The overall goal of this procedure is to use a yeast estrogen screen to quantify human estrogen receptor agonists extracted from personal care products. This method can help answer hey questions in the field of toxicology, endocrine disruption, and ecophysiology because it detects and quantifies environmental estrogens in water, foods, and personal care products. The main advantages of this in vitro technique are that it is inexpensive and widely used and provides an excellent platform for teaching technical laboratory skills.
Detection of environmental estrogens is critical because they can change hormonal signaling, particularly during fetal development and periods of physiological transition like puberty. The most difficult parts of this method are the calculations and data analysis, which we have simplified by developing an easy to use application. Also, accurate pipetting is essential.
This protocol begins with the making of reagents. Prepare glucose and galactose media, vehicle controls of 50%ethanol, and estradiol standards that will be used on day one of the assay, as well as LacZ buffer, dithiothreitol, and sodium carbonate for day two. Sterilize the glucose and galactose media by filtration through a 0.2 micrometer filter.
Up to 23 different personal care products or PCPs and extraction controls can be tested in triplicate on a single 96-well plate. PCPs include soap, hair cream, shampoo, sunscreen, makeup, shaving cream, and lotion. For the purposes of filming, samples will be prepared from only eight PCPs.
Combine one gram of each sample with 10 milliliters of anhydrous ethanol in a 15-milliliter conical tube. In addition, prepare a negative extraction control consisting of 11 milliliters of anhydrous ethanol in a 15-milliliter conical tube. Homogenize the contents of each conical tube for 30 minutes by a combination of manual shaking and vortexing.
Centrifuge the conical tubes at the maximum allowable speed in a bucket centrifuge for 10 minutes. Decant the supernatant from each tube through a 40-micrometer cell strainer into a 50-milliliter conical tube. Then transfer the contents of each 50-milliliter tube into a glass scintillation vial.
Allow the ethanol to evaporate completely by leaving the vials open in a ventilated hood for one week. Protect the drying samples from light to avoid degradation of light-sensitive constituents. When the samples are dry, reconstitute each sample in one milliliter of 50%ethanol and then vortex to homogenize.
Two nights or about 42 hours prior to preparing the yeast estrogen screen or YES plates, use sterile technique to subculture the yeast by adding 0.1 milliliters of each active yeast culture to 10 milliliters of filter-sterilized glucose media in a sterile Erlenmeyer flask. Incubate at 30 degrees Celsius and continue to incubate original cultures as stocks. Two days later, on day one of the YES assay, use sterile technique to dilute suspended yeast cultures and filter sterilized galactose media in a sterile beaker.
Confirm optical density by pipetting 120 microliters of each yeast sample in triplicate along with triplicate galactose media blanks into a clear polystyrene plate. Use a plate spectrophotometer to verify that the diluted yeast culture has a net optical density of 0.065 plus or minus 0.005 at a wavelength of 610 nanometers. Determine net OD610 of the yeast by subtracting the OD610 of galactose media blanks.
Using a repeating pipetter with a sterile syringe tip, add the diluted yeast suspension to the wells of a sterile polypropylene 96-well microplate according to this plate layout. During pipetting, keep the source yeast suspended by continuously swirling the beaker or mixing with the pipette. Lastly, add 120 microliters of filter-sterilized galactose media to wells H10 through H12, indicated in green in the plate layout, to serve as media controls that will account for absorbents contributed by the media alone.
Prepare separate plates for each combination of yeast strain and calorimetric substrate that will be tested. Next, construct a standard curve in each plate by serial dilution according to the plate layout. Use a single-channel pipetter with sterile tips to add five microliters of estradiol standard to wells A2, A2, and A3.Use a multichannel pipetter to mix the contents of the wells by pipetting, and then transfer 205 microliters from wells A1 to A3 into wells B1 to B3.Repeat the transfer of 205 microliters through row G.After the serial dilution is completed, discard 205 microliters from wells G1 to G3.At the end of this step, all standard wells should contain 120 microliters.
Add five microliters of 50%ethanol to each vehicle control well containing the yeast suspension, wells H1, H2, and H3, indicated in blue in this plate layout. The next step is to add five-microliter triplicates of samples or negative extraction controls to wells containing the yeast suspension, indicated in purple in the plate layout. Note which samples or extraction controls are placed in which wells.
