(593as) High-Throughput Screening System for the Determination of Estrogenic Properties of Small Molecules

Estrogenic compounds are an important group of chemicals with significant properties influencing human health.   They are used to treat several types of cancer, including breast, pancreatic and testicular cancer.  Estrogenic compounds are also linked to negative health effects, such as decreased age of onset in puberty in women.  Therefore, determining the estrogenic potential of a compound is of pharmaceutical and toxicological relevance.  Currently, estrogenic properties of a compound are determined using animal assays.  These assays involve the growth, treatment, sacrifice and autopsy of laboratory animals to determine the effect of the compound in question on animal health.  This is a costly procedure, making it infeasible as a high-throughput screening system.

We have developed an assay using thymidylate synthase (TS) knockout Escherchilia coli cells, in which we have inserted an intein containing the human estrogen receptor beta (ERβ) and thymidylate synthase, to create a bacterial biosensor.  The assay can discriminate between ERβ agonists and antagonists.  The current setup uses 96-well plates, but can be scaled to 384- or 1596-well plates for high-throughput screening.  To validate the assay as an effective method for non-animal testing, dose-response curves for thirty compounds were determined and compared to expected behaviors from literature.  These compounds have been recommended by the (ICCVAM) for the validation of non-animal endocrine disruptor assays.  Our results indicate that our bacterial strains are capable of reliably indicating the potential estrogenicity of most of the test compounds.  This method, which is much cheaper and more animal friendly then many conventional assays, can therefore provide an early screen for new therapeutic compounds, as well as the identification of potentially dangerous environmental estrogen disruptors.