A Practical Approach, Second Edition=Ronald D. Ho.pdf

THE U.S. EPA ENDOCRINE DISRUPTOR SCREENING PROGRAM 493androgen receptor because ligands for the androgen receptor may also interact with the glucocorticoidreceptor (GR) or progesterone receptor (PR). In this case, a compound such as triamcinoloneacetonide, which binds to and blocks PR and GR but not AR, can be added to the buffer to ensurespecific AR binding. 20,21 Limitations of this type of assay include the use of animals to acquire thetissue, use of radioactive ligand, and the fact that they are not easily amenable to a high-throughputsystem.The use of cultured cells and/or recombinant receptors eliminates the need to isolate receptorsfrom animal tissues, can be performed with human cells or receptors, and has the possibility ofadaptation for high throughput screening. With the use of recombinant receptors, the researcheralso has more control over the receptors that are present in the preparation. For example, transientexpression of AR in cells that do not express PR or GR eliminates the need for additional blockingcomponents in the buffer. 16,18 Assays can also be conducted with purified recombinant receptor. 17,19In this case, additional factors, such as heat shock proteins, may need to be added to help stabilizethe receptor in its proper conformation for binding. Cell-based assays also require cell cultureequipment, thorough training in cell culture techniques, and the use of radioactivity. Althoughpurified recombinant receptor techniques have not been used as extensively as receptor preparationsfrom tissues, they have the potential to provide an alternative method for the TIS battery.Data interpretation is an important component of steroid-receptor binding studies. Typically,the data from each competitive binding assay are plotted on a semilog graph so that a sigmoid plotis obtained. The data can be plotted as counts per minute bound or as percent of total specificbinding on the Y axis versus the concentration of the test chemical on the X axis. If the test chemicaland the radiolabeled ligand compete for a single common binding site, the competitive bindingcurve will have a shape (Figure 11.2A) determined by the law of mass action. 22 Specifically, thecurve should descend from 90% to 10% specific binding over approximately an 81-fold increasein the concentration of the test chemical (e.g., this portion of the curve will cover approximatelytwo log units, Figure 11.2B). A binding curve that drops dramatically (i.e., from 70% to 0%) overone order of magnitude should be questioned, as should one that is U-shaped (i.e., percent boundat first decreases with increasing concentration of competitor but then begins to increase, Figure 11.2C).In the both cases, something has happened to the dynamics of the binding assay, and the reactionis no longer following the law of mass action (e.g., the test chemical may be binding to multiplebinding sites or the test chemical may not be soluble at higher concentrations in the assay bufferand is precipitating from the solution). In these cases, an additional K i experiment may be requiredto determine if the test chemical is truly a competitive inhibitor. While it is possible to calculate aK i value from the IC 50 by use of the Cheng-Prusoff equation, 23 the use of this equation is only validif the competitive binding curve reflects a pure competition for a single binding site. 24 An experimentallyderived K i requires adding increasing concentrations of radiolabeled ligand in the presenceof fixed concentrations of the test chemical and then plotting the data on a double reciprocal plot. 25,26A pattern of lines that intersect on the ordinate axis is characteristic of competitive inhibition(Figure 11.3). Slopes obtained from each of the double reciprocal plots are then replotted as afunction of the inhibitor concentration. The slope and intercept of the replot can be used to calculatea K i value for the test chemical. 26Performance criteria for binding assays are being developed by the Office of Science, Coordinationand Policy (OSCP) of the U.S. EPA through an agreement with the ICCVAM. Even thoughsteroid receptor binding assays have been used for years, they have not been rigorously standardizedand validated as required by the EDSP. Currently, protocols for ER and AR competitive bindingassays are undergoing the standardization and validation process, under the direction of the OSCP.B. Androgen and Estrogen Receptor-Dependent Gene Transcription AssaysCell-based gene transcriptional activation assays were also proposed by EDSTAC, either to complementor as an alternative to the ER and AR competitive binding assays. These assays can be© 2006 by Taylor & Francis Group, LLC