Concentration and Inhibitor Removal Methods for Determination of Estrogenic Activity in River Water using Yeast Two-Hybrid Test

Abstract

To apply the yeast two-hybrid test for the evaluation of river water, an effective method of concentrating estrogenic substances and a method of removing inhibitors were developed. When 250 ml of river water was supplied to a Sep-Pak Plus PS-2 cartridge and then 10 ml of acetone was used for desorption, typical estrogenic substances were recovered by as much as 93-101% and were concentrated quickly to 500-fold. Moreover, when the concentrated water sample was supplied to a column containing 1.5g of silica gel and was eluted with 8 ml of 30% acetone in toluene, inhibitors of estrogenic activity were removed. The validity of these methods was confirmed using actual river water samples obtained from 19 sites of the Sagami River and 7 sites of the Ayase River.