Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

Impact of Human SULT1E1 Polymorphisms on the Sulfation of 17ß-Estradiol, 4-Hydroxytamoxifen, and Diethylstilbestrol by SULT1E1 Allozymes.

BACKGROUND AND OBJECTIVES: Previous studies have revealed that sulfation, as mediated by the estrogen-sulfating cytosolic sulfotransferase (SULT) SULT1E1, is involved in the metabolism of 17ß-estradiol (E2), 4-hydroxytamoxifen (4OH-tamoxifen), and diethylstilbestrol in humans. It is an interesting question whether the genetic polymorphisms of SULT1E1, the gene that encodes the SULT1E1 enzyme, may impact on the metabolism of E2 and these two drug compounds through sulfation. METHODS: In this study, five missense coding single nucleotide polymorphisms of the SULT1E1 gene were selected to investigate the sulfating activity of the coded SULT1E1 allozymes toward E2, 4OH-tamoxifen, and diethylstilbestrol. Corresponding cDNAs were generated by site-directed mutagenesis, and recombinant SULT1E1 allozymes were bacterially expressed, affinity-purified, and characterized using enzymatic assays. RESULTS: Purified SULT1E1 allozymes were shown to display differential sulfating activities toward E2, 4OH-tamoxifen, and diethylstilbestrol. Kinetic analysis revealed further distinct Km (reflecting substrate affinity) and Vmax (reflecting catalytic activity) values of the five SULT1E1 allozymes with E2, 4OH-tamoxifen, and diethylstilbestrol as substrates. CONCLUSIONS: Taken together, these findings highlighted the significant differences in E2-, as well as the drug-sulfating activities of SULT1E1 allozymes, which may have implications in the differential metabolism of E2, 4OH-tamoxifen, and diethylstilbestrol in individuals with different SULT1E1 genotypes.

Removal and Biodegradation of 17ß-Estradiol and Diethylstilbestrol by the Freshwater Microalgae Raphidocelis subcapitata.

Natural steroidal and synthetic non-steroidal estrogens such as 17ß-estradiol (E2) and diethylstilbestrol (DES) have been found in natural water, which can potentially endanger public health and aquatic ecosystems. The removal and biodegradation of E2 and DES by Raphidocelis subcapitata were studied in bacteria-free cultures exposed to single and mixture treatments at different concentrations for 96 h. The results showed that R. subcapitata exhibited a rapid and strong ability to remove E2 and DES in both single and mixture treatments by biodegradation. At the end of 96 h, the removal percentage of single E2 and DES achieved 82.0%, 80.4%, 74.6% and 89.9%, 73.4%, 54.1% in 0.1, 0.5, and 1.5 mg·L-1, respectively. With the exception of the 0.1 mg·L-1 treatment at 96 h, the removal capacity of E2 was more efficient than that of DES by R. subcapitata. Furthermore, the removal percentage of mixture E2 and DES achieved 88.5%, 82.9%, 84.3% and 87.2%, 71.8%, 51.1% in 0.1, 0.5, and 1.5 mg·L-1, respectively. The removal percentage of mixed E2 was significantly higher than that of the single E2. The presence of DES could accelerate the removal of E2 from the mixture treatments in equal concentrations. In addition, the removal was mainly attributed to the biodegradation or biotransformation process by the microalgae cells rather than simple sorption and accumulation in the cells. The microalgae R. subcapitata demonstrated a high capability for the removal of the E2 and DES indicating future prospects for its application.

Estrogen receptor affinity chromatography: a new method for characterization of novel estrogenic disinfection by-products.

To identify the unknown estrogenic disinfection by-products (DBPs) from the chlorination extract, an effective method based on affinity chromatography with immobilized human recombinant estrogen receptor α (ERα) was developed, which has an advantage in targeting different potential estrogenic compounds from mixed sample simultaneously by comparing their relative binding activities to ER. The new method worked well for six known environmental estrogens. To further test the validity of this method for unknown chemicals, six DBPs of diethylstilbestrol (DES) with relatively strong ER binding affinity after chlorination were isolated and identified. It was found that except for 2-chloro-DES which showed 1.36 times stronger binding affinity than DES, most of the by-products bound to ER much more weakly than DES. All these seven by-products induced a dose-dependent transcriptional activation in two-hybrid-yeast assays. Z,Z-dienestrol (DE) and 2-chloro-DES, which exhibiting the weakest and the strongest binding affinity, were further tested for their transcriptional potential as 0.00243 and 0.014 compared to DES, respectively. However, they were still potential harmful environmental estrogenic disruptors as their estrogenic activities were much stronger than that of bisphenol A (BPA). These results demonstrated that the new method can help to screen unknown estrogenic compounds from mixture more efficiently.

