Chemicals and Reagents.

LCA, cholic acid, dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sulfate (DHEA-S) were purchased from Steraloids, Inc. (Newport, RI), and LCA-S disodium salt was from Santa Cruz Biotechnology, Inc. (Dallas, TX). Adenosine 3′-phosphate 5′-phosphosulfate lithium salt hydrate (PAPS), sodium taurolithocholate, taurolithocholic acid 3-sulfate (TLCA-S) disodium salt, amoxicillin, clomifene citrate, tamoxifen, toremifene citrate, ospemifene, raloxifene, bazedoxifene acetate, DL-dithiothreitol, 2-mercaptoethanol, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Lithocholylglycine, 3-sulfoglycolithocholic acid (GLCA-S) disodium salt, lasofoxifene, 7-methoxylasofoxifene, nafoxidine hydrochloride, bazedoxifene-N-oxide, des(1-azepanyl)ethylbazedoxifene, cis-4-(1,2,3,4-tetrahydro-6-methoxy-2-phenyl-1-naphthalenyl)phenol, N-desmethyl-4-hydroxy-toremifene hydrochloride, N-desmethyltoremifene hydrochloride, 4-hydroxytoremifene, and 4′-hydroxytoremifene were bought from Toronto Research Chemicals (Toronto, ON). Droloxifene citrate was purchased from Abcam (Cambridge, UK), and arzoxifene hydrochloride and acolbifene were from AdooQ Bioscience (Irvine, CA). Minimum essential medium/Earle’s balanced salts (MEM/EBSS) culture medium (#SH30244.01), fetal bovine serum (no. SV30160.03), MEM nonessential amino acids (100×), trypsin-EDTA (0.25%), l-glutamine (200 mM), penicillin G-streptomycin (100×), and phosphate-buffered saline (pH 7.4) were of HyClone brand purchased from GE Healthcare Life Sciences (Buckinghamshire, UK). Coomassie (Bradford) Protein Assay kit (no. 23200) was bought from Thermo Fisher Scientific Inc. (Waltham, MA). All other commercially available chemicals were of analytical grade or HPLC grade.

Cytosol and Recombinant Enzymes.

Human liver cytosol (mixed gender; pool of 150 donors; catalog no. 452115, lot #38290 (ages 18–82) and #38291 (ages 18–77), Gentest brand; 75 women and 75 men) was purchased from Corning, Inc. (Corning, NY). Human recombinant SULT2A1 (catalog no. CYP104, lot no. INT044E2B) and SULT1E1 (catalog no. CYP103, lot no. INT044E1B) enzymes and control cytosol (isolated from Escherichia coli host cells) were purchased from Cypex Ltd. (Dundee, Scotland, UK). Human recombinant SULT2B1b enzyme (catalog no. 6174-ST-020, lot no. DADE0616011), which contained Met-1 to Glu-311 amino acids of SULT2B1b expressed in E. coli host cells and contained a C-terminal 6-histidine tag, was purchased from R&D Systems, Inc. (Minneapolis, MN). This recombinant SULT2B1b enzyme has been shown to be functional and has a high enzyme activity (>10 nmol/min per milligram protein) (R&D Systems, Inc.).

DHEA Sulfonation Assay and Quantification of DHEA-S by UPLC-MS/MS.

The DHEA sulfonation assay was conducted based on previously optimized method (Bansal and Lau, 2016b; Yip et al., 2018). Calibration standards were prepared by adding freshly prepared DHEA-S stock solutions (1–1000 μM in DMSO) to a standard incubation to give final concentrations of 1–1000 nM DHEA-S (equivalent to 0.2–200 pmol in 200 μl). The amount of DHEA-S and cortisol (internal standard) was quantified by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The UPLC-MS/MS method was previously validated and demonstrated to be more specific, easier, safer, and faster than radiometric-based sulfotransferase enzyme assays (Bansal and Lau, 2016b).

LCA, GLCA, and TLCA Sulfonation Assay.

The optimization of LCA sulfonation assay was performed as described previously (Bansal and Lau, 2016a). In general, each 200-µl standard incubation mixture contained potassium phosphate buffer (100 mM; pH 7.4), MgCl₂ (2.5 mM), substrate (LCA, GLCA, or TLCA), and various amounts of human liver cytosol or recombinant SULT2A1, SULT2B1b, or SULT1E1 enzymes, as specified in each figure legend. The final concentration of methanol was 0.5% v/v, which did not affect the catalytic activity of SULT2A1 (Ma et al., 2003). Each incubation mixture was prewarmed for 3 minutes at 37°C in a shaking water bath. PAPS, a cofactor (20 µM; a saturating concentration that resulted in maximum enzyme activity) (Bansal and Lau, 2016a), was added to initiate the enzyme reaction. After incubating the samples at 37°C for a specific duration, as specified in each figure legend, we terminated the reaction with 200 µl of ice-cold acetonitrile containing cholic acid (0.1 µM final concentration in LCA sulfonation assay or 0.3 µM final concentration in GLCA or TLCA sulfonation assay; internal standard). Each sample was mixed and placed immediately in an ice bath. The supernatant was transferred into a 96-well microplate after centrifugation of each sample at 16,000g for 15 minutes at 4°C. UPLC-MS/MS was used for the quantification of LCA-S, GLCA-S, and TLCA-S.

