DSSTox (KIERBL) EPA Estrogen Receptor Ki Binding Study (Laws et al.) Database
This study was conducted by EPA researchers to evaluate the validity of the rat uterine cytosolic (RUC) estrogen receptor (ER) competitive binding assay for use in the Endocrine Disruption Screening Program (EDSP). The assay measures the ability of radiolabeled 17-beta-estradiol (3H-E2) to bind with RUC ER in the presence of increasing concentrations of a test chemical. The goal is to employ this more ..
BioActive Compounds: 17
This study was conducted by EPA researchers to evaluate the validity of the rat uterine cytosolic (RUC) estrogen receptor (ER) competitive binding assay for use in the Endocrine Disruption Screening Program (EDSP). The assay measures the ability of radiolabeled 17-beta-estradiol (3H-E2) to bind with RUC ER in the presence of increasing concentrations of a test chemical. The goal is to employ this in vitro assay as a 1st tier screening tool for assessing the potential of structurally diverse environmental chemicals to bind to the ER, as well as to support research efforts to develop computational models to predict ER binding. A distinguishing feature of the present study was the use of secondary experimental analysis of binding curves and determination of Ki values to confirm that measured IC50 values corresponded to true positive results. The study concluded that a number of experimental conditions can confound interpretation of IC50 values obtained from the RUC ER-binding assay data and lead to false positive results. In particular, some chemical treatments likely alter the stability of the assay by changing the buffer pH, denaturing ER, or disrupting the ER binding kinetics. In addition, in some cases, neither an IC50 or Ki value could be determined due to solubility constraints (i.e., sufficient concentration of the chemical to produce greater than 40% inhibition could not be experimentally achieved). The chemicals used in this study were selected as part of a validation test for two Quantitative Structure-Activity Relationship (QSAR) models designed to predict ER binding [Fed. Reg. 67(250), 79618, Dec. 30, 2002]. Several hundred chemicals were initially screened, including approximately 100 chemicals selected from among the High Production Volume (HPV) chemicals included in EPA's Toxic Substances Control Act (TSCA) inventory that were predicted to be positive for ER binding, with the rest selected at random from the TSCA inventory. All chemicals were initially tested in ER competitive binding assays by Battelle Pacific Northwest Laboratories, Richland, Washington [EPA Contract Number 68-W-99-033, Task 6]. Approximately 50 chemicals produced binding curves that: (1) strongly implied competitive inhibition of ER binding, or (2) produced partial curves or irregular (non-sigmoid) binding curves. These chemicals were further evaluated in an ER competitive binding assay with secondary analysis to confirm true competitive inhibition and to determine an experimental Ki [U.S. EPA Contract 3D-5967-NTEX]. The results of RUC ER screening for these 50 structurally diverse chemicals, for which secondary confirmatory analyses were performed, were published in the Main Citation (Laws et al., 2006, Tox. Sci. 94:46-56). The remaining 228 chemicals were designated as non-binders based on initial screening studies.
The DSSTox KIERBL data files include all published IC50 and Ki experimental results for the 50 chemicals included in the Main Citation (Laws et al., 2006) (denoted Group 1), as well as previously unpublished results for an additional 228 structually diverse TSCA chemicals for which no ER binding was observed (denoted Group 2. IC50 values were determined for 32 compounds in Group 1 as the concentration of a test chemical that inhibits the maximal specific binding of 0.33 nM radiolabeled (3H) 17-beta-estradiol (E2) to rat uterine cytosolic (RUC) estrogen receptor (ER) by 50%. Confirmation of binding status was accomplished by secondary analysis using Lineweaver-Burk plots and slope replots to determine true competitive inhibition and an experimental Ki value. Ki is the inhibition constant for the test chemical, i.e., the concentration of the test chemical that will bind to half the binding sites at equilibrium (displacing half of the probe-bound receptors), in the absence of radioligand or other competitors. Ki reported as Mean +/- Standard Error from n=2 experiments (see StandardError_Ki_n2) and is determined from Lineweaver-Burk plots over the tested dose range (TestedRange_microM). For more information see BindingCurve_Group and BindingCurve_Details. If Ki is reported, ActivityOutcome value is "active" If true competitive binding and inhibition was confirmed by secondary Ki experiments and Ki value is reported, then ActivityOutcome value is "active" and ActivityScore ranges from 10-100. If no binding was observed when compound was tested to the concentration limit of 100uM, or else some binding was observed in initial screening but secondary analysis confirmed non-binder status, ActivityOutcome is "inactive" and ActivityScore=0. If some binding was observed, but Ki could not be determined due to solubility limitations that prevented the secondary Ki experiment, ActivityOutcome is "inconclusive" and ActivityScore=5. For further experimental details, see Main Citation, Laws et al. (2006). Binding curve classification (Complete/Partial/Limited/Incomplete/Irregular/) factored into final determination of Ki_microM_mean (i.e., true competitive binding determination required complete or partial binding curves), but even a complete binding curve did not necessarily correspond to true competitive binding as determined by secondary analysis. Confounding results may be due to chemicals that: alter the stability of the assay by changing the buffer pH, denature the estrogen receptor (ER), or disrupt ER-binding kinetics. The 228 compounds in Group 2 were tested in the RUC ER competitive binding assay and designated as non-binders (i.e., inactives) since these chemicals at concentrations up to 100 micromolar failed to inhibit the maximum binding of radiolabeled estradiol more than 20% (USEPA. 2002. EPA Contract No. 68-W-99-033, Work Assignment 3-04).
DSSTox File Version: KIERBL_v1a
To access complete SD file, documentation and Main Citation, refer to the corresponding DSSTox Download Page
Main Citation: Laws SC, Yavanhxay S, Copper RL, Eldridge JC. 2006. Nature of the binding interaction for 50 structurally diverse chemicals with rat estrogen receptors. Toxicological Sciences. 94(1), 46-56; doi:10.1093/toxsci/kfl092.
Data Table (Concise)