Liver X receptors interfere with the deleterious effect of diethylstilbestrol on testicular physiology.

Liver X receptors LXRα (NR1H3) and LXRß (NR1H2) are transcription factors belonging to the nuclear receptor superfamily, activated by specific oxysterols, oxidized derivatives of cholesterol. These receptors are involved in the regulation of testis physiology. Lxr-deficient mice pointed to the physiological roles of these nuclear receptors in steroid synthesis, lipid homeostasis and germ cell apoptosis and proliferation. Diethylstilbestrol (DES) is a synthetic estrogen considered as an endocrine disruptor that affects the functions of the testis. Various lines of evidences have made a clear link between estrogens, their nuclear receptors ERα (NR3A1) and ERß (NR3A2), and Lxrα/ß. As LXR activity could also be regulated by the nuclear receptor small heterodimer partner (SHP, NR0A2) and DES could act through SHP, we wondered whether LXR could be targeted by estrogen-like endocrine disruptors such as DES. For that purpose, wild-type and Lxr-deficient mice were daily treated with 0.75 µg DES from days 1 to 5 after birth. The effects of DES were investigated at 10 or 45 days of age. We demonstrated that DES induced a decrease of the body mass at 10 days only in the Lxr-deficient mice suggesting a protective effect of Lxr. We defined three categories of DES-target genes in testis: those whose accumulation is independent of Lxr; those whose accumulation is enhanced by the lack of both Lxrα/ß; those whose accumulation is repressed by the absence of Lxrα/ß. Lipid accumulation is also modified by neonatal DES injection. Lxr-deficient mice present different lipid profiles, demonstrating that DES could have its effects in part due to Lxrα/ß. Altogether, our study shows that both nuclear receptors Lxrα and Lxrß are not only basally important for testicular physiology but could also have a preventive effect against estrogen-like endocrine disruptors.

Structural insights into Resveratrol's antagonist and partial agonist actions on estrogen receptor alpha.

BACKGROUND: Resveratrol, a naturally occurring stilbene, has been categorized as a phytoestrogen due to its ability to compete with natural estrogens for binding to estrogen receptor alpha (ERα) and modulate the biological responses exerted by the receptor. Biological effects of resveratrol (RES) on estrogen receptor alpha (ERα) remain highly controversial, since both estrogenic and anti-estrogenic properties were observed. RESULTS: Here, we provide insight into the structural basis of the agonist/antagonist effects of RES on ERα ligand binding domain (LBD). Using atomistic simulation, we found that RES bound ERα monomer in antagonist conformation, where Helix 12 moves away from the ligand pocket and orients into the co-activator binding groove of LBD, is more stable than RES bound ERα in agonist conformation, where Helix 12 lays over the ligand binding pocket. Upon dimerization, the agonistic conformation of RES-ERα dimer becomes more stable compared to the corresponding monomer but still remains less stable compared to the corresponding dimer in antagonist conformation. Interestingly, while the binding pocket and the binding contacts of RES to ERα are similar to those of pure agonist diethylstilbestrol (DES), the binding energy is much less and the hydrogen bonding contacts also differ providing clues for the partial agonistic character of RES on ERα. CONCLUSIONS: Our Molecular Dynamics simulation of RES-ERα structures with agonist and antagonist orientations of Helix 12 suggests RES action is more similar to Selective Estrogen Receptor Modulator (SERM) opening up the importance of cellular environment and active roles of co-regulator proteins in a given system. Our study reveals that potential co-activators must compete with the Helix 12 and displace it away from the activator binding groove to enhance the agonistic activity.

Characterization of UDP-glucuronosyltransferases involved in glucuronidation of diethylstilbestrol in human liver and intestine.