Quantification of LCA-S, GLCA-S, and TLCA-S by UPLC-MS/MS.

Calibration standards were prepared by adding freshly prepared LCA-S, GLCA-S, or TLCA-S stock solutions (1–1000 μM in DMSO) to a standard incubation mixture to give final concentrations of 1–1000 nM LCA-S (equivalent to 0.2–200 pmol in 200 μl), 3–3000 nM GLCA-S (equivalent to 0.6–600 pmol in 200 μl), or 1–3000 nM TLCA-S (equivalent to 0.2–600 pmol in 200 μl). Quality-control samples were prepared in the same manner as that for the calibration standards. The final amount of GLCA-S in the low-, mid-, and high-quality control samples was 1.5, 10, and 100 pmol, respectively. The final amount of TLCA-S in the low-, mid-, and high-quality control samples was 1, 10, and 100 pmol, respectively. All the standard, quality-control, and unknown samples were centrifuged at 16,000g at 4°C for 15 minutes, and an aliquot of the supernatants was transferred to a 96-well polypropylene plate for UPLC-MS/MS analysis.

The method to quantify the amount of LCA-S was modified from a previously developed and validated UPLC-MS/MS method (Bansal and Lau, 2016a). New UPLC-MS/MS methods for the quantification of GLCA-S and TLCA-S were developed and validated. An Agilent Infinity1290 LC system (Agilent Technologies, Waldbronn, Germany) coupled to an AB Sciex Triple Quad 3500 triple-quadupole mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a TurboV ion source was used. Chromatographic separation was achieved on an ACQUITY UPLC BEH C₁₈ column (2.1 × 50 mm, 1.7 µm). The column and the autosampler compartment were maintained at 45°C and 4°C, respectively. The flow rate was 0.5 ml/min, and the sample injection volume was 5 µl. The mobile phases were (A) 10 mM ammonium acetate and (B) methanol. The gradient conditions were optimized as follows: 20% B at 0.0–1.0 minute, linear increase from 20% to 95% B at 1.0–2.5 minutes, 95% B at 2.5–5.0 minutes, linear decrease from 95% to 20% B at 5.0–5.1 minutes, and 20% B at 5.1–6.0 minutes. The total run time was 6 minutes. The UPLC effluent was introduced directly into the mass spectrometer from 1.1 to 5.0 minutes. The mass spectrometer was operated in the negative electrospray ionization mode. Compound-dependent and ion source-dependent mass spectrometric parameters were optimized to achieve maximal ion intensities in the multiple reaction monitoring mode. Nitrogen gas was used as the curtain gas, collision gas, and ion source gas. The optimized compound-dependent MS parameters and ion source parameters for LCA-S, GLCA-S, GLCA, TLCA-S, TLCA, and cholic acid are shown in Supplemental Table S1. Data acquisition and processing were performed using Analyst software version 1.6.2 (Applied Biosystems).

Enzyme Kinetic Analysis of LCA, GLCA, and TLCA Sulfonation.

The enzyme kinetics of LCA, GLCA, or TLCA sulfonation catalyzed by human liver cytosol and recombinant SULT2A1 enzyme were performed by conducting the sulfonation assay at various concentrations of LCA, GLCA, or TLCA. Each 200-µl standard incubation mixture contained potassium phosphate buffer (100 mM; pH 7.4), MgCl₂ (2.5 mM), human liver cytosol, or recombinant SULT2A1 enzyme, and varying concentrations of a substrate, at an amount or concentration stated in the figure legend. SigmaPlot version 12.5 Enzyme Kinetics Module (Systat Software, Inc., San Jose, CA) was used to calculate the values of apparent K_m and V_max by nonlinear least-squares regression analysis of the rate of enzyme activity (V) and substrate concentration (S) data. Various measures of goodness of fit, including Akaike information criterion, coefficient of determination (R²), S.D. of residuals (Sy.x), and visual inspection of the V versus [S] progress curve were considered in the selection of the best model, including the substrate inhibition model (eq. 1):(1)where V_max represents the apparent maximum reaction velocity, K_m represents the substrate concentration at which the reaction rate is half of V_max, and K_i represents the equilibrium dissociation constant between the substrate and the binding site of the enzyme.

