Employment of the human estrogen receptor beta ligand-binding domain and co-activator SRC1 nuclear receptor-binding domain for the construction of a yeast two-hybrid detection system for endocrine disrupters
Lee, H-Seog.; Miyauchi, K.; Nagata, Y.; Fukuda, R.; Sasagawa, S-ichi.; Endoh, H.; Kato, S.; Horiuchi, H.; Takagi, M.; Ohta, A.
Journal of Biochemistry 131(3): 399-405
ISSN/ISBN: 0021-924X PMID: 11872169 DOI: 10.1093/oxfordjournals.jbchem.a003115
To screen a wide variety of chemicals for endocrine disrupters, and to develop an effective microbial degradation system for them, a good system is needed for the rapid and accurate evaluation of the endocrine-disrupting activities of suspected chemicals and their degradation products. We constructed two-hybrid systems that co-express the Gal4p DNA binding domain/ligand-binding domain of human estrogen receptor (hER) alpha or beta and the Ga14p transactivation domain/nuclear receptor-binding domain of co-activator SRC1, TIF2, or AIB1 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of Gal4p-binding sites. We found that the combination of the hERbeta ligand-binding domain and SRC1 nuclear receptor-binding domain was most effective for the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by known xenoestrogens and phytoestrogens was found to correlate well with their estrogenic activities as measured by the previous system with rat ERalpha. This system detects estrogenic activity in some chemicals that have not been suspected of being positive. We also applied this assay system to test the microbial degradation products of gamma-hexachlorocyclohexane (gamma-HCH) by Sphingomonas paucimobilis. Among the gamma-HCH metabolites, 2,5-dichlorohydroquinone and chlorohydroquinone had estrogenic activities similar to the original chemical, while hydroquinone, a later stage metabolite, showed no activity, suggesting the necessity of evaluating intermediate metabolites in microbial degradation systems.