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

We use this assay to assess whether plant foods contain phytosteriods, which can affect physiological processes in animals, as well as behavior and reproductive success. This assay provides a simple and reliable method for determining whether plants produce estrogenic compounds with biological activity. This method was developed initially to screen drugs with xenoestrogen activity for applications in medicine.

For example, inhibiting cell proliferation. We are using it to provide insight into primate ecology and evolution. It is important to have practice working with aseptic technique in a clean environment prior to attempting this assay, as it is very sensitive to contamination.

Begin by freeze-drying the fresh plant items with the lyophilizer, then finely grind them using a grinding mill with a 0.85 millimeter mesh screen. Store the ground samples in bags in the dark until extraction. To extract any potential phytosteroids in the plants, add dried sample to an appropriately sized flask, then at HPLC grade methanol.

Cover the plant methanol solution with aluminum foil and set it to rotate at 100 RPM for three days on an orbital shaker. After three days, use filter paper to decant the supernate and into a drip filtration system. Dry the plant extract with a rotary evaporator until it is thickened, but pourable in a 300 milliliter round bottom flask.

Pour the sample into a 50 milliliter flask and rinse the large flask with a small amount of methanol, then continue to dry the sample in the small flask until the methanol is completely evaporated. When finished, weigh the sample residue with an analytical balance and record the mass. Dissolve the plant extract in DMSO at a concentration of 0.1 gram of extract to two milliliters of DMSO and vortex it until homogenized.

DMSO is not toxic on its own, but it is a vehicle that allows other substances to cross membranes. Since we do not know if our plants contain toxic chemicals, all are treated as if they're hazardous. Do not allow the solution to touch skin, replace gloves if contaminated, and wear thick, close-toed shoes and lab coats.

Store the sample at four degrees Celsius in amber glass vials until ready to use. Vortex the samples, then add four microliters of each sample to 496 microliters of compound screening medium to yield a 0.8%DMSO solution. Disinfect the outside surface of a pre-warmed cell recovery medium tube with 70%ethanol and transfer 10 milliliters of the medium into a tube of frozen reporter cells to thaw them.

Close the tube of reporter cells and place it in a 37 degrees Celsius water bath for five to 10 minutes. After retrieving the cells from the water bath, gently invert the tube several times to break up aggregates of cells and produce a homogenous suspension, then clean the surface of the tube with 70%ethanol. Use a multi-channel pipette to dispense 100 microliters of the reporter cell suspension into each well of a 96 well plate, then dispense 100 microliters of samples in triplicate into the appropriate wells.

Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 22 to 24 hours. Just prior to the end of the plate incubation remove detection substrate and detection buffer from the refrigerator and place them in a low light area until equilibrated to room temperature, then gently invert each tube to mix the solutions. Immediately before the plate incubation is complete, pour the entire contents of the detection buffer into the tube of detection substrate to create luciferase detection reagent.

Mix the tube gently so as not to produce foam. Discard the contents of the sample plate into inappropriate waste container and gently tap it on a clean absorbent paper towel to remove the last droplets from the wells. Add 100 microliters of the luciferase detection reagent to each well and allow the assay plate to rest at room temperature for 15 minutes, then quantify the luminescence using a 96 well plate reading luminometer.

22 extracts of fruits and vegetables commonly found in human diets were screened for the presence of estrogenic compounds. Estrogenic activity is presented in an ordinal qualitative manner of high, medium, low, or no activity. Organic and non-organic soybeans screened at high levels of activity, while all other fruit and vegetable items registered no activity.

Comparing soybean results to the standard curve also shows that they have high estrogenic activity levels at this concentration. Soybean extract, a known potent source of the isoflavones, daidzein and genistein, was further used to determine the dilution yielding a 50%signal to the maximum. This extract requires 422 times more dilution to produce half the signal of our standard dilution protocol.

Samples identified with activity could be sent for analysis by mass spectrometry to identify the active chemicals. Using an antagonist method, we could also determine if the plant food contains chemicals that reduce the binding and activity of endogenous estrogens. This technique allows us to further our understanding of how prevalent estrogenic foods are in primate diets, as well as how consumption of phytoestrogens affects physiology and behavior when the assay is combined with fecal hormone analyses and field data.