Enzyme Inhibition Experiments.

Inhibition of LCA, GLCA, or TLCA sulfonation was determined by conducting enzyme activity assay in the presence a test chemical (SERM or a metabolite/analog) or methanol (0.5% v/v; vehicle), a substrate (LCA, GLCA, or TLCA), and human liver cytosol or recombinant SULT2A1 enzyme at an amount or concentration stated in each figure legend. Amoxicillin (1 mM), which did not inhibit DHEA sulfonation in a previous study (Bamforth et al., 1992), was used as a negative control. Concentration-response experiments for each of the SERMs were conducted in the presence of varying concentrations of a test chemical or methanol (0.5% v/v; vehicle), a substrate (LCA, GLCA, or TLCA), and human liver cytosol at an amount or concentration stated in each figure legend. The IC₅₀ was determined by nonlinear regression analysis (SigmaPlot 12.5) using the sigmoidal dose-response (variable slope) model (eq. 2):(2)where min is the minimum inhibitory effect, max is the maximum inhibitory effect, x is the inhibitor concentration, and Hill slope is the Hill coefficient.

Enzyme Kinetic Analysis of the Inhibition of LCA Sulfonation by SERMs.

To determine the inhibition kinetics, LCA sulfonation assay was conducted in the presence varying concentrations (0.2, 0.4, 0.6, or 0.8 µM) of LCA and varying concentrations of a SERM, as stated in the figure legend. The apparent K_i value (equilibrium dissociation constant for the enzyme-inhibitor complex) and the mode of inhibition of each SERM were determined by nonlinear regression analysis of the rate of LCA-S formation at varying concentrations of LCA and a SERM, using equations for full and partial competitive, noncompetitive, and mixed-mode inhibition (SigmaPlot 12.5). The goodness of fit for each model was evaluated by considering Akaike information criterion, R², and visual inspection of the data in the Lineweaver-Burk plot. The K_i value was determined using various inhibition models, including the full competitive inhibition model (eq. 3), partial mixed inhibition model (eq. 4), full mixed inhibition model (eq. 5), and full noncompetitive inhibition model (eq. 6):(3)(4)(5)(6)where S represents the substrate concentration, I represents the inhibitor concentration, V_max represents the apparent maximum reaction velocity, K_m represents the substrate concentration at which the reaction rate is half of V_max, and K_i represents the apparent equilibrium dissociation constant for the enzyme-inhibitor complex.

Enzyme Inactivation Experiments.

Each 200 µl of primary incubation mixture contained potassium phosphate buffer (100 mM; pH 7.4), MgCl₂ (2.5 mM), human liver cytosol (200 µg cytosolic protein), and a SERM (each at 10 µM) or methanol (0.5% v/v; vehicle). The mixture was prewarmed for 3 minutes at 37°C in a shaking water bath. Enzymatic reaction was initiated by adding PAPS (20 µM), and the mixture was preincubated at 37°C for 0, 30, 60, or 90 minutes. An aliquot (10 µl) of the primary incubation mixture was transferred to a prewarmed secondary incubation mixture (190 µl) containing phosphate buffer (pH 7.4), MgCl₂ (2.5 mM), LCA (2.5 µM), and PAPS (20 µM). This secondary incubation mixture was incubated at 37°C for 30 minutes. The reaction was terminated with 200 µl of ice-cold acetonitrile containing cholic acid (0.1 µM final concentration). After mixing thoroughly, each sample was placed immediately in an ice bath and centrifuged at 16,000g for 15 minutes at 4°C. The supernatant was then transferred to a 96-well microplate for the quantification of LCA-S using UPLC-MS/MS.

Cell Culture.

HepG2 human hepatocellular carcinoma cells (ATCC HB-8065) were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and authenticated by ATCC. HepG2 cells were cultured in MEM/EBSS culture medium supplemented with MEM nonessential amino acids (1×), 2 mM l-glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin, and 10% v/v fetal bovine serum. HepG2 cells were cultured as described previously (Seow and Lau, 2017). The passage number of HepG2 cells was kept between 10 and 15.

In Situ Lithocholic Sulfonation Assay in HepG2 Cells.

In the first experimental design, cultured HepG2 cells were seeded onto 12-well plates at a cell density of 200,000 cells per well. At 96 hours after plating, HepG2 cells were washed with phosphate-buffered saline (pH 7.4). To each well, 200 μl of phosphate-buffered saline containing substrate (0.5 μM LCA) and a SERM (each at 10 µM), substrate (0.5 μM LCA) and amoxicillin (1 mM), substrate (0.5 μM LCA), and DMSO (0.1% v/v; vehicle) or DMSO alone (0.1% v/v; vehicle) was added. The cells were incubated for 15 minutes at 37°C in a humidified incubator with 95% air and 5% carbon dioxide. At the end of the incubation period, 160 μl of the incubation mixture was collected from each well and transferred into a microcentrifuge tube. The remaining incubation mixture in each well was aspirated and washed with phosphate-buffered saline. To each well, 200 μl of a lysis buffer (1% v/v Triton X-100 and 20 mM EDTA in phosphate-buffered saline) was then added. The content in each plate was mixed on a plate shaker for 1 hour to ensure complete cell lysis. At the end of cell lysis, 160 μl of the cell lysates was collected from each well and transferred into a microcentrifuge tube.

