Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

1. Making Reagents

1. Prepare glucose and galactose media by mixing 6.7 g yeast nitrogen base, 20 g dextrose or galactose, 20 mg adenine sulfate (added as 10 mL of a 200 mg/100 mL aqueous stock solution), 20 mg uracil (added as 10 mL of a 200 mg/100 mL aqueous stock solution), 60 mg leucine (added as 6 mL of a 1 g/100 mL aqueous stock solution), and 20 mg histidine (added as 2 mL of a 1 g/100 mL aqueous stock solution) in deionized water to a final volume of 1 L. Sterilize by filtration (0.2 µm filter). Media can be stored at 4 °C for several weeks.
2. Prepare estradiol (E2) standards by dissolving powdered E2 in anhydrous ethanol and diluting to the appropriate working concentrations (227.5 nM & 9.75 nM) in 50% ethanol.
   Note: If estradiol is purchased as a vial of 1 mg, add 1 mL ethanol directly to the vial to dissolve estradiol powder. This yields a 3.67 mM stock #1.  Dilute 10 μL of stock #1 in 990 μL ethanol to make 1 mL of 36.71 μM stock #2.  Then, dilute 10 μL of stock #2 in 990 μL 50% ethanol to make 1 mL of 367.13 nM stock #3.  To make working stocks, dilute 619.7 μL stock #3 in 380.3 μL 50% ethanol to make 1 mL of 227.5 nM working stock used to create standard curves with yeast expressing ERα.  Dilute 26.56 μL stock #3 in 973.44 μL 50% ethanol to make 1 mL of 9.75 nM working stock used to create standard curves with yeast expressing ERβ. Seal standards with parafilm and store at -20 or -70 °C.
   CAUTION: E2 is a suspected carcinogen and reproductive toxicant. It is harmful if inhaled, swallowed, or absorbed through the skin. E2 may cause harm to breast-feeding children and fetuses. E2 is very toxic to aquatic life. Use appropriate personal protection (gloves, fume hood, dust mask) and avoid exposure during pregnancy and lactation. Dispose E2 as hazardous waste and do not release into the environment.
3. Prepare LacZ buffer by mixing 8.52 g Na₂HPO₄, 5.52 g NaH₂PO₄•H₂O, 95.21 mg MgCl₂, 745.5 mg KCl, 2 g N-lauroylsarcosine sodium salt, and either ONPG (400 mg) or CPRG (77.7 mg) in deionized water to a final volume of 1 L. Store LacZ buffer in 20 mL aliquots at -20 °C.
   NOTE: A 20 mL aliquot is sufficient for one 96-well plate.
4. Prepare sodium carbonate by mixing 105.9 g sodium carbonate in deionized water to a final volume of 1 L. Store at RT.
5. Prepare 1 M dithiothreitol (DTT) in water. Freeze 25 µL aliquots of DTT at -20 °C.
   CAUTION: DTT is an acute skin and eye irritant. Use appropriate personal protection equipment (gloves, fume hood, dust mask) to avoid skin and eye contact, inhalation and ingestion.
   NOTE: Each aliquot is sufficient for one 96-well plate.

2. Preparing Samples and Extraction Controls

1. Select samples to test.
   NOTE: This article presents data for fifteen PCPs, including soap, hair cream, shampoo, sunscreen, lip balm, foundation, shaving cream, nail polish, and lotion.
2. Combine 1 g of sample with 10 mL of anhydrous ethanol in a 15 mL conical tube. Prepare a negative extraction control consisting of 11 mL of ethanol in a 15 mL conical tube. Prepare replicates as needed.
   NOTE: For the presented experimental design, three aliquots of all 15 PCPs and triplicate extraction control solutions were prepared, yielding a total of 48 samples, each of which was analyzed in triplicate using the protocol in section 4. A single plate can accommodate up to 23 samples in triplicates. Anhydrous ethanol is used in this step because it evaporates readily. Use negative extraction controls to verify that estrogens are not leaching into samples from the conical tubes.
3. Homogenize tube contents for 30 min by a combination of manual shaking and vortexing or by shaking tubes on a multi-tube vortexer.
4. Centrifuge conical tubes at the maximal allowable speed in a bucket centrifuge for 10 min. Decant the supernatant from each tube through a 40 µm cell strainer into a 50 mL conical tube. Empty contents of each 50 mL tube into a glass scintillation vial.
5. Leave vials open in a ventilated hood for one week to allow ethanol to evaporate completely. Alternatively, dry samples in a ventilated hood under nitrogen or with a rotary evaporator. To avoid degradation of light sensitive constituents, protect drying samples from light (e.g., place opaque fabric over ventilated hood door).
6. Reconstitute samples in 1.0 mL of 50% ethanol and vortex to homogenize.

