Eight phthalate monoesters (the structures of phthalate monoesters are given in Figure 1 ) were purchased from J&K Chemical (Beijing, China). Human SULT isoforms (SULT1A1, 1A3, 1B1, and 1E1) were obtained from BD Gentest Corp (Woburn, MA, USA). 4-Nitrophenol (PNP) and its sulfate PNP-S,3′-phosphoadenosine-5′-phosphosulfate (PAPS), Tris-HCl, and MgCl₂ were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultra-performance liquid chromatography (UPLC) grade acetonitrile was purchased from Tianjin Saifurui Technology Ltd. Millipore Elix 5 UV and Milli-Q Gradient Ultra-Pure Water System were used for the preparation of ultra-pure water. Other reagents were of ultra-performance liquid chromatography (UPLC) grade or of the highest grade commercially available.

The incubation system (total volume = 200 μl) containing 100 mM of Tris-HCl buffer (pH 7.4), 5 mM of MgCl₂, 40 μM of PAPS, SULTs, and PNP. The concentrations of SULT 1A1, SULT1A3, SULT1B1, and SULT1E1 were 10 μg/ml, 10 μg/ml, 20 μg/ml, and 10 μg/ml, respectively. In the incubation process, a 3-minute pre-incubation was performed, and then PAPS was added to initiate the metabolic reaction. The reaction temperature was 37°C and the reaction time was 30–60 min. At the end of the reaction, 200 μl of ice-cold acetonitrile was added for termination. An incubation solution without phthalate monoesters was used as a control. Then, after centrifugation at 12,000 rpm, 10 μl of supernatant was taken for analysis with liquid chromatography (UPLC)-UV instrument. The column used for UPLC separation is a C18 column (4.6*200 mm, 5 μm, Kromasil) with a flow rate of 0.4 ml/min and a column temperature of 25°C. The mobile phase is 0.5% formic acid aqueous solution for phase A and acetonitrile for phase B. The gradient condition was used as follows: 0–6 min, 5% B; 6–8 min, 95% B; 8–14 min, 95% B; 14–17 min, 5% B. The detection wavelength was 280 nm. The incubation system (total volume = 200 μl) containing 100 μM of phthalate monoesters, Tris-HCl buffer (50 mM, pH 7.4), MgCl₂ (5 mM), PAPS (40 μM), SULTs, and PNP was used to determine the Michaelis constant (K _m) of PNP metabolites. The used concentration range was 1–400 μM for SULT1A1, 0.1–7 mM for SULT1A3, 0.1–1 mM for SULT1B1, and 0.1–1.2 mM for SULT1E1. The kinetic parameters (V _max and K _m) were determined through fitting the data to the Michaelis–Menten equation or the substrate inhibition equation using Prism 4 software.

4-Nitrophenol was employed as a nonselective probe substrate for human SULT isoforms to investigate the inhibition of phthalate monoesters on SULT isoforms. The incubation system (total volume = 200 μl) containing 100 μM of phthalate monoesters,100 mM of Tris-HCl buffer (pH 7.4), 5 mM of MgCl₂, and 40 μM of PAPS, and the concentrations of 4-nitrophenol and SULTs are different. The concentrations of 4-nitrophenol were 40, 1,200, 40, and 120 μM for SULT1A1, SULT1A3, SULT1B1, and SULT1E1, respectively. In addition, the concentrations of SULT isoforms were 10, 10, 20, and 10 μg/ml for SULT1A1, SULT1A3, SULT1B1, and SULT1E1, respectively. The absence of phthalate monoesters was set as the negative control. After pre-incubation at 37°C for 3 min, 40 μM PAPS were added for the reaction. The metabolic reaction is terminated by the addition of acetonitrile of the same volume and ensures that the incubation time can reach 30–60 min according to different SULTs. The optimized microsome protein concentration and incubation time were used to ensure linear velocity (v). The final mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant of 10 μl was then used for ultra-performance liquid chromatography (UPLC)-UV instrument. All experiments were repeated in two independent experiments. Screening of phthalate monoesters was carried out with a SULT inhibition rate greater than 80% for subsequent experiments.

The concentration-dependent inhibition effect of phthalate monoesters on SULTs was investigated using different concentrations of phthalate monoesters (ranging from 0 μM to 100 μM), and the half inhibition concentration (IC₅₀) was calculated. Based on different SULT isoforms, multiple concentrations of 4-Nitrophenol (covering the K _m value) and phthalate monoesters (covering the IC₅₀ values) were used to determine the inhibition kinetics. Lineweaver–Burk plots are used to determine the type of inhibition kinetics. The second plot was used to calculate the inhibition kinetic parameters (K _i), using the linear slope of the Lineweaver–Burk double reciprocal plot and the concentration of the inhibitor phthalate monoesters.

In vivo inhibition magnitude of SULTs was determined through IVIVE. The following equation was used:

The terms are defined as follows: AUC_i/AUC was the predicted ratio of in vivo exposure of xenobiotics or endogenous substances with or without the co-exposure of phthalate monoesters. [I] was the in vivo exposure concentration of phthalate monoesters, and the K _i was the in vitro inhibition constant. The standard used was as follows: [I]/K _i < 0.1, low possibility; 0.1 < [I]/K _i < 1, medium possibility; [I]/K _i > 1, high possibility.

