ER binding assay

We compared the assay results for 80 common chemicals from both the Nishihara et al. and NCTR data sets inconsistent assay results were observed for 12 chemicals. Specifically, of 30 active chemicals in the Nishihara et al. data set, one chemical was found inactive in the NCTR data set of 50 inactive chemicals in the Nishihara et al. data set, 11 chemicals were found active in the NCTR data set. These observations show that even using the experimental data from the ER binding assay (the NCTR data set) to predict the experimental results from the yeast two-hybrid assay (the Nishihara et al. data set), there may be about a 15% (12/80) discrepancy, or 3.3% (1/30) false negative rate and 22% (11/50) false positive rate. Care should be taken in interpreting the QSAR validation results using this data set (Hong et al., 2002). 

JEPA examined the literature on SDs and STs to determine whether risk assessment was really necessary. Except for the estrogenic effects, it was decided that the assessment was not necessary. Additional examinations were performed on ER binding assay, proliferation of MCF-7 human breast cancer cells (E-screen assay), yeast two hybrid assay and yeast estrogen selective (YES) assay [12]. From the results of these examinations, it was judged that estimating the risk of SDs and STs is not necessary at the present time. JEPA officially announced a revised edition of SPEED 98 in 2000, in which SDs and STs had been deleted from the list of EDs along with n-butylbenzene. 

Test compounds used in this study are shown in Figure 2. Methoxychlor (1) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Synthesis of all other conqxrunds was previously rqwrted (7). No contaminants in tested conqtounds were observed by thin-4ayer chromatography. Conqxtunds were dissolved in dimethyl sulfoxide and diluted to 0.1 mM for the inhibition of ER binding assays (final concentration 38 pM). Metabolite mixtures of each test conqmund were obtained as follows. Each compound (final concentration 0.S > mM) was incubated for 60 min at 37 C with 5% rat liver S9 fraction (Kikkoman... 

Branham et al. [11] used an ER-ligand binding assay to measure the RBA for 46 chemicals considered to be potential phytoestrogens and mycoestrogens. The classes of chemicals examined (flavones, isoflavones, flavanones, coumarins, chalcones, and mycoestrogens) are shown in Figure 18.8. Representative chemicals from each of these classes showed some affinity for ER, ranging widely in RBAs from 43 to 0.00008 E2 = 100). No further work was done to describe specific SARs for these classes. 

Note that controversies over the technical and clinical validation of immunohistochemistry have not been completely resolved. Whether or not this method should completely replace biochemical ligand-binding assays remains controversial. Despite this cautionary statement, it is true that the specificity of immunohistochemistry is theoretically valid because it is based on the use of well-characterized monoclonal antibodies raised against epitopes restricted to the ERs. 

Figure 13.10 Depiction of the recursive process used by NCTR to develop QSAR models for predicting ER binding. The process starts with an initial set of chemicals from the literature for QSAR modeling. Next, the preliminary QSAR models are used prospectively to define a set of chemicals that will further improve the model s robustness and predictive capability. The new chemicals are assayed, and these data are then used to challenge and refine the QSAR models. Validation of the model is critical. The process emphasizes the living model concept.

Experimental agents were evaluated for their ability to compete with the reference 17-fi-estradiol using the radioligand binding assay described by Miller (1) and to determine the relative binding affinities of selected experimental agent for both ERa and ER(3 receptors. Testing results are provided in Table 2. 

Estrogen Receptor (ER) competitive binding assays, which measures the binding affinity of a chemical for the estrogen receptor (Fang et al., 2000). 

Reporter gene assays that measure ER binding-dependant transcriptional and translational activity. The yeast-based assays fall within this group. The vast majority of yeast-based assays are based on S. cerevisiae containing a stably transfected human estrogen, receptor (hER), although there are several variations in strain and detection method (NICEATM, 2002)... 

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