To keep track of which wells have samples and which do not, start with a fresh box of tips and use tips from the same locations in the box as the locations of the corresponding wells in the plate. Mix the contents of the sample, extraction control, and vehicle control wells by pipetting. Adjust the volumes of these wells to 120 microliters by removing 205 microliters from each.
Seal each plate with a sterile adhesive porous film, label, and incubate at 30 degrees Celsius for 17 hours. After the 17-hour incubation, remove the plates from the incubator. If plates cannot be processed immediately, wrap them in plastic wrap and refrigerate the wrapped plates at four degrees Celsius for six days.
When ready to process the plates, unwrap them. Use a multichannel pipetter to mix the contents of each row of wells, and then transfer 50 microliters of yeast suspension from each well to the corresponding well of a clear polystyrene 96-well microplate. Label the new plate.
To avoid cross contaminating wells, use new pipette tips for each well, but the tips do not need to be sterile. In a fume hood, add 20 microliters of one molar DTT to 20 milliliters of thawed, room temperature LacZ buffer for a final concentration of one millimolar DTT. Mix the LacZ buffer well.
Using a repeating pipetter, add 200 microliters of LacZ buffer containing DTT and either ONPG or CPRG to all wells of the polystyrene plate, and immediately measure and record the optical density at 610 nanometers of all wells using a plate reader. Repeat as needed for additional plates. Cover the plates with a lid and incubate them at 30 degrees Celsius for the indicated times.
When the incubation is complete, remove plates from the incubator. Use a repeating pipetter to add 100 microliters of sodium carbonate to each well to hold the beta-galactosidase reaction. Measure and record the optical density at 405 nanometers for ONPG or 574 nanometers for CPRG of all wells using a plate spectrophotometer.
A calculation of LacZ values can be done using an automated application that requires Java to be installed. To begin, open the LacZ automated application described in appendix one. Paste in the optical density readings at 405 nanometers for ONPG or 574 nanometers for CPRG of all wells using a plate layout identical to that shown in figure one.
Enter the amount of incubation time and hours for the assay to produce color and click Next. Paste in the optical density readings at 610 nanometers for all wells. Click Submit to display the LacZ results.
LacZ results can be copied using keyboard commands and pasted to a spreadsheet or other files as needed. Begin this analysis by opening the online YES assay data analysis application. Upload a CSV data file formatted as described on the first tab.
Digitalize the estradiol standard curve on the second tab and estimate the regression parameters. View the regression model outputs on the third tab, and then find the interpolated x-axis values corresponding to the LacZ values of the samples on the fourth tab. Click on the fifth tab to see the estrogen equivalent or EEQ values of the samples.
Finally, standardize the EEQ values to the original sample mass as described in the text protocol. Shown are examples of standard curves after a 17-hour incubation of yeast with 17 beta-estradiol. For reasons discussed in the manuscript, CPRG is often a better substrate choice than ONPG.
Consistent with previous observations, assays with yeast-expressing ER Beta were more than an order of magnitude more sensitive than assays with yeast-expressing ER Alpha. Therefore, estrogenic activity was more often detected with ER Beta-expressing yeast. An additional aim of the study was to test incubation duration times that are compatible with the schedule of an undergraduate laboratory course.
When a six-day refrigeration period after the 17-hour incubation was introduced, standard curves from refrigerated plates were comparable to those from non-refrigerated plates. The standard errors of parameters estimated using four-parameter logistic regression models were all smaller for refrigerated plates than for non-refrigerated plates. Thus, refrigerating plates for six days reduces error and improves the accuracy of EEQ estimates.
Once mastered, this technique can be completed in two three to four-hour sessions. While attempting the procedure, it's important to remember to employ sterile technique for all the steps performed before the 17-hour incubation period, to pipette carefully and accurately, and to follow the plate layout presented in figure one. Following this procedure, other methods like in-vivo animal assays can be performed to investigate additional mechanisms of estrogenic action and whole organism effects.
After its development, the yeast estrogen screen became accepted as a low-cost measure to measure estrogenic ligands in environmental samples such as food, plant tissues, and personal care products. After watching this video, you should have a good understanding of how to perform simple extractions of personal care products, culture yeast, prepare standard dilution curves, run the YES assay in a 96-well plate, and complete associated calculations. And don't forget that working with estradiol can be extremely hazardous, and precautions such as wearing gloves and other personal protective equipment, as well as ensuring appropriate disposal should always be taken while performing this procedure.
