A fractionation system, combined with an in vitro assay for detecting estrogenic activity, was developed in order to isolate and identify the major estrogenic chemicals present in seven sewage-treatment works (STW) effluents, receiving primarily domestic effluent, discharging into British rivers. Three sterols were isolated from estrogenic fractions of sewage extracts; these were the natural hormones 17 -estradiol and estrone and the synthetic hormone 17R-ethynylestradiol. 17 -Estradiol and estrone were present in all the effluents at measured concentrations ranging from 1 ng/L to almost 50 and 80 ng/L, respectively. The concentration of 17R-ethynylestradiol was generally below the limit of detection but was positively identified in three of the effluent samples at concentrations ranging from 0.2 to 7.0 ng/L. These data suggest that natural and synthetic hormones may be responsible for the observed induction of vitellogenin synthesis in male fish placed downstream of effluent discharges from STWs that receive mainly domestic inputs.

Abstract-An estrogen-inducible screen was developed in yeast (Saccharomyces cerevisiae) in order to assess whether surfactants and their major degradation products are estrogenic. The DNA sequence of the human estrogen receptor (hER) was integrated into the yeast genome, which also contained expression plasmids carrying estrogen-responsive sequences (ERE) controlling the expression of the reporter gene lac-Z (encoding the enzyme ␤-galactosidase). Thus, in the presence of estrogens, ␤-galactosidase is synthesized and secreted into the medium, where it causes a color change from yellow to red. This recombinant strain was used to determine whether representatives of major surfactant classes and some of their principal degradation products possess estrogenic activity. The results were compared to the effects of the main natural estrogen 17␤-estradiol. None of the parent surfactants tested possessed estrogenic activity. However, one class of surfactants, the alkylphenol polyethoxylates, degrade to persistent metabolites that were weakly estrogenic. Another group of degradation products, the sulfophenyl carboxylates, which are derived from the biodegradation of linear alkylbenzene sulfonates, do not appear to possess estrogenic activity.

The occurrence of certain natural and synthetic steroidal estrogens in the final effluent from STW has been demonstrated. 17β-Estradiol and estrone were present at concentrations in the tens of nanograms per liter range, and the synthetic estrogen 17R-ethynylestradiol was also identified, albeit in the low nanogram per liter range. The findings from subsequent in vivo tank trial experiments, in which adult male rainbow trout (Oncorhynchus mykiss) and adult roach (Rutilus rutilus) were exposed for 21 days via the water to environmentally relevant concentrations of 17β-estradiol and estrone are presented. In addition, the response of adult male and female roach following exposure to 17β-estradiol (1, 10, and 100 ng/L) was compared to the response to the alkylphenolic xenoestrogen, 4-tert-octylphenol (1, 10 and 100 µg/L). Plasma levels of vitellogenin were determined using previously validated radioimmunoassays in order to measure the estrogenic response of the fish to the varying concentrations of the compounds tested. The results indicate that environmentally relevant concentrations of natural steroidal estrogens are sufficient to account for the levels of vitellogenin synthesis observed in caged male fish placed downstream of certain STW effluent discharges in British rivers.

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Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.