In the second experimental design, cultured HepG2 cells were seeded onto 12-well plates at a cell density of 400,000 cells per well. At 48 hours after plating, cells were treated with 1 ml of culture medium (with 5% fetal bovine serum) containing substrate (0.5 μM LCA) and a SERM (each at 10 µM), substrate (0.5 μM LCA), and amoxicillin (1 mM), substrate (0.5 μM LCA) and DMSO (0.1% v/v; vehicle) or DMSO alone (0.1% v/v; vehicle). At 24 hours after the chemical treatment period, 160 μl of the culture medium was collected from each well and transferred into a microcentrifuge tube. The remaining culture medium was aspirated, and cells were washed with phosphate-buffered saline. The lysates were prepared as described above.

In the third experimental design, cultured HepG2 cells were seeded onto 12-well plates at a cell density of 400,000 cells per well. At 48 hours after plating, cells were treated with 1 ml of culture medium (with 5% fetal bovine serum) containing substrate (0.5 μM LCA) and a SERM (each at 10 µM), substrate (0.5 μM LCA) and amoxicillin (1 mM), substrate (0.5 μM LCA) and DMSO (0.1% v/v; vehicle), or DMSO alone (0.1% v/v; vehicle) for 24 hours. At the end of the treatment period, 200 μl of phosphate-buffered saline containing substrate (0.5 μM LCA) and a SERM (each at 10 µM), substrate (0.5 μM LCA) and amoxicillin (1 mM), substrate (0.5 μM LCA) and DMSO (0.1% v/v; vehicle), or DMSO alone (0.1% v/v; vehicle) was added. The cells were incubated for 15 minutes at 37°C in a humidified incubator with 95% air and 5% carbon dioxide. At the end of the incubation period, 160 μl of the incubation mixture was collected from each well and transferred into a microcentrifuge tube. The remaining incubation mixture in each well was aspirated and washed with phosphate-buffered saline. The lysates were prepared as described above.

To each tube of incubation mixture, culture medium, or cell lysates, 80 μl of acetonitrile containing 0.2 μM cholic acid (internal standard; final concentration of 0.1 μM in a total sample volume of 240 μl) was added and stored at −20°C before sample analysis. To prepare the calibration standards, LCA-S was serially diluted with DMSO (1–1000 μM) and further diluted by 1000× with phosphate-buffered saline, and LCA substrate (0.5 μM) was added. The final DMSO concentration was 0.2% v/v. A 160 μl of LCA-S standard solution was mixed with 80 μl of acetonitrile containing 0.2 μM cholic acid (internal standard; final concentration of 0.1 μM in a total sample volume of 240 μl). The final concentration of LCA-S was 1–1000 nM (equivalent to 0.16–160 pmol in 160 μl). The standard and sample mixtures were centrifuged at 16,000g for 15 minutes at 4°C. The supernatants were transferred into a 96-well microplate for analysis by the UPLC-MS/MS method as described herein already.

Total Protein Quantification Assay.

At the end of the treatment/incubation period and cell lysis, the total amount of cellular protein in each well was quantified using the Coomassie (Bradford) Protein Assay kit (Thermo Fisher Scientific Inc.), according to the manufacturer’s protocol. The cell lysate samples were diluted 10× using phosphate-buffered saline. The calibration curve was constructed using bovine serum albumin diluted with 10% v/v lysis buffer in phosphate-buffered saline (solvent composition similar that of the samples) to give a final concentration of 25–1000 μg/ml. A 5-μl aliquot of each sample or standard was transferred into a 96-well clear microplate, and 250 μl of the Coomassie reagent was added to each well. The content in each plate was mixed and incubated at room temperature for 10 minutes. The absorbance was measured at 600 nM using a plate reader (GloMax Explorer; Promega Corporation, Madison, WI). The net absorbance was calculated by subtracting the absorbance of blank sample (diluent) from that of the samples/standard sample. The amount of total cellular protein was calculated based on the calibration curve freshly prepared in each experiment.

Statistical Analysis.

Data were analyzed by one-way or two-way analysis of variance and, where appropriate, was followed by the Student-Newman-Keuls post hoc test (SigmaPlot 12.5). The level of statistical significance was set a priori at P < 0.05.