Yeast Estrogen Screen Research Articles

Applicability of a yeast oestrogen screen for the detection of oestrogen-like activities in environmental samples

A (xeno)oestrogen bioassay was introduced, using a genetically modified yeast strain which produces a fusion protein encompassing the human oestrogen receptor hormone binding domain and the yeast GAL4-DNA binding domain. Upon binding of appropriate substances this fusion protein recognises the respective DNA sequence thereby enhancing the transcription of a β-galactosidase reporter gene. The bioassay procedure was evaluated by screening 30 compounds, including some known or suspected (xeno)oestrogens and determining EC 50-values for 17β-oestradiol, 1.5 nM, 4-tert.-octylphenol, 6.7 μM and bisphenol A, 104 μM. Toluene extracts from different environmental matrices were tested for their oestragenic activity. The positive test results obtained with a sewage sludge extract indicated the applicability of this bioassay for environmental monitoring.

Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen

We have used the yeast estrogen screen (YES) consisting of the human estrogen receptor and a reporter containing two estrogen response elements linked to the lacZ gene to evaluate the interaction between ovarian, phyto-, and synthetic estrogens with extracellular binding proteins. YES was incubated with charcoal-stripped human serum, human sex hormone-binding globulin, or human α-fetoprotein in the presence of concentrations of various estrogens that induced a 100% estrogenic response, as measured by β-galactosidase activity. The activity of estradiol and coumestrol, a phytoestrogen, was reduced 75% with physiological levels of serum, sex hormone-binding globulin, or α-fetoprotein. The β-galactosidase activity of genistein, another phytoestrogen, also decreased with extracellular proteins but to a lower extent than estradiol. In contrast, the activity of the synthetic estrogens diethylstilbestrol, kepone, and p,p′-DDD was only minimally reduced with extracellular proteins. These results indicate a potential fundamental difference in the interaction of estrogens from diverse sources with extracellular binding proteins. This suggests that the capacity for various estrogens to induce estrogen-associated responses is in part regulated by their affinity for extracellular binding proteins.

Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays.

Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans. To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity. Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES). This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast. Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay. Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene. Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity. Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models. Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays. The antifungal agent benomyl had no estrogenic activity. We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.

Identification of Environmental Chemicals with Estrogenic Activity Using a Combination of In Vitro Assays

Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans. To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity. Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES). This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast. Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay. Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene. Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity. Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models. Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays. The antifungal agent benomyl had no estrogenic activity. We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.

A yeast estrogen screen for examining the relative exposure of cells to natural and xenoestrogens.

Xenoestrogens, such as o,p'-DDT and octyl phenol (OP), have been associated with reproductive abnormalities in various wildlife species. Xenoestrogens mimic the natural estrogen 17 beta-estradiol and compete for binding to the estrogen receptor. Even though the affinity of o,p'-DDT and OP for the estrogen receptor is approximately 1000-fold lower than 17 beta-estradiol, the actions of xenoestrogens could be enhanced if their bioavailability in serum were greater than 17 beta-estradiol. To test this hypothesis, the yeast estrogen screen (YES) was created by expressing human estrogen receptor (hER) and two estrogen response elements (ERE) linked to the lacZ gene. The beta-galactosidase activity of the YES system was significantly increased after treatment with 17 beta-estradiol or the xenoestrogens diethylstilbestrol (DES), o,p'-DDT, and OP but not with vehicle, antiestrogen ICI 164,384, dexamethasone, or testosterone. To determine whether serum proteins affected the bioavailability of natural estrogens compared to xenoestrogens, albumin, sex hormone binding globulin (SHBG), or charcoal-stripped serum were added to the YES system and beta-galactosidase activity assayed. Albumin and SHBG decreased beta-galactosidase activity in the presence of estradiol to a greater extent than DES, o,p'-DDT, and OP. Human and alligator charcoal-stripped serum were also effective at selectively reducing beta-galactosidase activity in the presence of estradiol compared to xenoestrogens. Human serum was more effective than alligator serum in reducing beta-galactosidase activity in the presence of xenoestrogens, indicating that serum may serve as a biomarker for sensitivity to xenoestrogens. Selective binding of 17 beta-estradiol by proteins in serum indicates that certain xenoestrogens may exert greater estrogenicity than originally predicted. The estrogenic potency of a compound involves its binding affinity, bioavailability in serum, and persistence in the environment. Our data demonstrate the utility of the YES system for identifying and characterizing environmental estrogens.

