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

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.