Diethylstilbestrol (DES), a synthetic estrogen, is famous for its carcinogenic effects. Human exposure to this compound can occur frequently through dietary ingestion and medical treatment. Glucuronidation has been demonstrated to be a predominant metabolic pathway for DES in human. Therefore, glucuronidation metabolism may have a significant impact on its toxicities, and it is essential to clarify this metabolic pathway. Accordingly, this in vitro study is designed to characterize the UGTs involved in DES glucuronidation and, furthermore, to identify the roles of individual isoforms in the reaction in liver and intestine. Human liver microsomes (HLM) displayed much higher potential for DES glucuronidation than human intestinal microsomes (HIM). The intrinsic clearances in HLM and HIM were demonstrated to be 459 and 14 µL/min/mg protein, respectively. Assays with recombinant UGTs demonstrated that UGT1A1, -1A3, -1A8, and -2B7 could catalyze DES glucuronidation, among which UGT2B7 showed the highest affinity. Chemical inhibitors of UGT2B7 and UGT1A1/1A3 both displayed similar inhibition against the reaction in UGT2B7 and HLM. In addition, DES glucuronidation in individual HLM exhibited a large individual variability and strongly correlated to UGT2B7 activity. All evidence indicates that UGT2B7 may act as a major enzyme responsible for DES glucuronidation in human liver. For HIM, both UGT2B7 inhibitor and UGT1A1/1A3/1A8 inhibitor exerted moderate inhibition. It is suggested that although UGT2B7 contributes to DES glucuronidation in intestine, other UGTs may contribute equally. In summary, this study characterizes human UGTs involved in DES glucuronidation in human liver and intestine, which may be helpful for further study about DES-related toxicities.

Occurrence and removal efficiencies of eight EDCs and estrogenicity in a STP.

The occurrence and removal of eight endocrine disrupting compounds (EDCs), including estrone (E(1)), 17ß-estradiol (E(2)), estriol (E(3)), 17α-ethinylestradiol (EE(2)), diethylstilbestrol (DES), bisphenol A (BPA), nonylphenol (NP) and octylphenol (OP), and their estrogenicities were investigated in a sewage treatment plant in Harbin city, China. The EDCs were extracted from wastewater samples by solid phase extraction (SPE) method and analyzed with gas chromatography coupled with mass spectrometry (GC-MS). The average concentrations in the influents and effluents ranged from 6.3 (EE(2)) to 1725.8 ng L(-1) (NP) and from

Is bisphenol A a weak carcinogen like the natural estrogens and diethylstilbestrol?

Bisphenol A (BPA) displays weak estrogenic properties and could be a weak carcinogen by a mechanism similar to that of estrone (E(1)), estradiol (E(2)) and the synthetic estrogen diethylstilbestrol, a human carcinogen. A wide variety of scientific evidence supports the hypothesis that certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, react with DNA to cause mutations that can lead to the initiation of cancer. One of the major pathways of estrogen metabolism leads to the 4-catechol estrogens, 4-OHE(1)(E(2)), which are oxidized to their quinones, E(1)(E(2))-3,4-Q. The quinones react with DNA to form predominantly the depurinating adducts 4-OHE(1)(E(2))-1-N3Ade and 4-OHE(1)(E(2))-1-N7Gua. This process constitutes the predominant pathway in the initiation of cancer by estrogens. One pathway of BPA metabolism is hydroxylation of one of its symmetric benzene rings to form its catechol, 3-OHBPA. Subsequent oxidation to BPA-3,4-quinone would lead to reaction with DNA to form predominantly the depurinating adducts 3-OHBPA-6-N3Ade and 3-OHBPA-6-N7Gua. The resulting apurinic sites in the DNA could generate mutations in critical genes that can initiate human cancers. The catechol of BPA may also alter expression of estrogen-activating and deactivating enzymes, and/or compete with methoxylation of 4-OHE(1)(E(2)) by catechol-O-methyltransferase, thereby unbalancing the metabolism of estrogens to increase formation of E(1)(E(2))-3,4-Q and the depurinating estrogen-DNA adducts leading to cancer initiation. Thus, exposure to BPA could increase the risk of developing cancer by direct and/or indirect mechanisms. Knowledge of these mechanisms would allow us to begin to understand how BPA may act as a weak carcinogen and would be useful for regulating its use.

Melatonin modulates the effects of diethylstilbestrol (DES) on the anterior pituitary of the female Wistar rat.

