Electrostatic interactions of androgens and progesterone derivatives with rainbow trout estrogen receptor

In primary cultures of immature male rainbow trout (rt) hepatocytes, vitellogenin (Vg) gene expression is regulated by E(2) via the estrogen receptor (ER). However, steroids other than estrogens can also stimulate Vg gene expression. These steroids are hardly converted into E(2) during incubation and their stimulatory activity is completely inhibited by tamoxifen implying rtER involvement. These steroids have no or a slightly positive charge on the Connolly surface. In contrast, steroids that failed to stimulate Vg gene expression had a strong positive or negative charge around rings C and D due to polarization. The amino acid sequences of the ligand binding domains (LBD) of rtER and human ER alpha have 57.7% homology; only one amino acid differs in the presumed steroid binding site. We modeled the three-dimensional structure of the LBD of rtER using X-ray crystallographic data for hER alpha in order to investigate the fit (structural and electrostatic) between steroid and rtER. Two factors are essential for binding to rtER: (i) hydroxyl or carbonyl groups near C3 and C17 of the steroids (hydrophilic regions) that can form hydrogen bonds with His(489), Arg(359), and Glu(318), (ii) a hydrophobic steroid nucleus that interacts with a hydrophobic region of the rtER LBD through van der Waals forces. If polar functional groups are present, the hydrophobic interaction between steroid and the rtER LBD is considerably weakened.     

Induction of vitellogenin production in male tilapia (Oreochromis mossambicus) by commercial fish diets

Mozambique tilapia, (Oreochromis mossambicus), are a euryhaline teleost and an important biological model species. Captive male tilapia frequently have high levels of the estrogen-induced yolk precursor protein vitellogenin (Vg), a common indicator of exposure to estrogenic compounds. Sex steroids are found in commercial fish diets, but relatively few studies have examined the relationship between commercial diets and Vg production. In a fasting experiment to ascertain a dietary role in male Vg production, plasma Vg was reduced to negligible levels after 2 weeks of fasting, while no change in estrogen receptor (ER) expression was seen. When male tilapia were fed a squid-based diet that replaced the commercial trout diet, plasma Vg was reduced to undetectable levels over 40 days, concomitant with significant reductions in hepatic expression of Vgs A, B, and C, and ERβ, compared with control fish fed commercial trout diet. Female tilapia fed the squid-based for 20 days had no change in these parameters. When male tilapia were fed a defined, soy-based diet, plasma Vg reduced to 20% of levels in fish given either commercial trout diet or a defined, fishmeal-based diet. Overall, results from these studies suggest that estrogens in a commercial trout diet induce vitellogenin production by increasing expression of Vg, but not ER genes in male tilapia.     

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.     

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).     

Limited species differences in estrogen receptor alpha-medicated reporter gene transactivation by xenoestrogens

Estrogen receptors (ERs) play an important role in estrogen function. However, it is well known that there are species differences in amino acid sequences of the ligand binding domains. Here, we report on the analysis of species differences in ER-dependent transactivation with some chemicals using reporter gene assays. Full-length ER cDNAs from human, rat, chicken, alligator (Caiman), whiptail lizard, African clawed frog and rainbow trout were prepared from hepatic mRNA by the RT-PCR method and inserted into expression plasmids. Both expression and reporter plasmids were transiently transfected into HeLa cells, and then the estrogenic effects of chemicals were analyzed in terms of induction of luciferase activity. No species differences in transactivation were found among human, rat, chicken, alligator, whiptail lizard and African clawed frog ERs. However, thermo-dependent alteration in susceptibility to 17-beta-estradiol was observed with the rainbow trout ER because of thermo-dependence of estrogen binding.     

Induction of estrogen-regulated genes differs in immortal and tumorigenic human mammary epithelial cells expressing a recombinant estrogen receptor.

Studies on estrogen receptor (ER)-positive human breast cancer cell lines have shown that estrogen treatment positively modulates the expression of the genes encoding transforming growth factor-alpha (TGF alpha), 52-kDa cathepsin-D, and pS2. To determine whether these genes would be similarly regulated by estrogens in normal human mammary epithelial cells, we stably transfected immortal nontumorigenic human mammary epithelial cells with an ER-encoding expression vector. ER-negative tumor cells were also transfected for comparison. Levels of TGF alpha and 52-kDa cathepsin-D mRNA were enhanced by estrogen treatment of both ER-transfected immortal and tumorigenic cells, demonstrating that the ER by itself is sufficient to elicit estrogenic regulation of the expression of these genes. In contrast, expression of the pS2 gene was detected only in the ER-transfected tumor cells. The ER in both cell lines is capable of recognizing the pS2 promoter, however, since estrogen enhanced the activity of an introduced pS2-CAT reporter plasmid in transient expression analyses. These and other experiments with somatic cell hybrids between the immortal cells and ER+/pS2+ MCF-7 tumor cells, where pS2 gene expression is extinguished, support the conclusion that the immortal nontumorigenic cells encode gene products that block endogenous pS2 expression. These results also imply that such repressors are not active in the tumor cells.     

雌激素受体亚型及其配体调节基因转录机制的研究

本文综述雌激素受体亚型（ERα和ERβ）的结构,功能,组织分布,生理作用及雌激素受体配体调节基因转录的机制,目的是深入系统地了解植物雌激素和选择性雌激素受体调节剂的作用路径及其组织特异性的发生机制,最终为提高雌激素类药物的选择性,优化以临床为基础的药物设计提供一条较为系统的思路。结果表明,ERα和ERβ对不同雌激素类化合物产生不同应答,配体的结构不同,调节基因转录的路径不同和募集的辅调节蛋白的不同是雌激素受体两种亚型组织特异性激活或抑制的主要原因。     

Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicals

Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats.