Screening for Phytoestrogens using a Cell-based Estrogen Receptor β Reporter Assay

Summary

We have optimized a commercially available estrogen receptor β reporter assay for screening human and nonhuman primate foods for estrogenic activity. We validated this assay by showing that the known estrogenic human food soy registers high, while other foods show no activity.

Abstract

Plants are a source of food for many animals, and they can produce thousands of chemicals. Some of these compounds affect physiological processes in the vertebrates that consume them, such as endocrine function. Phytoestrogens, the most well studied endocrine-active phytochemicals, directly interact with the hypothalamo-pituitary gonadal axis of the vertebrate endocrine system. Here we present the novel use of a cell-based assay to screen plant extracts for the presence of compounds that have estrogenic biological activity. This assay uses mammalian cells engineered to highly express estrogen receptor beta (ERβ) and that have been transfected with a luciferase gene. Exposure to compounds with estrogenic activity results in the cells producing light. This assay is a reliable and simple way to test for biological estrogenic activity. It has several improvements over transient transfection assays, most notably, ease of use, the stability of the cells, and the sensitivity of the assay.

Introduction

Plants are a necessary source of food for many animals, providing calories and nutrients critical to survival, reproduction, growth, development, and behavior¹. Plants produce thousands of chemicals, many as adaptations for their own growth, stomatic maintenance, and reproduction. Other compounds, deemed plant secondary metabolites (PSMs), have functions that are less clear, though some are toxic and likely used as a defense against herbivory and parasitism (e.g., alkaloids, tannins)^2,3. Some of these chemicals have the ability to affect long term physiological processes in animals, suc....

Protocol

1. Preparation of plant materials

1. Freeze dry plant items that were collected fresh using a lyophilizer.
   1. To protect samples from light, cover chambers with aluminum foil during drying process.
   2. To ensure that samples are completely dry, lyophilize until chambers no longer feel cold to touch and plant materials no longer lose mass when weighed.
   3. Store dried plants in sterile low residue bags in absence of light until grinding.
2. Finely grind samples using a grinding mill with 0.85 mm mesh screen.
   1. Store the ground samples in the bags in the absence of light until extraction.
....

Results

Twenty-two extracts of fruits and vegetables commonly found in human diets were screened for the presence of estrogenic compounds. A variety of foods were assayed, including legumes, such as soybeans, snow peas, and snap peas, as the pea family is a known source of phytoestrogens¹⁶, as well as figs, dates, corn, carrots, apples, bananas, strawberries, tomato, kale, and cabbage. Endocrine disrupting compounds are found in common substances (e.g., plastics and pesticides) and some are biologically a.......

Discussion

The ERβ reporter assay developed to individually screen pharmaceutical agents is also suitable for screening plant foods for phytoestrogens biologically active through the ERβ. Important considerations in the protocol include treating the plant samples with care: fresh plant material needs to be dried swiftly to prevent molding or other biological degradation, and it needs to be kept away from light to prevent photolysis of the compounds¹⁸. The assay protocol¹²

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1000 µL pipette
20 µL pipette
200 µL pipette
37 ° water bath
37 °, humidified 5% CO2 incubator
70% ethanol
analytical balance
cell culture-rated laminar flow hood
dimethyl sulfoxide
disposable media basin, sterile
drip filtration system
Erlenmeyer flasks			125 mL and 250 mL
HPLC grade methanol
Human ERβ Reporter Assay System, 1 x 96-well format assays	Indigo Biosciences	IB00411	Assay kit - analyzes 24 samples plus standard curve
lyophilizer
multi-channel pipette
orbital shaker
plate-reading luminometer			ex. Bioteck Synergy HTX
rotory evaporator
round bottom flasks			50 mL and 300 mL
sterile microcentrifuge tubes or sterile multi-channel media basins
sterile tips			200 µL and 1000 µL
Whatman grade 1 paper
whirl-pak bags			sterile polyethylene bags