Direct determination of estrogenic and antiestrogenic activities using an enhanced plant two-hybrid system

This paper reports a simple, low-cost, and extremely sensitive reporter-gene assay system for comprehensive analysis of estrogenic activity using transgenic Arabidopsis thaliana: the EPTH system. It had the capability to detect 17 beta-estradiol at a concentration of 10 pM. The system was rendered 5 times more sensitive than a previous system [Tojo, T.; Tsuda, K.; Wada, T.; Yamazaki, K. Ecotoxicol. Environ. Saf. 2006, 64, 106-114) (1)] by increasing the copy number of the transactivation domain fused to a nuclear receptor co-activator. The system can efficiently detect other estrogenic and antiestrogenic substances. Estrogenic activities were determined in treated sewage samples from four distinct sewage farms using the system. Results showed that the system can detect estrogenic activity directly and more efficiently than a yeast two-hybrid system without any manipulation for extraction and condensation of hydrophobic compounds and aseptic treatment. Furthermore, the system also is useful as a powerful tool for discovery of a new category of natural estrogenic substances that are undetectable by previous plant and yeast systems.