Employment of the human estrogen receptor α ligand-binding domain and co-activator SRC1 nuclear receptor-binding domain for the construction of a yeast two-hybrid detection system for endocrine disrupters

Haeng Seog Lee, Keisuke Miyauchi, Yuji Nagata, Ryouichi Fukuda, Shin Ichi Sasagawa, Hideki Endoh, Shigeaki Kato, Hiroyuki Horiuchi, Masamichi Takagi, Akinori Ohta

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)

Abstract

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) α or α and the Gal4p 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 hERα 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 ERα. 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 γ-hexachlorocyclohexane (γ-HCH) by Sphingomonas paucimobilis. Among the γ-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.

Original languageEnglish
Pages (from-to)399-405
Number of pages7
JournalJournal of biochemistry
Volume131
Issue number3
DOIs
Publication statusPublished - 2002 Jan 1

Keywords

  • Co-activator SRC-1
  • Endocrine disrupter
  • HERβ
  • Yeast two-hybrid system
  • γ-HCH

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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