Differential estrogen receptor binding of estrogenic substances: a species comparison

The study investigated the ability of 34 natural and synthetic chemicals to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed glutathione-S-transferase (GST)-estrogen receptors (ER) fusion proteins from five different species. Fusion proteins consisted of the ER D, E and F domains of human alpha (GST-hERalphadef), mouse alpha (GST-mERalphadef), chicken (GST-cERdef), green anole (GST-aERdef) and rainbow trout ERs (GST-rtERdef). All five fusion proteins displayed high affinity for E2 with dissociation constants (K(d)) ranging from 0.3 to 0.9 nM. Although, the fusion proteins exhibited similar binding preferences and binding affinities for many of the chemicals, several differences were observed. For example, alpha-zearalenol bound with greater affinity to GST-rtERdef than E2, which was in contrast to other GST-ERdef fusion proteins examined. Coumestrol, genistein and naringenin bound with higher affinity to the GST-aERdef, than to the other GST-ERdef fusion proteins. Many of the industrial chemicals examined preferentially bound to GST-rtERdef. Bisphenol A, 4-t-octylphenol and o,p' DDT bound with approximately a ten-fold greater affinity to GST-rtERdef than to other GST-ERdefs. Methoxychlor, p,p'-DDT, o,p'-DDE, p,p'-DDE, alpha-endosulfan and dieldrin weakly bound to the ERs from the human, mouse, chicken and green anole. In contrast, these compounds completely displaced [3H]E2 from GST-rtERdef. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for estrogenic compounds and that these differences may be due to the variability in the amino acid sequence within their respective ER ligand binding domains.     

Ability of structurally diverse natural products and synthetic chemicals to induce gene expression mediated by estrogen receptors from various species

The ability of 14 structurally diverse estrogenic compounds to induce reporter gene expression mediated by estrogen receptors (ERs) from different species was examined. MCF-7 cells were transiently transfected with a Gal4-regulated luciferase reporter gene (17m5-G-Luc) and Gal4-ER chimeric receptors containing the D, E and F domains of the human alpha (Gal4-hERalphadef), mouse alpha (Gal4-mERalphadef), mouse beta (Gal4-mERbetadef), chicken (Gal4-cERalphadef), green anole (Gal4-aERalphadef), Xenopus (Gal4-xERdef) or rainbow trout alpha ERs (Gal4-rtERalphadef). The efficacy of 17beta-estradiol (E2) in inducing reporter gene expression was similar among the different constructs overall, with EC(50) values ranging from 0.05 to 0.7nM. However, Gal4-rtERalphadef had an EC(50) value at 37 degrees C of 28nM, though at 20 degrees C an EC(50) value of 1nM was observed. Despite a similar response to E2 treatment among the ERs, many differences were observed in the magnitude of the response to other structurally diverse chemicals. For example, coumestrol induced Gal4-mERbetadef- and Gal4-aERdef-mediated reporter gene expression 164- and 8-fold greater, respectively, than mediated with the other Gal4-ERs. As well, in contrast to results with other Gal4-ERs, alpha-zearalenol consistently induced Gal4-rtERalphadef-mediated reporter gene activity at lower concentrations than did E2. Overall, the results demonstrate that selected estrogenic compounds exhibit a differential ability to induce reporter gene activity mediated by ERs from different vertebrate species. These data also highlight the importance of incubation temperature when examining rtERalpha-mediated activity.     

Development of methods for extraction and in vitro quantification of estrogenic and androgenic activity of wastewater samples

Chemicals released into the environment by anthropogenic activities have been linked to estrogenic or androgenic effects in exposed wildlife, and there is a need to develop and validate rapid and cost-effective methods to quantify the total estrogenic and androgenic activity of environmental water samples. In this study, estrogen receptors (ER) were isolated from sheep (Ovis aries) uteri and rainbow trout (Oncorhynchus mykiss) livers and androgen receptors (AR) were isolated from rainbow trout brains. The isolated receptors were used in competitive receptor binding assays to test the affinity of known estrogenic and androgenic chemicals for the receptor binding site, and results were compared with literature values for the rat uterine ER binding assay and the E-Screen. The relative binding affinities of the tested compounds to ER from different species were similar, and binding to the ER was a more responsive endpoint than the cellular effect measured in the E-Screen. Using the sheep ER binding assay in combination with solid-phase extraction, the estrogenic activity in a raw sewage sample from a municipal treatment plant in Brisbane (Queensland, Australia) was measured at 51-73 ng/L estradiol equivalents (EEq).     

The N-terminal transactivation domain of rat estrogen receptor is localized in a hydrophobic domain of eighty amino acids

In order to determine the transactivation domain(s) of the rat estrogen receptor (rat ER), we made a number of rat ER deletion mutants and transfected the mutant plasmids into COS7 cells together with an estrogen-responsive reporter plasmid, ERE-tk(197)-CAT, which contained the estrogen response element of Xenopus vitellogenin A2 gene. We have identified and localized the N-terminal transactivation domain of the rat ER to a hydrophobic region extending from Ala 59 to Glu 140.     

