Issues Arising When Interpreting Results from an in Vitro Assay for Estrogenic Activity

Abstract

Concern about possible adverse effects caused by the inadvertent exposure of humans and wildlife to endocrine active chemicals, has led some countries to develop an in vitro-in vivo screening programme for endocrine effects.In this paper, a previously described estrogen-inducible recombinant yeast strain (Saccharomyces cerevisiae), is used to investigate a number of issues that could potentially lead to the mislabelling of chemicals as endocrine disruptors. The chemicals studied were; 17 -estradiol, dihydrotestosterone, testosterone, estradiol-3-sulfate, 4-nonylphenol, 4-tertoctylphenol, 4-tert-butylphenol, bisphenol-A, methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, butyl benzyl phthalate, 4-hydroxytamoxifen, and ICI 182,780. Many of the issues raised will also affect other in vitro assays for endocrine activity, and some will be relevant to the interpretation of data from in vivo assays. These examples illustrate that considerable care and thought must be applied when interpreting results derived from any single assay. Only by using a suite of assays will we minimise the chances of wrongly labelling chemicals as endocrine disruptors.2

Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters

“…The BLYES and BLYAS assays are consistent with previously published yeast-based reporter assays (Sanseverino et al, 2009). The 40 -50% variability of the EC 50 values shown in Figure 3 reaffirms the suggestion that no single assay should be used to determine an absolute EC 50 value but rather as a first step in estimating the hormonal activity of a chemical (Beresford et al, 2000).…”

Re-evaluation of the first synthetic estrogen, 1-keto-1,2,3,4-tetrahydrophenanthrene, and bisphenol A, using both the ovariectomised rat model used in 1933 and additional assays

“…This showed reduced turbidity in the wells containing high concentrations of both test chemicals, indicating that the yeast cells did not divide as rapidly in these wells. As yeast cell number affects the degree and rate of response of the assay to active chemicals (see Beresford et al, 2000, for a full discussion of this point), the results probably do not suggest that the test chemicals are weakly anti-oestrogenic, but rather that they are toxic (to yeast cells) at concentrations above about 1x10 -10 M. In contrast, THP-1 was clearly androgenic and THP-4 showed marginal androgenic activity ( Figure 4A). Both THP-1 and THP-4 showed dose-related anti-androgenic activity comparable with that of the positive control, flutamide ( Figure 4B).…”

Optimization of a yeast estrogen screen and its applicability to study the release of estrogenic isoflavones from a soygerm powder.

“…Routledge and Sumpter (16) investigated the estrogenic potency of pure chemicals by means of such a transformed yeast. Recently, Beresford et al (25) reported that, upon longer incubation of this recombinant yeast, the color of the blank wells changed slowly. They contributed this increase of the 540-nm absorbance values to the constitutive expression of the reporter enzyme β-galactosidase.…”