3. Culturing and Subculturing Yeast¹⁴

1. Maintain active yeast cultures (recombinant W303a strain of S. cerevisiae¹⁴) by incubating yeast in filter-sterilized glucose media at 30 °C. Subculture yeast weekly in fresh filter-sterilized glucose media.
2. Two nights (about 42 h) prior to preparing the YES plates, subculture yeast by adding 0.1 mL of active yeast cultures to 10 mL of filter-sterilized glucose media using sterile technique.  Incubate at 30 °C for two nights.  Shaking is not required if yeast are grown as shallow cultures in sterile 250 mL Erlenmeyer flasks.
   NOTE: Yeast can also be stored on refrigerated agar plates for several months. For longer term storage (years), combine O/N cultures with 15 – 50% sterile glycerol, vortex, and freeze at -70 °C. To prepare sterile glycerol, use a syringe to transfer 300 µL glycerol into a cryovial and autoclave with the cap loosened. Then add 1,200 µL O/N yeast culture using sterile technique.

4. Preparing YES Plates (Day 1 of the Assay)

1. In a sterile beaker and using sterile technique, dilute yeast from step 3.2 to an optical density of 0.065 ±0.005 at 610 nm (OD₆₁₀) in filter-sterilized galactose media.
   1. Confirm optical density by pipetting 120 μL of each yeast sample in triplicate along with triplicate galactose blanks into a clear polystyrene plate (plate does not need to be sterile).  Use a plate spectrophotometer to verify that the diluted yeast culture has a net OD₆₁₀ of 0.065 ±0.005   Subtract OD610 readings of galactose blanks from yeast OD₆₁₀ readings to determine net OD₆₁₀ of the yeast. Prepare a final volume of at least 30 mL diluted yeast for each 96-well plate. 
      NOTE: Spectrophotometer filters measuring absorbance between 595 and 610 nm can be used for this measurement. An approximate 1:6 v:v ratio of yeast:media typically yields an OD₆₁₀ close to 0.065, so start by adding 4.5 mL yeast to 30 mL media and add more yeast or media as appropriate to achieve a netOD₆₁₀ between 0.060 and 0.070.
2. Using sterile technique, add this diluted yeast suspension to the wells of a sterile, polypropylene, 96-well microplate according to the plate layout (Figure 1). During pipetting, keep the source yeast suspended by continuously swirling the container or mixing with the pipette. For this step, it is helpful to use a repeating pipettor with a sterile syringe tip.
   Note:  Polypropylene plates are sterilized by autoclaving two stacked plates wrapped in foil.  The top plate serves as a lid for the sample plate and can be washed, re-autoclaved, and reused. 
3. Add 120 µL filter-sterilized galactose media to 3 additional wells (H10 – 12) according to the plate layout (Figure 1). These wells serve as media controls to account for absorbance contributed by the media alone.
4. Construct a standard curve in each plate by serially diluting triplicates of E2 working standards (227.5 nM for ERα, 9.75 nM for ERβ) in wells containing the yeast suspension (A1 – G3) according to the plate layout (Figure 1).  Begin by by adding 5 µL of E2 standards to wells A1 through A3. Mix microwell contents by pipetting, and then serially dilute this suspension by moving 205 µL from wells A1 – 3 into wells B1 – 3. Repeat the mixing and transfer of 205 µL through row G.
   Note: After the serial dilution is completed, discard 205 μL from wells G1 - 3. At the end of this step, all standard wells should contain 120 μL. The serial dilution will yield the final E2 concentrations listed in Table 1. For the serial dilution steps, it is helpful to use a multichannel pipettor with sterile tips.
5. Add 5 µL of vehicle (50% ethanol) to vehicle control wells containing the yeast suspension (H1 – 3) according to the plate layout (Figure 1).
   NOTE: Vehicle control wells are used to quantify background absorbance associated with yeast cells and media. Additionally, cytotoxicity or growth promoting effects of test samples can be detected by comparing the OD₆₁₀ values of test wells with those of vehicle control wells.
6. Add 5 µL triplicates of samples and negative extraction controls to wells containing the yeast suspension according to the plate layout (Figure 1).
   Note: Make a note of which samples or negative extraction controls are added to each well.  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. One 96-well plate can accommodate up to 23 samples in triplicate.
7. Mix the contents of the sample, extraction control, and vehicle control wells by pipetting. Adjust volumes of these wells to 120 µL by removing 205 µL from each as described in the legend for  Figure 1.
8. Seal the plate(s) with a sterile, adhesive, porous film. Label plates and incubate for 17 h at 30 °C. The plate does not need to be shaken during incubation

