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

THE U.S. EPA ENDOCRINE DISRUPTOR SCREENING PROGRAM 4951/Bound (nM)2015105(A)050 µM100 µM200 µM-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.51/Total (nM)9(B)7Slope531-100 0 100 200Competitor (µM)Figure 11.3Experiment demonstrating competitive inhibition of steroid receptor binding by a test chemical.(A) Double-reciprocal plots of inhibition of [ 3 H]-ligand specific binding. (B) Slope-replot analysisfor the determination of the inhibition constant (x intercept = K i ). These data represent a testchemical that is a competitive inhibitor of estrogen receptor binding with a K i = 89 µM.There are benefits as well as limitations to the use of mammalian versus yeast cells for theseassays. Yeast-based assays are simple, sensitive, and generally less expensive to conduct than assaysusing mammalian cells. However, with yeasts there are rank-order differences in potencies ofendocrine active test chemicals, 27–29 as well as differences in permeability of compounds throughthe yeast cell wall relative to the plasmalemma of mammalian cells. In this respect, assays utilizingmammalian cells are thought to be more representative of risk to humans.A number of in vitro transcriptional activation assays using mammalian cells have been developedthat assess either estrogen- or androgen-dependent gene transcription. Transient transfectionassays in which both the receptor and a hormone-responsive reporter are cotransformed intocells 18,30–34 have been widely used. However, in transient transfection assays, target DNA sequencesare over expressed and therefore do not reflect physiological levels of receptor. In addition, transfectionefficiencies (i.e., percentage of cells expressing the receptor) may vary greatly from assayto assay. Unfortunately, the responsiveness is limited in time, as the transgenes are usually lostwithin 72 h, so transfections must be repeated with each new set of assays.Adenoviral transduction, which uses a replication-defective virus to deliver biologically competentgenes, has also been used to develop androgen receptor transcriptional activation assays. 35Since the virus is replication defective, it represents no hazard of infection. Both the AR and areporter gene are introduced into either CV-1 or MDA-MB-453 cells. As with transient transfectionassays, transduction requires that the procedure be repeated for each new set of assays. However,viral infection appears to be much more efficient than transfection because induction is consistentlyhigh, with much lower variability. Transduction and transfection techniques require similar facilities,and both can be easily accomplished with basic cell culture supplies and equipment (culture hoodand cell culture incubator). Stably transformed cells eliminate the need for repeated transient© 2006 by Taylor & Francis Group, LLC