The ability of certain man-made chemicals to mimic the effects of natural steroid hormones and their potential to disrupt the delicate balance of the endocrine system in animals are of increasing concern. The growing list of reported hormone-mimics includes the alkylphenolic (AP) compounds, a small number of which have been reported to be weakly estrogenic. In their most basic form, APs are composed of an alkyl group, which can vary in size, branching, and position, joined to a phenolic ring. The aim of this project was to identify the important structural features responsible for the estrogenic activity of AP chemicals. This was achieved by incubating APs with different structural features in a medium containing a previously described estrogen-inducible strain of yeast (Saccharomyces cerevisiae) expressing the human estrogen receptor and comparing their activity spectrophotometrically by the resulting color change of the medium. The results were compared to the effects of the main natural estrogen 17␤-estradiol. The data indicate that both the position (para > meta > ortho) and branching (tertiary > secondary ‫؍‬ normal) of the alkyl group affect estrogenicity. Optimal estrogenic activity requires a single tertiary branched alkyl group composed of between 6 and 8 carbons located at the para position on an otherwise unhindered phenol ring. The results are discussed in relation to the purity and composition of the chemicals tested.In the early 1930s, scientists began synthesizing compounds based on the phenanthrene nucleus of steroidal estrogens in an attempt to produce substances with similar properties and considerable clinical value. Estrogenic activity was assessed by subcutaneous administration of chemicals (dissolved in sesame oil) to ovariectomized rats that were then observed for the onset of estrus (1). It soon became apparent that the phenanthrene condensed ring structure was not required for estrogenic activity, with the discovery that diphenyl and diphenyl methane derivatives (2) and stilbene derivatives (3-6) containing two hydroxyl groups in the para positions (e.g. 4,4Ј-dihydroxy diphenyl) were also active. The substituted derivative of stilbene (4,4Ј-dihydroxystilbene) was renamed stilbestrol and was used as the parent compound in the production of another series of estrogenic compounds, including 4,4Ј-dihydroxydiethylstilbene

It has been proposed that tissue-specific estrogenic and/or antiestrogenic actions of certain xenoestrogens may be associated with alterations in the tertiary structure of estrogen receptor (ER) ␣ and/or ER␤ following ligand binding; changes which are sensed by cellular factors (coactivators) required for normal gene expression. However, it is still unclear whether xenoestrogens affect the normal behavior of ER␣ and/or ER␤ subsequent to receptor binding. In view of the wide range of structural forms now recognized to mimic the actions of the natural estrogens, we have assessed the ability of ER␣ and ER␤ to recruit TIF2 and SRC-1a in the presence of 17␤-estradiol, genistein, diethylstilbestrol, 4-tert-octylphenol, 2,3,4,5-tetrachlorobiphenyl-ol, and bisphenol A. We show that ligand-dependent differences exist in the ability of ER␣ and ER␤ to bind coactivator proteins in vitro, despite the similarity in binding affinity of the various ligands for both ER subtypes. The enhanced ability of ER␤ (over ER␣) to recruit coactivators in the presence of xenoestrogens was consistent with a greater ability of ER␤ to potentiate reporter gene activity in transiently transfected HeLa cells expressing SRC-1e and TIF2. We conclude that ligand-dependent differences in the ability of ER␣ and ER␤ to recruit coactivator proteins may contribute to the complex tissue-dependent agonistic/antagonistic responses observed with certain xenoestrogens.One of the greatest challenges in understanding the mechanisms of estrogen action has been to determine how different estrogen receptor (ER) 1 ligands (steroidal estrogens, antiestrogens, xenoestrogens) produce such diverse biological effects. The recent discovery of a second subtype of the estrogen receptor, named estrogen receptor-␤ (ER␤) to distinguish it from the classical ER (now renamed ER␣), adds another level of complexity to the mechanism of estrogen action and has opened new possibilities by which estrogens might exert tissue-and cell-specific effects (1). Indeed, it has been shown that ⌭R␣ and ER␤ differ in terms of their ability to activate gene expression from either the consensus estrogen response element (ERE) from the VTG gene or the divergent ERE from the luteinizing hormone ␤ gene in transiently transfected Cos-1 cells (2). Moreover, ⌭R␣ and ER␤ activate and inhibit, respectively, transcription from an AP1 enhancer site when complexed to 17␤-estradiol (E2), whereas ER␤ was a transcriptional activator on AP1 sites when complexed to antiestrogens (3). Studies in rodents have revealed that the distribution and relative levels of ⌭R␣ and ER␤ expression differ among tissues. For example, ⌭R␣ is predominantly expressed in the pituitary, uterus, ovary (oviduct and germinal epithelium), mammary gland, testis, epididymis, and kidney, whereas ER␤ is the predominant form in regions of the hypothalamus, ovary (granulosa cells), prostate gland, lung, and bladder (4 -8). The coexpression of ⌭R␣ and ER␤ in certain tissues and cells, and the ability of ⌭R␣ and ER␤ to form heterodimers and bind...

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