Estrogenic effects of environmental chemicals: an interspecies comparison

The development of various in vitro screening methods has led to identification of novel estrogenic chemicals of natural and anthropogenic origin. In this study, the (anti)estrogenic potential of several environmental chemicals were compared in an array of in vitro test systems comprising: (i) competitive binding to estrogen receptors derived from the human breast cancer cell line MCF-7 (hER) and rainbow trout (Oncorhynchus mykiss) (rtER), (ii) a proliferation assay with MCF-7 cells (E-SCREEN), and iii) induction of vitellogenin (rtVtg) in isolated rainbow trout hepatocytes. The results showed substantial differences in assay sensitivity for potent estrogens like 17beta-estradiol, diethylstilbestrol and zearalenone (ranking order of sensitivity: E-SCREEN > hER approximately rtER approximately rtVtg). Chemicals like 4-n-nonylphenol and bisphenol A had higher relative binding affinity to the hER, whereas 4-t-butylphenol and 4-n-butylphenol showed highest affinity to the rtER. Zearalenone and the novel estrogen 4-t-butylhexanol displayed a considerable higher relative potency in the E-SCREEN than the rtVtg assay, whereas alkylphenols and the novel estrogen mimic 4-t-butyl-nitrobenzene were most potent in fish cells. Correlation analysis of data from the test systems suggest that interspecies differences is largely due to inter-assay variation of the ER-dependent cellular responses, whereas binding to the ER are fairly similar in the two species tested.     

PES1与雌激素受体存在相互作用

雌激素(E2)和雌激素受体(ER)在E2诱发的肿瘤中起着极其重要的作用.ER共调节因子通过与ER相互作用调节其生物学功能.PES1主要表达于E2的重要靶器官如乳腺、卵巢等组织中,并在乳腺癌细胞中高表达.用PCR技术构建HA标签的PES1全长以及1～322aa、312 ～588aa和414～588aa三个不同功能区片段的重组质粒.将不同的重组质粒与FLAG-ERα和或FLAGC-ERβ共转染293T细胞后进行免疫共沉淀,以验证PES1与ER是否有相互作用以及相互作用的区域.用含雌激素受体作用元件的荧光素酶报告基因( ERE-LUC)检测PES1对ERα和ERβ转录激活活性的影响.结果表明,PES1与ERα和ERβ均相互作用,且PES1的1～ 322aa区域与ERα和ERβ相结合.PES1能特异地、E2非依赖性抑制ERβ的转录激活活性.实验结果显示,PES1是一个新的ER共调节因子,需要进一步研究其在ERβ信号通路及其在E2诱发的肿瘤的作用.     

The complete nuclear estrogen receptor family in the rainbow trout: discovery of the novel ERalpha2 and both ERbeta isoforms

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERalpha (NR3A1) and ERbeta (NR3A2). The ERbeta subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERalpha have not been demonstrated in any fish species. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ERalpha subtype (i.e. ERalpha1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERalpha isoform, ERalpha2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERalpha1. Both the rainbow trout ERbeta subtypes, ERbeta1 [NR3A2a] and ERbeta2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERalphas (approximately 40% similarity). ERbeta1 and ERbeta2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERalpha subtype clade, with the closest relative to rainbow trout ERalpha2 being rainbow trout ERalpha1, suggesting a recent, unique duplication event to create these two isoforms. For the ERbeta subtype there are two distinct subclades, one represented by the ERbeta1 isoform and the other by the ERbeta2 isoform. The rainbow trout ERbeta1 and ERbeta2 are not closely associated with each other, but instead fall into their respective ERbeta subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ERalpha1, ERalpha2, ERbeta1, and ERbeta2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.     

Molecular evidence for a clade of turtles.

Although turtles have been generally grouped with the most primitive reptile species, the origin and phylogenetic relationships of turtles have remained unresolved to date. To confirm the phylogenetic position of turtles in amniotes, we have cloned and determined the cDNA sequences encoding for skink lactate dehydrogenase (LDH)-A and LDH-B, snake LDH-A, and African clawed frog LDH-A; four alpha-enolase cDNA sequences from turtle, alligator, skink, and snake were also cloned and determined. All of these eight cDNA sequences, as well as the previously published LDH-A, LDH-B, and alpha-enolase of mammals, birds, reptiles, and African clawed frog, were analyzed by the phylogenetic tree reconstruction methods of neighbor-joining, maximum parsimony, and maximum likelihood. In the phylogenetic analyses, the turtle was found to be closely related to the alligator. Also, we found that the turtle had diverged after the divergence of squamates and birds. This departs from previous hypotheses of turtle evolution and further suggests that turtles are the latest of divergent reptiles, having been derived from an ancestor of crocodilian lineage within the last 200 million years.     

Molecular cloning of estrogen receptor alpha of the Nile crocodile

Estrogens are essential for normal reproductive activity in female and male vertebrates. In female reptiles, they are essential for ovarian differentiation during a critical developmental stage. To understand the molecular mechanisms of estrogen action in the Nile crocodile (Crocodylus niloticus), we have isolated cDNA encoding the estrogen receptor alpha (ERalpha) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify Nile crocodile cDNA from the ovary. The full-length Nile crocodile ERalpha cDNA was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the Nile crocodile ERalpha showed high identity to the American alligator ERalpha (98%), caiman ER (98%), lizard ER (82%) and chicken ERalpha (92%), although phylogenetic analysis suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. Expression of ERalpha was observed in the ovary and testis of juvenile Nile crocodiles. These data provide a novel tool allowing future studies examining the regulation and ontogenic expression of ERalpha in crocodiles and expands our knowledge of estrogen receptor evolution.     

Plasma binding of 1-butanol, phenol, nitrobenzene and pentachlorophenol in the rainbow trout and rat: a comparative study

1. The in vitro binding of 1-butanol, phenol, nitrobenzene, and pentachlorophenol in trout plasma and rat plasma was determined. 2. Binding to rainbow trout plasma proteins agreed within 9% of that observed in rat plasma. 3. Percentage bound to rainbow trout (2-99%) or rat (10-99%) plasma proteins increased as the log octanol/water partition coefficient of the chemicals increased within the Log P 1-3 range, and was suggestive of hydrophobic interactions in binding.