We studied the anti-tumorigenic effect of melatonin in diethylstilbestrol (DES)-treated anterior pituitaries in rats. Twenty-one female Wistar rats were randomly allocated into three groups: vehicle control rats, DES-treated rats, and DES-treated rats co-administrated with melatonin beginning at week 13. At the end of 16 weeks, rats were weighed and decapitated for morphological studies, including an H+E staining-based score evaluation in regard to cell proliferation, angiogenesis, immunostaining for VEGF, MMP-9, and AQP-1, and electron microscopy. Compared with vehicle, long-term treatment of DES significantly reduced rat body weight and increased H+E score, both of which were counteracted by melatonin. Administration of melatonin also reduced the expression of VEGF and MMP-9, although no changes were detected in AQP-1 expression. In rats cotreated with melatonin, the RER loosened and accumulated more secretion granules. We thus concluded that melatonin can modulate the effects of DES on the rat anterior pituitary by downregulating expression of VEGF and MMP-9 and suppressing the release of secretion granules, suggesting a therapeutic potential in estrogen-induced pituitary malfunctions.

Xeno-oestrogens and phyto-oestrogens are alternative ligands for the androgen receptor.

The androgen receptor (AR) plays a critical role in prostate cancer development and progression. This study aimed to use a computerized docking approach to examine the interactions between the human AR and phyto-oestrogens (genistein, daidzein, and flavone) and xeno-oestrogens (bisphenol A, 4-nonylphenol, dichlorodiphenyl trichloroethane [DDT], diethylstilbestrol [DES]). The predicted three-dimensional structure of AR and androgens was established using X-ray diffraction. The binding of four xeno-oestrogens and three phyto-oestrogens to AR was analysed. The steroids estradiol and dihydrotestosterone (DHT) were used as positive controls and thyroxine as negative control. All the ligands shared the same binding site except for thyroxine. The endogenous hormones DHT and 17beta-oestradiol showed the strongest binding with the lowest affinity energy (< -10 kcal mol(-1)). All three phyto-oestrogens and two xeno-oestrogens (bisphenol A and DES) showed strong binding to AR. The affinities of flavone, genistein, and daidzein were between -8.8 and -8.5 kcal mol(-1), while that of bisphenol A was -8.1 kcal mol(-1) and DES -8.3 kcal mol(-1). Another two xeno-oestrogens, 4-nonylphenol and DDT, although they fit within the binding domain of AR, showed weak affinity (-6.4 and -6.7 kcal mol(-1), respectively). The phyto-oestrogens genistein, daidzein and flavone, and the xeno-oestrogens bisphenol A and DES can be regarded as androgenic effectors. The xeno-oestrogens DDT and 4-nonylphenol bind only weakly to AR.

Meta-analysis of supramaximal effects in in vitro estrogenicity assays.

In scientific literature, several estrogenic compounds are reported to induce responses in vitro that are significantly higher than that of estradiol (E2). These supramaximal (SPMX) estrogenic effects do not occur consistently and seem to differ depending on the cellular models applied. This study analyzes the possible underlying causes, mechanisms, and drivers for SPMX estrogenic effects in in vitro functional assays reported in the peer-reviewed literature. For the 21 natural and industrial chemicals identified as SPMX inducers, the culture and exposure conditions varied greatly among and between the assays. Detailed information on assay characteristics, however, sometimes lacked. Diethylstilbestrol, genistein, and bisphenol A were selected to build a database. The meta-analysis revealed that the occurrence of SPMX effects could be related to a number of specific assay characteristics: (1) the type of serum used to supplement the exposure medium, (2) the end point used to quantify the estrogenic potency (endogenous or transfected), (3) the number of estrogen response elements, and (4) and the promoter's nature. An SPMX response was not reported for expression of endogenous genes, assays that used African green monkey kidney (COS-1) cell line or with chloramphenicol transferase as the reporter gene. There were no indications that solvent concentration in culture, exposure period, or cell model influenced the occurrence of an SPMX effect. It is important to understand the mechanism behind this phenomenon because in vitro assays for estrogenicity are used extensively to characterize and quantify the estrogenic potency of compounds, mixtures and environmental extracts.

Accessibility of the extracellular loops of follicle stimulating hormone receptor and their role in hormone-receptor interaction.

