beta-estradiol (E2; 10 and 50 nM) for 96 h. Follicle diameters were measured at 24-h intervals. After culture, follicles were morphologically evaluated for atresia. Both mono-OH and HPTE (10 microg/ml) inhibited follicle growth and increased follicle atresia. The antiestrogen, ICI, did not protect antral follicles from MXC or metabolite toxicity in regard to follicle growth or atresia, but E2 decreased MXC- and mono-OH-induced atresia in small antral follicles. These data suggest that MXC metabolites inhibit follicle growth and induce atresia and that ER-regulated pathways may mediate the ovarian toxicity of MXC and its metabolites.     

Modulation of estrogen receptor-dependent reporter construct activation and G0/G1-S-phase transition by polycyclic aromatic hydrocarbons in human breast carcinoma MCF-7 cells.

It has been suggested that the estrogenicity of PAHs could contribute to their carcinogenic effects via increased tissue-specific cell proliferation. Both benzo[a]pyrene (BaP) and benz[a]anthracene (BaA) are known to weakly activate estrogen receptor (ER)-dependent reporter constructs. In this study, several other PAHs, including fluorene, fluoranthene, pyrene, chrysene, phenanthrene and anthracene, were found to act as very weak inducers of ER-mediated activity in the MCF-7 cell line stably transfected with a luciferase reporter gene. The effects of PAHs were time-dependent and they were not completely inhibited by antiestrogen ICI 182,780. In addition, BaP and BaA, as well as weakly estrogenic fluoranthene, significantly potentiated the maximum ER-mediated activity of 17beta-estradiol. Therefore, the effects of inhibitors of several types of protein kinases known to activate ERalpha in a ligand-independent manner were investigated. However, neither inhibitors nor inducers of extracellular signal-regulated kinases 1 and 2 (ERK1/2), phosphatidylinositol-3 kinase, protein kinase C, c-Src, or protein kinase A modified ER-mediated activity in this model. Neither estradiol nor BaA activated ERK1/2, two kinases suggested to play significant roles in ER signaling, suggesting that another kinase is involved in the observed phosphorylation of ERalpha. Similar to 17beta-estradiol, BaA stimulated G(0)/G(1)-S-phase transition in MCF-7 cells, which was fully suppressed by ICI 182,780. In conclusion, some PAHs can potentiate 17beta-estradiol-induced ER activation and stimulate cell cycle entry in vitro. However, their exact mode(s) of action and whether this phenomenon is of in vivo relevance remains to be elucidated.     

Estrogen receptor antagonist fulvestrant (ICI 182,780) inhibits the anti-inflammatory effect of glucocorticoids

The glucocorticoid receptor (GR) and estrogen receptor (ER) play important roles in both physiological and pathological conditions involving cell growth and differentiation, lipolysis, control of glucose metabolism, immunity, and inflammation. In fact, recent studies suggest that 17beta-estradiol, like glucocorticoids, may also have anti-inflammatory properties, even if the molecular mechanisms responsible for these activities have not yet been completely clarified. The present study was designed to gain a better understanding of the possible cross-talk between GR and ER in a model of lung inflammation (carrageenan-induced pleurisy). In particular, we have investigated whether fulvestrant (ICI 182,780), a selective ER-alpha antagonist, is able to attenuate the well known anti-inflammatory effect of dexamethasone (DEX), a synthetic glucocorticoid, in ovariectomized rats. We show that ICI 182,780, a selective ER-alpha antagonist, reverses the anti-inflammatory activity exhibited by DEX. Moreover, the coadministration of ICI 182,780 significantly inhibited the ability of DEX to reduce: 1) the degree of lung injury, 2) the rise in myeloperoxidase activity, 3) the increase of poly-(ADP-ribose) polymerase activity, tumor necrosis factor alpha, and interleukin-1beta levels, 4) inducible nitric-oxide synthase, 5) lipid peroxidation, 6) nitrotyrosine formation, 7) cyclooxygenase expression, and 8) the IkappaB-alpha degradation caused by carrageenan administration. In addition, quantitative PCR shows that DEX down-regulates GR and up-regulates glucocorticoid-induced leucine zipper levels, whereas ICI 182,780 does not counteract these effects. In conclusion, these results suggest that the in vivo anti-inflammatory property of DEX is also related to the ER-alpha.     

Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor-alpha reporter gene assay.

A human breast cancer cell line, MCF-7, transiently transfected with a chimeric estrogen receptor (Gal4-HEG0) and a luciferase reporter plasmid (17m5-G-Luc), was used to investigate the estrogenic activity of benzo[a]pyrene (B[a]P), a prototypical polyaromatic hydrocarbon (PAH). B[a]P at concentrations > or = 1 microM produced responses comparable to that of 0.1 nM 17beta-estradiol (E2). The ER antagonist ICI 182,780 (ICI) completely inhibited the response to both E2 and B[a]P, indicating that the responses were ER-mediated. However, 2 microM alpha-napthoflavone (alpha-NF), an Ah receptor antagonist and P450 inhibitor, also decreased the response to B[a]P but not to E2. Analysis of the profile of B[a]P metabolites in the transfected MCF-7 cultures indicated that alpha-NF inhibited the production of the 3- and 9-hydroxy (3-OH and 9-OH), as well as the 7, 8- and 9,10-dihydroxy (7,8-OH and 9,10-OH) B[a]P species. In the ER-alpha reporter assay, the 3-OH and 9-OH metabolites produced maximal responses comparable to E2, with EC50 values of 1.2 microM and 0.7 microM, respectively. The 9,10-OH metabolite exhibited minimal activity in the assay. These responses were inhibited by ICI for both the 3-OH and the 9-OH species; however, alpha-NF inhibited only the response to the 9-OH metabolite. The 7,8-OH metabolite did not exhibit significant estrogenic activity. Furthermore, 7,8-OH B[a]P displayed observable cytotoxicity at concentrations > or = 10(-7) M. This cytotoxic response was completely inhibited by alpha-NF, suggesting that 7,8-OH B[a]P was being further metabolized to one or more cytotoxic metabolites.     

