Development of a Competitive Enzyme Immunoassay for Detection of Capacity of Chemicals to Bind Quail Estrogen Receptor .ALPHA. and .BETA.

Abstract

In vitro binding assays are useful in the initial screening of endocrine disrupting chemicals. Such assays should be applied to the estrogen receptors (ER) of not only humans but also wildlife. As a system for birds is yet to established, we expressed the ligand binding domain (LBD) of quail ERα and ERβ as a fusion protein with glutathione S-transferase and using these proteins, developed two systems (a competitive enzyme immunoassay and a fluorescence polarization assay) for assaying the capacity to bind ERs in vitro. Moreover, 20 test chemicals selected by Ministry of the Environment of Japan were evaluated in terms of binding ability. Both systems worked well, the competitive enzyme immunoassay proving especially powerful, since it needs no special equipment. This system is applicable to other species including fish, amphibians and reptiles when information on the LBD of ER is available.Key words ---endocrine disrupting chemicals, estrogen receptor, enzyme immunoassay, quail, endocrine disruptor, in vitro binding assay completed. 5)For the initial screening in vitro, the receptor binding assay is often utilized. The yeast two-hybrid assay is particularly useful, since it is easy and relatively cheap to perform, and also no special equipment is needed. 6,7) This method is based on the interaction between the ligand binding domain (LBD) in the hormone receptor and the coactivator in the ligand-dependent manner. 6) Recently, it was proposed that the effect of EDCs should be considered not only in humans but also in wildlife, and indeed adverse effects on humans and wildlife were reported. However, information on coactivators is restricted to specific species, such as humans, rats and mice.

Evaluation of Ability of Chemicals to Bind Frog (<I>Xenopus laevis</I>) Estrogen Receptor by <I>in Vitro</I> Binding Assay

“…The results for the 20 chemicals are summarized by showing the IC 50 and RBA (%) or REC20 in Table 1. Among those chemicals tested, 4-nonylphenol, poctylphenol and bisphenol A exhibited relatively strong binding to Xenopus ERα as observed for quail ERα, 5) although in each case the IC 50 was 10 -7 M and the RBA was less than 2%. Nine chemicals including 4-nonylphenol, p-octylphenol and bisphenol A gave an IC 50 , and 7 chemicals showed very weak binding and gave a REC20.…”

“…We previously established a competitive enzyme immunoassay (EIA) for analysis of the capacity to bind quail ERα and ERβ, and then tested the chemicals selected by MOE. 5) Although it has recently been proposed that the effects of EDCs are considered not only in humans but also in wildlife, 3,6,7) such as birds, frogs and fish, and indeed adverse effects on human and wildlife were reported (Ministry of Environment, Japan; http://www.env.go.jp/en/topic/edcs.html), 7) a standardized method is not yet established. Moreover, a method for use with amphibian ERs such as frog ER had not been developed.…”

Development of Standardized in Vitro Assay System for Estrogen Receptors and Species Specificity of Binding Ability of 4-Nonylphenol and p-Octylphenol

“…The construction of pGEX-4T-1-ERα-and ERβ-LBD for human and quail was described previously. 5,9) pGEX-4T-1-ERα-and ERβ-LBDs for medaka were a gift from Dr. Nakai at Chemicals Evaluation and Research Institute, Japan. Thus, pGEX-4T-1 was used for all constructs for the expression of ER-LBDs.…”

“…5) Using this competitive enzyme immunoassay system, we next tested the capacity of 4-nonylphenol and p-octylphenol to bind 7 kinds of ERs. The results are shown in Figs.…”

“…We previously established the competitive enzyme immunoassay (EIA) for analysis of the capacity to bind quail ERα and ERβ, and then tested the chemicals selected by MoE. 5) Recently, it has been proposed that the effects of EDCs are considered not only in humans but also in wildlife, such as birds, frogs and fish, and indeed adverse effects on human and wildlife were reported. However, a standardized method is not yet established.…”