A Yeast Estrogen Screen for Examining the Relative Exposure of Cells to Natural and Xenoestrogens

Xenoestrogens, such as o,p'-DDT and octyl phenol (OP), have been associated with reproductive abnormalities in various wildlife species. Xenoestrogens mimic the natural estrogen 17 beta-estradiol and compete for binding to the estrogen receptor. Even though the affinity of o,p'-DDT and OP for the estrogen receptor is approximately 1000-fold lower than 17 beta-estradiol, the actions of xenoestrogens could be enhanced if their bioavailability in serum were greater than 17 beta-estradiol. To test this hypothesis, the yeast estrogen screen (YES) was created by expressing human estrogen receptor (hER) and two estrogen response elements (ERE) linked to the lacZ gene. The beta-galactosidase activity of the YES system was significantly increased after treatment with 17 beta-estradiol or the xenoestrogens diethylstilbestrol (DES), o,p'-DDT, and OP but not with vehicle, antiestrogen ICI 164,384, dexamethasone, or testosterone. To determine whether serum proteins affected the bioavailability of natural estrogens compared to xenoestrogens, albumin, sex hormone binding globulin (SHBG), or charcoal-stripped serum were added to the YES system and beta-galactosidase activity assayed. Albumin and SHBG decreased beta-galactosidase activity in the presence of estradiol to a greater extent than DES, o,p'-DDT, and OP. Human and alligator charcoal-stripped serum were also effective at selectively reducing beta-galactosidase activity in the presence of estradiol compared to xenoestrogens. Human serum was more effective than alligator serum in reducing beta-galactosidase activity in the presence of xenoestrogens, indicating that serum may serve as a biomarker for sensitivity to xenoestrogens. Selective binding of 17 beta-estradiol by proteins in serum indicates that certain xenoestrogens may exert greater estrogenicity than originally predicted. The estrogenic potency of a compound involves its binding affinity, bioavailability in serum, and persistence in the environment. Our data demonstrate the utility of the YES system for identifying and characterizing environmental estrogens.

Symposium on estrogens in the environment, III.

Symposium on estrogens in the environment, III.

Handbook of environmental fate and exposure data for organic chemicals: Vol. 2. Solvents

The oxidative degradation of the oral contraceptive 17α-ethinylestradiol (EE2) in water by a new advanced catalytic oxidation process was investigated. The oxidant employed was hydrogen peroxide in aqueous solution and the catalyst was the iron tetra-amido macrocyclic ligand (FeIII-TAML) complex that has been designated Na[Fe(H2O)(B*)] (FeIII-B*). EE2 (10 μM) was oxidised rapidly by the FeIII-B*/H2O2 (5 nM/4 mM) catalytic oxidation system at 25 °C, and for reactions at pH 8.40–11.00, no unchanged EE2 was detected in the reaction mixtures after 60 min. No oxidation of EE2 was detected in blank reactions using either H2O2 or FeIII-B* alone. The maximum rate of EE2 loss occurred at pH 10.21. At this pH the half-life of EE2 was 2.1 min and the oxidised products showed around 30% estrogenicity removal, as determined by the yeast estrogen screen (YES) bioassay. At pH 11.00, partial oxidation of EE2 by FeIII-B*/H2O2 (5 nM/4 mM) was studied (half-life of EE2 was 14.5 min) and in this case the initial intermediates formed were a mixture of the epimers 17α-ethynyl-1,4-estradiene-10α,17β-diol-3-one (1a) and 17α-ethynyl-1,4-estradiene-10β,17β-diol-3-one (1b) (identified by LC-ToF-MS and 1H NMR spectroscopy). Significantly, this product mixture displayed a slightly higher estrogenicity than EE2 itself, as determined by the YES bioassay. Upon the addition of further aliquots of FeIII-B* (to give a FeIII-B* concentration of 500 nM) and H2O2 (to bring the concentration up to 4 mM assuming the final concentration had dropped to zero) to this reaction mixture the amounts of 1a and 1b slowly decreased to zero over a 60 min period as they were oxidised to unidentified products that showed no estrogenicity. Thus, partial oxidation of EE2 gave products that have slightly increased estrogenicity, whereas more extensive oxidation by the advanced catalytic oxidation system completely removed all estrogenicity. These results underscore the importance of controlling the level of oxidation during the removal of EE2 from water by oxidative processes.