Assessment of phytoestrogen and mycoestrogen recognition by recombinant human estrogen receptor-α using ligand titration arrays

Abstract

Introduction: Exposure to phytoestrogens and mycoestrogens has emerged as a public health issue due to their potentially endocrine disruption activities resulting from direct interaction with sex-steroid hormone receptors. There is a significant requirement for comprehensive, reproducible methods to determine the extent of estrogen mimicry by compounds encountered in the environment to estimate risk:benefit ratios, particularly in humans.

Objective: To develop a systematic approach for assessing recognition of chemically diverse compounds by human estrogen receptor proteins to aid in their assessment as endocrine disruptor compounds (EDCs).

Methods: Recombinant human estrogen receptor-α protein (rhERα) was expressed in Saccharomyces cervisiae as an ubiquitin fusion under control of a CUP1 promoter and partially purified with heparin affinity chromatography in the unliganded state. A novel radio-ligand binding array was developed to evaluate structurally diverse compounds, both naturally occurring and synthetic, for estrogen binding activity and affinity.

Results: Binding affinities of suspected estrogen mimics for rhERα were calculated over a range of [(3) H]estradiol-17β concentrations using Lundon OneSite® and Compete® software.

Conclusion: β-zearalanol, a mycoestrogen similar to zearalenone used as an ICCVAM validation substance for the in vitro estrogen receptor binding assays (ICCAM report), was employed as a model estrogen mimic to illustrate the approach, methods and calculations using these techniques.