The follicle stimulating hormone receptor (FSHR), a member of the G-protein coupled receptor family, has a large extracellular domain (ECD) which is responsible for hormone binding specificity. Whether the extracellular loops (ELs) of FSHR which are outward projections of its transmembrane domain have any role in receptor function is not yet well understood. Here, we use antipeptide antibodies to peptides corresponding to the FSHR-ELs to check the surface accessibility of the loops. These antibodies were further used to understand the involvement of the loops in hormone binding and signaling. The results demonstrate that EL1 and EL3 are surface accessible on the mature receptor in spite of the presence of a large ECD. It is observed that the EL1 and EL3 serve as secondary binding sites and they possibly interact with the ECD-bound hormone's alpha subunit which is common to the gonadotropins.

Role of estrogenic compounds (diethylstibestrol, 17beta-estradiol, and bisphenol A) in the phosphorylation of substrate by protein kinase Calpha.

Estrogenic compounds can activate protein kinase C (PKC), which is a calcium and phospholipid-dependent serine/threonine kinase. In the present study, we investigated the role of 17beta-estradiol (E2), diethylstibestrol (DES), and bisphenol A (BPA) in the phosphorylation of substrate by PKCalpha using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The level of phosphorylated peptide was low in the absence of phosphatidylserine (PS). Moreover, reduction of phosphorylation ratios was identified in the presence of diacylglycerol (DAG) and Ca(2+) or PS and Ca(2+) after adding E2, DES, and BPA. However, no change in phosphorylation ratios was found in the presence of DAG and PS. Addition of E2, DES, and BPA also had no influence on the phosphorylation reaction of substrate by cell or tissue lysate samples. Our study suggests that E2, DES, and BPA can bind to the C2 domain of PKCalpha but have no effects on the phosphorylation reaction of substrates in the presence of DAG and PS.

Isoflavone reduces body weight by decreasing food intake in ovariectomized rats.

BACKGROUND/AIMS: The primary objective of this study was to further determine the mechanisms by which isoflavone prevents obesity induced by ovariectomy. METHODS: Female 8-week-old Wistar rats were randomly assigned to 6 groups: a sham-operated group; an ovariectomized (OVX) control group; 3 OVX groups orally administered 400 ppm (L-SI), 1,200 ppm (M-SI) and 3,600 ppm (H-SI) of an isoflavone preparation, respectively, and an OVX group receiving 0.45 ppm of diethylstilbestrol. All animals were allowed free access to a high-fat diet and water for 4 weeks. Some neuropeptides, including ghrelin, neuropeptide Y (NPY), alpha-melanocyte-stimulating hormone (alpha-MSH), cholecystokinin (CCK), peptide YY (PYY), insulin and estradiol (E2), were measured by radioimmunoassay. RESULTS: Compared with the OVX control group, body weight, total abdominal fat, food intake and food availability of the M-SI and H-SI groups were significantly reduced. The results also showed that isoflavone and diethylstilbestrol could decrease ghrelin and NPY levels and increase CCK, PYY and E2 levels. The level of alpha-MSH was not changed. CONCLUSIONS: These findings showed that isoflavone could reduce obesity by decreasing food intake, possibly by (1) reducing ghrelin and NPY levels, thereby decreasing food intake, and (2) increasing CCK and PYY levels, which can induce satiety by irritating the vagal center.

Mechanism of metabolic activation and DNA adduct formation by the human carcinogen diethylstilbestrol: the defining link to natural estrogens.