In order to better clarify the molecular interaction between phthalate monoesters and SULTs, an in silico docking method was used to dock the chemical structure of phthalate monoesters into the activity cavity of SULTs. Three-dimensional (3D) structure of SULT isoforms was established using a homology modeling method. Autodock Version 4.2 was employed to dock the flexible small molecule of phthalate monoesters into the rigid protein of SULTs. The non-polar hydrogen atoms of SULT enzymes were merged. The gridbox was generated with 60 × 60 × 60 in X, Y, and Z coordinates, covering the entire ligand-binding site. Lamarckian Genetic Algorithm (LGA) method was selected to possess molecular docking study for the binding of phthalate monoesters towards SULTs. The interactions between phthalate monoesters and SULTs were analyzed, including hydrogen bonds and hydrophobic contacts.

Experimental data were expressed as the mean value plus standard deviation (S.D.). Statistical analysis between two groups using a two-tailed unpaired Student’s t-test. Multiple groups were compared using the one-way ANOVA, and p < 0.05 was considered to be significant.

The inhibition of phthalate monoesters on SULT1A1, SULT1B1, and SULT1E1 is shown in Figure 2 , and multiple phthalate monoesters have strong inhibition towards SULT1A1, SULT1B1, and SULT1E1. MHP, MOP, MBZP, and MEHP significantly inhibited the activity of SULT1A1. MBP, MHP, MOP, MCHP, and MEHP significantly inhibited the activity of SULT1B1. MHP, MOP, and MEHP significantly inhibited the activity of SULT1E1. As shown in Figure 2B , relatively more phthalate monoesters exhibited strong inhibition towards SULT1B1, with more than 80% activity inhibited by 100 μM of phthalate monoesters. All phthalate monoesters have no significant inhibition on the activity of SULT1A3 ( Supplementary Figure 1 ). According to the preliminary screening results, we can find some structure–activity relationship for the inhibition of SULTs by phthalate monoesters. Phthalate monoesters with long chains inhibit the activity of SULTs relatively strongly, while phthalate monoesters with short chains (e.g., MMP, MEP, etc.) showed no significant inhibition towards all SULTs isoforms.

Some phthalate monoesters significantly inhibited SULT1A1, SULT1B1, and SULT1E1, and IC₅₀ was further determined. The concentration-dependent inhibition of MHP and MOP towards SULT1A1, SULT1B1, and SULT1E1 was exhibited ( Figure 3 ). The concentration-dependent inhibition curve for other phthalate monoesters towards other SULTs isoforms are given in Supplementary Figure 2 . The IC₅₀ values for the inhibition of MHP, MOP, MBZP, and MEHP towards SULT1A1 were calculated to be 17.1, 5.6, 0.3, and 2.3 μM, respectively; IC₅₀ values for the inhibition of MBP, MHP, MOP, MCHP, and MEHP towards SULT1B1 were calculated to be 8.5, 11.6, 2.7, 12.8, and 13.9 μM, respectively; IC₅₀ values for the inhibition of MHP, MOP, and MEHP towards SULT1E1 were calculated to be 4.5, 1.8, and 1.7 μM, respectively ( Table 1 ). In addition, inhibition kinetics were further determined, including kinetic types and parameters (K _i). As shown in Figures 4A, B , the intersection point was located in the vertical axis in the Lineweaver–Burk plot for the inhibition of SULT1B1 and SULT1E1 by MOP, indicating the competitive inhibition of MOP towards SULT1B1 and SULT1E1. The slopes of the lines in the Lineweaver–Burk plot were calculated and drawn versus the concentrations of MOP. The second plot was used to calculate the inhibition kinetic parameters (K _i). According to Figures 4C, D , the inhibition kinetic parameters (K _i) were calculated to be 2.2 μM and 5.5 μM for the inhibition of MOP on SULT1B1 and SULT1E1, respectively.

Since phthalate monoesters showed broad inhibition on SULT1B1, SULT1B1 was selected as the representative SULT isoform. We used the in silico docking method to analyze the mechanism of its inhibition of SULT1B1. The in silico docking method was used to dock the chemical structure of phthalate monoesters into the activity cavities of SULT1B1, and the representative docking results of MHP and MOP were given. The active site where SULT1B1 binds to MHP consists of amino acid residues ARG-131, SER-49, TRP-53, GLY-50, THR-52, THR-51, PHE-256, LYS-48, PRO-255, SER-139, ARG-258, HIS-142, and PHE-143, as shown in Figure 5A . The active site where SULT1B1 binds to MOP consists of amino acid residues MET-233, THR-52, MET-257, PHE-256, TRP-53, SER-139, LYS-48, SER-49, GLY-50, PHE-230, GLY-260, LYS-259, TYR-194, ARG-258, and ARG-131, as shown in Figure 5B . In the binding pocket of SULT1B1, MHP formed eight hydrogen bonds to SER-139, ARG-131, LYS-48, SER-49, GLY-50, and THR-51 ( Figure 6A ), and 4 hydrophobic contacts were formed between MHP and the active cavity of SULT1B1 ( Figure 7A ). MOP formed three hydrogen bonds to SER-139, ARG-131, and LYS-48 ( Figure 6B ), and 4 hydrophobic contacts were formed between MOP and the active cavity of SULT1B1 ( Figure 7B ). The binding free energy of MBP, MHP, MOP, MCHP, and MEHP towards SULT1B1 were −7.66, −8.05, −8.23, −8.92, and −8.43 kcal/mol, respectively. The other phthalate monoester docking results are given in Supplementary Figures 3A–C, 4A–C, and 5A–C .