5. Processing YES Plates (Day 2 of the Assay)

1. After the 17 h incubation at 30 °C, remove plates from the incubator. If plates will be processed immediately, proceed to step 5.1.1. If plates will be processed at a later date, proceed to step 5.1.2.
   1. Use a multichannel pipettor to mix the contents of each row of wells, and then transfer 50 µL of yeast suspension from each well to the corresponding well of a clear, polystyrene, 96-well microplate.
      Note: Polystyrene plates do not need to be sterile but should have a lid. Use new pipet tips for each row of wells to avoid cross-contaminating wells, although if pipetting across triplicates with an 8-tip multichannel pipette, tips only need to be changed between triplicate sets.
      1. Label the new plate. Proceed to step 5.2.
   2. To store plates before processing, wrap them in plastic wrap. Refrigerate wrapped plates at 4 °C for 6 d. Afterward, remove the plates from the refrigerator, unwrap them, and transfer the contents of all wells by pipetting as in step 5.1.1. Proceed to step 5.2.
2. Add 20 µL of 1 M DTT to 20 mL of thawed, room-temperature LacZ buffer for a final concentration of 1 mM DTT. Mix LacZ buffer well. If using a multi-channel pipette, dispense into a reagent reservoir. 
   CAUTION: Wear gloves and work with DTT in a hood, if possible
3. Using either a multichannel pipette or repeating pipettor, add 200 µL of LacZ buffer containing DTT and either ONPG or CPRG to all wells of the polystyrene plate, and immediately measure and record the OD₆₁₀ of all wells using a plate spectrophotometer. Repeat as needed for additional plates.
   Note: The OD₆₁₀ readings are used in the denominator of the LacZ calculation (step 6.1). For this reason, obtain the readings immediately after pipetting and before cells settle or are lysed by LacZ buffer. If OD₆₁₀ values for sample wells are noticeably higher or lower than OD₆₁₀ values for vehicle control wells, the sample extracts are either growth-promoting or cytotoxic to yeast, respectively. The LacZ calculation will correct for small differences in cell densities across wells, but if differences exceed 30% in either direction, then dilute the reconstituted sample (from step 2.6) in additional 50% ethanol and retest the sample by returning to step 3.2¹².
4. Cover plates with a lid. Incubate plates containing yeast that express ERα¹⁴ at 30 °C for 40 min (for ONPG) or 3 h (for CPRG). Incubate plates containing yeast that express ERβ¹⁴ at 30 °C for 70 min (for ONPG) or 4 h (for CPRG).
   Note: When working with CPRG, incubation times are flexible; incubating for 2 – 4 h yields suitable results with both receptors, with longer incubations increasing assay sensitivity.
5. After incubating plates, use a multichannel pipette or repeating pipettor to add 100 µL of sodium carbonate to each well. Sodium carbonate raises the pH and halts the β-galactosidase reaction.
6. Measure and record the OD₄₀₅ (for ONPG) or OD₅₇₄ (for CPRG) of all wells using a plate spectrophotometer.

6. Calculating LacZ Values

NOTE: LacZ values quantify the degree of color change for each sample and offer a normalized method of comparing values among separate assays by accounting for several variables (e.g., yeast optical density, media optical density, and incubation time if this differs among wells on the same plate)¹².

1. Calculate means of triplicate OD₆₁₀ values for media (galactose) control wells (H10-12), and calculate means of triplicate OD₄₀₅ or OD₅₇₄ values for vehicle (50% ethanol) controls (H1-3). Use these means and the incubation time (t) in hours for the plate to change color (step 5.5) to calculate LacZ values (corresponding to the degree of color change in each well) for all standard and sample wells using the following equations:
   
   
   1. Alternatively, use the automated application in Appendix 1 to calculate LacZ values for standards and samples.
      Note: The plate layout used with the application must be identical to the plate layout presented in Figure 1. If some sample wells were not used, retain absorbance readings from the empty wells as place holders in the absorbance dataset being pasted into the Appendix 1 LacZ application.  This preserves the spatial layout of the plate in the application and ensures that media control wells are in their required location.  Additionally, the application will not execute if there are empty cells. 
2. Calculate means of triplicate LacZ values for each E2 standard and each sample. Report mean LacZ values as µL^-1h^-1. Log-transform the concentrations of each E2 standard, and generate a spreadsheet document formatted as in the template.csv file (Appendix 2).

7. Interpolating Sample Estradiol Equivalents (EEQs) Using 4-Parameter Logistic Regression

NOTE: EEQs relate sample LacZ values (color change) to the LacZ values of the standard curve created with E2. EEQs thus determine how much sample is required to elicit the same color change response as a known concentration of E2.

1. To calculate EEQs for test samples, first plot the standard curve. Fit mean LacZ values for E2 standards (y-axis) and the corresponding log-transformed E2 concentrations (x-axis) to a four-parameter logistic regression model.
2. Use the model to interpolate EEQs for test sample LacZ values. Conduct logistic regression calculations of EEQs using statistical software as outlined in Appendix 2.

8. Standardizing EEQs (ng/mL) to PCP Sample Mass

1. To relate EEQs to the amount of PCP sample added to each well in step 4.5, multiply EEQ (ng/mL) by the total volume plated into the sample wells (325 µL) and then divide by the volume of extracted sample added to each well (5 µL).
   NOTE: These values represent EEQ per mL of extracted sample. If samples were prepared using 1 g of PCP, these values also represent EEQ per gram of PCP (ng/g). Expressed in this way, EEQ values (ng/g) can be compared across different PCPs. EEQ (ng/g) values for the personal care products tested in this study are shown in Table 2, and sample data collected throughout the entire protocol are included in Appendix 3.