Diethylstilbestrol (DES) is a human carcinogen, based on sufficient epidemiological evidence. DES is mainly metabolized to its catechol, 3'-hydroxyDES (3'-OH-DES), which can further oxidize to DES-3',4'-quinone (DES-3',4'-Q). Similarly to estradiol-3,4-quinone, the reaction of DES-3',4'-Q with DNA would form the depurinating 3'-OH-DES-6'-N3Ade and 3'-OH-DES-6'-N7Gua adducts. To prove this hypothesis, synthesis of DES-3',4'-Q by oxidation of 3'-OH-DES with Ag(2)O was tried; this failed due to instantaneous formation of a spiro-quinone. Oxidation of 3'-OH-DES by lactoperoxidase or tyrosinase in the presence of DNA led to the formation of 3'-OH-DES-6'-N3Ade and 3'-OH-DES-6'-N7Gua adducts. These adducts were tentatively identified by LC-MS/MS as 3'-OH-DES-6'-N3Ade, m/z = 418 [M+H](+), and 3'-OH-DES-6'-N7Gua, m/z = 434 [M+H](+). Demonstration of their structures derived from their oxidation by MnO(2) to the DES quinone adducts and subsequent tautomerization to the dienestrol (DIES) catechol adducts, which are identical to the standard 3'-OH-DIES-6'-N3Ade, m/z = 416 [M+H](+), and 3'-OH-DIES-6'-N7Gua, m/z = 432 [M+H](+), adducts. The reaction of DIES-3',4'-Q or lactoperoxidase-activated 3'-OH-DIES with DNA did not produce any depurinating adducts, due to the dienic chain being perpendicular to the phenyl planes, which impedes the intercalation of DIES into the DNA. Enzymic oxidation of 3'-OH-DES suggests that the catechol of DES intercalates into DNA and is then oxidized to its quinone to yield N3Ade and N7Gua adducts. These results suggest that the common denominator of tumor initiation by the synthetic estrogen DES and the natural estrogen estradiol is formation of their catechol quinones, which react with DNA to afford the depurinating N3Ade and N7Gua adducts.

The environmental impact of growth-promoting compounds employed by the United States beef cattle industry: history, current knowledge, and future directions.

The current state of knowledge regarding the environmental impact of growth-promoting compounds associated with the U.S. beef cattle industry is extensive in some areas but virtually nonexistent in others. The compounds administered to the cattle are quite well understood, as are bovine metabolism and excretion. If the sex and age of the cattle on the feedlot are known, the metabolites excreted by the cattle should be predictable with a great deal of accuracy. The fate, transport, and biological effects of growth-promoting compounds are just beginning to be studied. Most of the research conducted on the fate and transport of growth-promoting compounds has focused on 17beta-E2; however, much of this research was not conducted using feedlot runoff or manure. Studies are needed that focus specifically on manures and runoff from experimental or commercial feedlots. To date, the degree to which growth-promoting compounds are released from feedlots in a bioavailable form remains a point of speculation. The environmental fate and transport of TBA, P, and MGA have not been well studied. Comparisons between the fate and transport of T and 17beta-E2, however, make it clear that compounds with similar structure may behave very differently once released into the environment. Considering that 17beta-E2 is a naturally occurring estrogen and that TBA is a nonaromatizable androgen, it is not surprising that these compounds directly impact the reproductive physiology of fishes. The effects of these two compounds have been well documented, as has been described here; however, the effects of P and MGA exposures have gone largely uninvestigated. This is a serious critical gap in our knowledge base because progestogins play an important role in sex steroid synthesis and reproduction. Clearly, additional research on the consequences of exposures to P and MGA is warranted. The majority of research investigating the effects of 17beta-E2 and TBA metabolites on fish has been conducted in the laboratory and has typically focused on continuous, pharmacological exposures to single compounds. These exposures may not bear much similarity to environmentally relevant exposures, and as such may offer little information regarding biological effects seen in nature. Cattle feedlot runoff is likely to contain a suite of growth-promoting compounds rather than any single compound. Clearly, deciphering the biological effects of exposure to complex mixtures containing androgenic, estrogenic, and progestogenic compounds will remain an important area of study for the next few years. A second complexity associated with the biological runoff from cattle feedlots is the discontinuous nature of the release. It is likely that inadvertent entry of growth-promoting compounds will follow spring snowmelt or rainstorm events. These events will result in intermittent, pulsed exposures to high concentrations of these compounds interspersed by long-term exposures to lower concentrations. The effects of exposure timing and duration should be considered to generate a clearer understanding of the biological consequences of exposures to growth-promoting compounds. To date, a very limited number of studies (only one!) have sought to determine whether fish living in waterways receiving runoff from cattle feedlots are adversely affected by growth-promoting compounds associated with the runoff. Clearly, more field studies need to be conducted before a relationship between cattle feedlot effluent and biological consequences can be elucidated.

Regulation of ER alpha signaling pathway in neuronal HN10 cells: role of protein acetylation and Hsp90.