Estrogenic activity of environmental polycyclic aromatic hydrocarbons in uterus of immature Wistar rats

Polycyclic aromatic hydrocarbons (PAHs) are an important group of environmental pollutants, known for their mutagenic and carcinogenic activities. Many PAHs are aryl hydrocarbon receptor (AhR) ligands and several recent studies have suggested that PAHs or their metabolites may activate estrogen receptors (ER). The present study investigated possible estrogenic/antiestrogenic effects of abundant environmental contaminants benzo[a]pyrene (BaP), benz[a]anthracene (BaA), fluoranthene (Fla) and benzo[k]fluoranthene (BkF) in vivo, using the immature rat uterotrophic assay. The present results suggest that BaA, BaP and Fla behaved as estrogen-like compounds in immature Wistar rats, when applied for 3 consecutive days at 10mg/kg/day, as documented by a significant increase of uterine weight and hypertrophy of luminal epithelium. These effects were likely to be mediated by ERalpha, a major subtype of ER present in uterus, as they were inhibited by treatment with ER antagonist ICI 182,780. BaA, the most potent of studied PAHs, induced a significant estrogenic effect within a concentration range 0.1-50mg/kg/day; however, it did not reach the maximum level induced by reference estrogens. The proposed antiestrogenicity of the potent AhR agonist BkF was not confirmed in the present in vivo study; the exposure to BkF did not significantly affect the uterine weight, although a weak suppression of ERalpha immunostaining was observed in luminal and glandular epithelium, possibly related to its AhR-mediated activity. The PAHs under study did not induce marked genotoxic damage in uterine tissues, as documented by the lack of Ser-15-phoshorylated p53 protein staining. With the exception of Fla, all three remaining compounds increased CYP1-dependent monooxygenation activities in liver at the doses used, suggesting that the potential tissue-specific antiestrogenic effects of PAHs mediated by metabolization of 17beta-estradiol also cannot be excluded. Taken together, these environmentally relevant PAHs induced estrogenic effects in vivo, which might affect their toxic impact and carcinogenicity.     

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.     

Effect of single and repeated in vitro exposure of ovarian follicles to o,p'-DDT and p,p'-DDT and their metabolites

The aim of the presented study was to compare the effect of o,p'-DDT [1,1-dichloro-2,2-bis-(p,p'-chlorophenyl)-ethylene] and p,p'-DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane] and their metabolites DDE and DDD on estradiol secretion by ovarian follicles, the target organs of environmental estrogens. Theca interna (Tc) and granulosa cells (Gc) were collected from medium size porcine follicles and cultured as a monolayer. The cells were initially cultured for 24 h to allow attachment to the plates and then media were changed for the new ones and o,p'-DDT and p,p'-DDT and their metabolites: o,p'-DDE, p,p'-DDE and o,p'-DDD were added at doses of 4, 40, 400 ng and 4 microg/ml medium to investigate dose-dependent effects. Media were collected after 24 h and frozen for estradiol content determination. When the effect of single and repeated exposure was investigated, the lowest dose of 4 ng/ml and the highest one of 4 microg/ml were chosen on the basis of the results of Experiment 1. o,p'-DDT exerted antiestrogenic action at all doses used while its metabolites and p,p'-DDT and its metabolites decreased estradiol secretion only when present in the medium at a dose of 4 ng/ml. The highest doses caused the increase in estradiol secretion. Parent o,p'-DDT and its metabolites showed antiestrogenic action after single exposure to 4 ng/ml while parent p,p'-DDT and its metabolites caused estrogenic action. All investigated compounds, except o,p'-DDT, increased estradiol secretion after single exposure to the dose of 4 microg/ml. Repeated exposure resulted in a massive antiestrogenic action of all investigated chemicals. In conclusion, our study points to time-dependent effect of DDT and its metabolites on ovarian follicles with the strongest estrogenic properties observed after single exposure and antiestrogenic action caused by repeated exposure. Given the duration of folliculogenesis, one can imagine many different potential mechanisms by which DDT could influence steroidogenesis.     

Acute actions of 3 alpha-hydroxy-tibolone on factors influencing contraction in guinea-pig ventricular myocytes

We investigated whether one of the main estrogenic metabolites of the postmenopausal agent, tibolone (Org OD14), exerts direct cardiac actions in a similar way to 17beta-estradiol. 3alpha-OH-tibolone (40 microM) decreased both cell shortening and Ca2+ transient amplitude of field-stimulated (1 Hz, 37 degrees C) guinea-pig ventricular myocytes. These effects were still observed in cells that had been incubated with the specific estrogen receptor antagonist, ICI 182,780 (C(32)H(47)F(5)O(3)S), suggesting an estrogen receptor-independent mechanism of action. In addition, 3alpha-OH-tibolone inhibited the L-type Ca2+ current and shortened action potential durations (APD). This mechanism may contribute to a potential cardiovascular action of tibolone.