Ioxynil and diethylstilbestrol impair cardiac performance and shell growth in the mussel Mytilus coruscus.

The herbicide ioxynil (IOX) and the synthetic estrogen diethylstilbestrol (DES) are environmentally relevant contaminants that act as endocrine disruptors (EDCs) and have recently been shown to be cardiovascular disruptors in vertebrates. Mussels, Mytilus coruscus, were exposed to low doses of IOX (0.37, 0.037 and 0.0037 mg/L) and DES (0.27, 0.027 and 0.0027 mg/L) via the water and the effect monitored by generating whole animal transcriptomes and measuring cardiac performance and shell growth. One day after IOX (0.37 and 0.037 mg/L) and DES (0.27 and 0.027 mg/L) exposure heart rate frequency was decreased in both groups and 0.27 mg/L DES significantly reduced heart rate frequency with increasing time of exposure (P < 0.05) and no acclimatization occurred. The functional effects were coupled to significant differential expression of genes of the serotonergic synapse pathway and cardiac-related genes at 0.027 mg/L DES, which suggests that impaired heart function may be due to interference with neuroendocrine regulation and direct cardiac effect genes. Multiple genes related to detoxifying xenobiotic substances were up regulated and genes related to immune function were down regulated in the DES group (vs. control), indicating that detoxification processes were enhanced, and the immune response was depressed. In contrast, IOX had a minor disrupting effect at a molecular level. Of note was a significant suppression (P < 0.05) by DES of shell growth in juveniles and lower doses (< 0.0027 mg/L) had a more severe effect. The shell growth depression in 0.0027 mg/L DES-treated juveniles was not accompanied by abundant differential gene expression, suggesting that the effect of 0.0027 mg/L DES on shell growth may be direct. The results obtained in the present study reveal for the first time that IOX and DES may act as neuroendocrine disrupters with a broad spectrum of effects on cardiac performance and shell growth, and that DES exposure had a much more pronounced effect than IOX in a marine bivalve.

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.

Multiplug filtration cleanup method with multi-walled carbon nanotubes for the analysis of malachite green, diethylstilbestrol residues, and their metabolites in aquatic products by liquid chromatography-tandem mass spectrometry.

The food safety supervision in aquatic products has raised public concern in recent years. In this study, a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the simultaneous quantification and identification of four residues of the ever widely used analytes (including malachite green, leucomalachite green, diethylstilbestrol, and dienestrol) in aquaculture samples was developed. For sample preparation, a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was used, which was initially developed for pesticide residue analysis. For cleanup procedure, low-temperature cleanup method was combined with multiplug filtration cleanup (m-PFC) method based on multi-walled carbon nanotubes (MWCNTs). The volume of water, extraction solvent, cleanup sorbents, and m-PFC procedure were optimized for carp, striped bass, and giant salamander matrices. It was validated by analyzing four residues in each matrix spiked at three concentration levels of 0.5, 5, and 50 µg/kg (n = 5). The method was successfully validated according to the 2002/657/EC guidelines. After optimization, spike recoveries were within 73-106 % and <15 % relative standard deviations (RSDs) for all analytes in the tested matrices. Limits of quantification (LOQs) for the proposed method ranged from 0.10 to 0.50 µg/kg. Matrix-matched calibrations were performed with the coefficients of determination >0.998 between concentration levels of 0.5 and 200 µg/kg. The developed method was successfully applied to the determination of residues in market samples. Graphical abstract Flow chart of multi-plug filtration cleanup combined with low-temperature cleanup method.

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.

Isolation and characterization of Pseudomonas sp. strain capable of degrading diethylstilbestrol.

Since diethylstilbestrol (DES) interrupts endocrine systems and generates reproductive abnormalities in both wildlife and human beings, methods to remove DES from the environments are urgently recommended. In this study, bacterial strain J51 was isolated and tested to effectively degrade DES. J51 was identified as Pseudomonas sp. based on its nucleotide sequence of 16S rRNA. The quinoprotein alcohol dehydrogenase and isocitrate lyase were identified to be involved in DES degradation by MALDI-TOF-TOF MS/MS analysis. In the presence of 40 mg/l DES, increase of the genes encoding quinoprotein alcohol dehydrogenase and isocitrate lyase in both RNA and protein levels was determined. The HPLC/MS analysis showed that DES was hydrolyzed to a major degrading metabolite DES-4-semiquinone. It was the first time to demonstrate the characteristics of DES degradation by specific bacterial strain and the higher degradation efficiency indicated the potential application of Pseudomonas sp. strain J51 in the treatment of DES-contaminated freshwater and seawater environments.

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.

Modeling of three dimensional structure of human alpha-fetoprotein complexed with diethylstilbestrol: docking and molecular dynamics simulation study.

It has been long experimentally demonstrated that human alpha-fetoprotein (HAFP) has an ability to bind immobilized estrogens with the most efficiency for synthetic estrogen analog - diethylstilbestrol (DES). However, the question remains why the human AFP (HAFP), unlike rodent AFP, cannot bind free estrogens. Moreover, despite the fact that AFP was first discovered more than 50 years ago and is presently recognized as a "golden standard" among onco-biomarkers, its three-dimensional (3D) structure has not been experimentally solved yet. In this work using MODELLER program, we generated 3D model of HAFP on the basis of homology with human serum albumin (HSA) and Vitamin D-binding protein (VTDB) with subsequent molecular docking of DES to the model structure and molecular dynamics (MD) simulation study of the complex obtained. The model constructed has U-shaped structure in which a cavity may be distinguished. In this cavity the putative estrogen-binding site is localized. Validation by RMSD calculation and with the use of PROCHECK program showed good quality of the model and stability of extended region of four alpha-helical structures that contains putative hormone-binding residues. Data extracted from MD simulation trajectory allow proposing two types of interactions between amino acid residues of HAFP and DES molecule: (1) hydrogen bonding with involvement of residues S445, R452, and E551; (2) hydrophobic interactions with participation of L138, M448, and M548 residues. A suggestion is made that immobilization of the hormone using a long spacer provides delivery of the estrogen molecule to the binding site and, thereby, facilitates interaction between HAFP and the hormone.

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.

Environmental estrogens and obesity.

Many chemicals in the environment, in particular those with estrogenic activity, can disrupt the programming of endocrine signaling pathways that are established during development and result in adverse consequences that may not be apparent until much later in life. Most recently, obesity and diabetes join the growing list of adverse consequences that have been associated with developmental exposure to environmental estrogens during critical stages of differentiation. These diseases are quickly becoming significant public health issues and are fast reaching epidemic proportions worldwide. In this review, we summarize the literature from experimental animal studies documenting an association of environmental estrogens and the development of obesity, and further describe an animal model of exposure to diethylstilbestrol (DES) that has proven useful in studying mechanisms involved in abnormal programming of various differentiating estrogen-target tissues. Other examples of environmental estrogens including the phytoestrogen genistein and the environmental contaminant Bisphenol A are also discussed. Together, these data suggest new targets (i.e., adipocyte differentiation and molecular mechanisms involved in weight homeostasis) for abnormal programming by estrogenic chemicals, and provide evidence that support the scientific hypothesis termed "the developmental origins of adult disease". The proposal of an association of environmental estrogens with obesity and diabetes expands the focus on the diseases from intervention/treatment to include prevention/avoidance of chemical modifiers especially during critical windows of development.