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Avoiding false positives and optimizing identification of true negatives in estrogen receptor binding and agonist/antagonist assays
Citation:
Hornung, M., M. Tapper, J. Denny, B. Sheedy, R. Erickson, T. Sulerud, Rick Kolanczyk, AND P. Schmieder. Avoiding false positives and optimizing identification of true negatives in estrogen receptor binding and agonist/antagonist assays. Applied In Vitro Toxicology. Mary Ann Liebert, Inc., Larchmont, NY, 3(2):163-181, (2017).
Impact/Purpose:
In vitro receptor binding and gene activation assays are tools in common use to identify and prioritize chemicals for their potential to affect endocrine hormone signaling. This paper addresses methods to avoid false assignment of chemicals as hormone receptor antagonists due to artifacts inherent in this type of loss-of-signal assay. This is an important issue as in vitro assays are being implemented to screen chemicals for antagonism activity and these chemicals are subsequently prioritized for further testing. How to best conduct assays to correctly interpret antagonism responses is a current topic of international interest. This has come up for discussion within the OECD’s Validation Management Group- Non-Animal (VMG-NA) which is involved in validation of in vitro methods for screening chemicals for potential endocrine disruption. The information presented in this paper will also help inform the EPAs EDSP screening efforts where other antagonism assays such as those for the androgen receptor or other loss of signal assays are in use.
Description:
The potential for chemicals to affect endocrine signaling is commonly evaluated via in vitro receptor binding and gene activation, but these assays, especially antagonism assays, have potential artifacts that must be addressed for accurate interpretation. Results are presented from screening 94 chemicals from 54 chemical groups for estrogen receptor (ER) activation in a competitive rainbow trout ER (rtER) binding assay and a trout liver slice vitellogenin mRNA expression assay. Results from true competitive agonists and antagonists, and inactive chemicals with little or no indication of ER binding or gene activation were easily interpreted. However, results for numerous industrial chemicals were more challenging to interpret, including chemicals with: (1) apparent competitive binding curves but no gene activation, (2) apparent binding and gene inhibition with evidence of either cytotoxicity or changes in assay media pH, (3) apparent binding but non-competitive gene inhibition of unknown cause, or (4) no rtER binding and gene inhibition not due to competitive ER interaction but due to toxicity, pH change, or some unknown cause. The use of endpoints such as toxicity, pH, precipitate formation, and determination of inhibitor dissociation constants (Ki) for interpreting the results of antagonism and binding assays for diverse chemicals is presented. Of the 94 chemicals tested for antagonism only two, tamoxifen and ICI-182,780, were found to be true competitive antagonists. This report highlights the use of two different concentrations of estradiol tested in combination with graded concentrations of test chemical to provide the confirmatory evidence to distinguish true competitive antagonism from apparent antagonism.