Estrogen has a variety of neuroprotective effects but the molecular basis of its function is still mainly unclear. Estrogen receptor (ER) signaling is highly dependent on posttranslational modifications and the assembly of coactivator and corepressor complexes. Several proteins involved in ER alpha signaling have recently been found to be acetylated, including ER alpha itself and Hsp90, a key chaperone in the functional regulation of ER alpha. ER alpha complexes also contain histone deacetylases (HDAC) which repress transactivation. Our purpose was to clarify the role of protein acetylation and Hsp90 function in the ERE-mediated ER alpha signaling in neuronal HN10 cells. We observed that increasing protein/histone acetylation status with trichostatin A, a potent HDAC inhibitor, increased the 17beta-estradiol (E2)-induced transactivation of ERE-driven luciferase in non-transfected cells, and even more extensively in pER alpha-transfected cells. E2-induced ERE-driven transactivation was blocked by ICI 182.780. Several ER antagonists, such as raloxifene and tamoxifen, were unresponsive. Valproate, an antiepileptic drug which is recently characterized as a HDAC inhibitor, was also able to potentiate the E2-induced ERE-transactivation. Inhibition of the function of Hsp90 chaperone with geldanamycin strongly inhibited the E2-induced ERE-transactivation. Overexpression of SIRT2 protein deacetylase did not inhibit the acetylation-potentiated ERE-driven transactivation indicating that SIRT2 deacetylase is not involved in ER alpha signaling. Our results reveal that ER alpha signaling is dependent on protein acetylation and epigenetic regulation.

Interactions of diethylstilbestrol (DES) and DES analogs with membrane progestin receptor-alpha and the correlation with their nongenomic progestin activities.

Progestin induction of oocyte maturation (OM) in fish is a useful model for investigating endocrine disruption of nongenomic steroid actions. Although diethylstilbestrol (DES) analogs have been shown to mimic the actions of progestins to induce meiotic maturation of goldfish and zebrafish oocytes, their molecular mechanisms of action remain unclear. The ability of these endocrine-disrupting chemicals (EDCs) to interact with the progestin receptor mediating OM was investigated in receptor binding assays using plasma membranes from goldfish ovaries and breast cancer cells transfected with goldfish membrane progestin receptor (mPR)-alpha. Membranes prepared from both ovaries and mPRalpha-transfected cells showed high-affinity, saturable, displaceable, single binding sites specific for the goldfish maturation-inducing steroid, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP). DES and DES analogs (dipropionate-DES and hexestrol), which induce OM in goldfish, bound to the receptor and caused concentration-dependent displacement of [3H]-17,20beta-DHP, whereas dimethyl ether-DES had no affinity for the receptor. Scatchard plot analysis of specific 17,20beta-DHP binding in the presence of different amounts of DES showed that DES binding is of the noncompetitive type. The activities of DES and DES analogs to induce meiotic maturation of goldfish oocytes were examined in an in vitro bioassay. Whereas a concentration-dependent induction of OM was observed in response to DES, dipropionate-DES, and hexestrol, dimethyl ether-DES did not show any OM-inducing activity. The close correspondence between binding of DES and its analogs to the mPRalpha protein and their OM-inducing activities suggests a mechanism of endocrine disruption mediated by binding to mPRalpha resulting in its activation, thereby mimicking the nongenomic action of the progestin 17,20beta-DHP.

Critical evaluation of methods to incorporate entropy loss upon binding in high-throughput docking.

Proper accounting of the positional/orientational/conformational entropy loss associated with protein-ligand binding is important to obtain reliable predictions of binding affinity. Herein, we critically examine two simplified statistical mechanics-based approaches, namely a constant penalty per rotor method, and a more rigorous method, referred to here as the partition function-based scoring (PFS) method, to account for such entropy losses in high-throughput docking calculations. Our results on the estrogen receptor beta and dihydrofolate reductase proteins demonstrate that, while the constant penalty method over-penalizes molecules for their conformational flexibility, the PFS method behaves in a more "DeltaG-like" manner by penalizing different rotors differently depending on their residual entropy in the bound state. Furthermore, in contrast to no entropic penalty or the constant penalty approximation, the PFS method does not exhibit any bias towards either rigid or flexible molecules in the hit list. Preliminary enrichment studies using a lead-like random molecular database suggest that an accurate representation of the "true" energy landscape of the protein-ligand complex is critical for reliable predictions of relative binding affinities by the PFS method.