Structure-dependent activity of phthalate esters and phthalate monoesters binding to human constitutive androstane receptor.

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Structure-Dependent Activity of Phthalate Esters and Phthalate Monoesters Binding to Human Constitutive Androstane Receptor Hong Zhang,† Zhaobin Zhang,† Tsuyoshi Nakanishi,‡ Yi Wan,† Youhei Hiromori,‡ Hisamistu Nagase,‡ and Jianying Hu*,† †

MOE Laboratory for Earth Surface Process, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, Gifu 501-1196, Japan

‡

S Supporting Information *

ABSTRACT: The present study investigated the human constitutive androstane receptor (CAR) binding activities of 23 phthalate esters and 10 phthalate monoesters using a fast and sensitive human CAR yeast twohybrid assay. Of 23 phthalate esters, 16 were evaluated as positive, and the 10% relative effective concentrations (REC10) ranged from 0.28 (BBP) to 29.51 μM (DEHP), whereas no obvious binding activities were found for the phthalate esters having alkyl chains more than six carbons in length. Of 10 phthalate monoesters, only monoethyl phthalate (MEP), monoisobutyl phthalate (MIBP), and mono-(2-ethyhexyl) tetrabromophthalate (TBMEHP) elicited human CAR binding activities. The REC10 values of MEP and MIBP were 4.27 and 14.13 μM, respectively, higher than those of their corresponding phthalate esters (1.45 μM for DEP and 0.83 μM for DIBP), whereas TBMEHP (0.66 μM) was much lower than TBHP (>102 μM). A molecular docking method was performed to simulate the interaction modes between phthalates and human CAR, and active phthalates were found to lie at almost the same site in the human CAR pocket. The docking results suggest that the strong binding of phthalates to human CAR arises primarily from hydrophobic interactions, π−π interactions, and steric effects and that weak hydrogen bonds and weak halogen bonds greatly contribute to the high binding activity of TBMEHP. In conclusion, the current study clarified that an extensive array of phthalates are activators of human CAR. cytochrome P450s (CYP2B, CYP2C, CYP3A).14 It has been reported that, by regulating CAR through an adenosine monophosphate kinase phosphorylation cascade, phenobarbital can induce the expression of known CAR target genes to promote the metabolism of diethylnitrosamine (DEN), thereby leading to liver tumorigenesis.15 Triclosan can stimulate liver cell proliferation and fibrotic responses by activating CAR and therefore can accelerate hepatocellular carcinoma development, as demonstrated in a tumor promotion study in which triclosan treatment was preceded by administration of DEN in mice.16 Ligand-elicited CAR activators such as 4-bis-[2-(3, 5-dichloropyridyloxy)] benzene can also promote DEN to induce hepatocarcinogenesis in mice.17 These lines of evidence have increasingly confirmed that CAR regulators can activate the metabolism of some xenobiotics to induce liver turmorigenesis. In addition, liver tumors have also been observed in mice exposed to phthalates, such as di(2-ethylhexyl) phthalate (DEHP).11 The highly induced expression of CYP2B10 and CYP2B6, target genes of CAR, suggested the bioactivation of CAR in mice after DEHP exposure.18 Since CAR plays potential roles in the toxicity of phthalate esters, there is a

1. INTRODUCTION Phthalate esters are a group of plasticizers with an annual production of over 470 million pounds1 that have been widely used in numerous industrial and consumer products, including food packaging materials, building materials, medical devices, children’s toys, and personal-care products.2 Phthalate esters are frequently detected in water, sediment, air, and dust,3,4 and humans are exposed to phthalate esters through multiple routes and media.5 Many studies have reported that phthalate esters and their metabolites are ubiquitous in human body fluids and matrices, including human urine, serum, seminal fluid, breast milk, amniotic fluid, and placenta.6−10 Additionally, the health impairments from phthalates exposure, including hepatocarcinogenesis, endocrine disruption, and reproductive and developmental toxicity, have received wide attention.5,11−13 Metabolism can protect from the effects of xenobiotics or can bioactivate chemicals to induce toxicity. Nuclear receptors including pregnane X receptor (PXR), peroxisome proliferator activated receptors (PPARα, β, and γ), aryl hydrocarbon receptor (AhR), and constitutive androstane receptor (CAR) play essential roles in a number of xenobiotic metabolism pathways.14 CAR is increasingly thought to be an important mediator of xenobiotic induction responses since it upregulates genes that encode xenobiotic-metabolizing enzymes, such as © XXXX American Chemical Society

Received: January 18, 2015

A

DOI: 10.1021/acs.chemrestox.5b00028 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology Table 1. Structures of Target Compoundsa

a

A, phthalate esters; B, phthalate monoesters.

The present study, for the first time, determined the human CAR binding activities of 23 phthalate esters with diverse

need to screen other phthalates with diverse structures to determine their CAR binding activities. B


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Chemical Research in Toxicology

Figure 1. Dose−response curves for the human CAR binding activities of phthalates assessed by a yeast two-hybrid assay. (A) CITCO. (B) BBP, DnBP, DAP, and DBzP. (C) DPRP, DIBP, DIPRP, and DIPP. (D) DnPP, DEP, DPHP, and DHXP. (E) BMPP, DCHP, DMP, and DEHP. (F) MIBP, MEP, and TBMEHP. Data are presented as the mean ± SD of triplicate assays. CITCO was used as the positive control. The fitted sigmoidal dose−response curves REC10 calculation were calculated using GraphPad Prism 5. added to 200 μL of fresh medium. After incubation at 30 °C for 4 h, 150 μL aliquots of the culture mixtures were taken out to determine the absorbance at 595 nm. The residual culture was centrifuged (12 000 rpm) for 5 min at 4 °C, and the collected cells were digested enzymatically by resuspension in 200 μL of Z buffer (0.1 M sodium phosphate, 10 mM KCl, and 1 mM MgSO4) containing 1 mg/mL Zymolyase 20T at 30 °C for 20 min. The lysate was mixed with 40 μL of 2-nitrophenyl-β-D-galactoside (ONPG) (4 mg/mL) at 30 °C and reacted until development of a yellow color was observed (usually 20 min). Then, 100 μL of Na2CO3 (1 M) was added to stop the enzymatic reaction, and 150 μL aliquots were transferred into the wells of a 96-well microplate. Absorbance at 415 and 570 nm was read on a microplate reader, and based on the absorbances at 595, 415, and 570 nm, the units (U) of β-galactosidase activity at each concentration were calculated by the following equation

structures and 10 phthalate monoesters by a yeast two-hybrid assay and further explored the interaction between the tested chemicals and human CAR by molecular docking. These results will expand the growing list of environmental contaminants capable of activating human CAR.

2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. Information regarding chemicals and reagents is provided in the Supporting Information. The names and structures of 23 target phthalate esters and 10 phthalate monoesters are shown in Table 1. 2.2. Yeast Two-Hybrid Assay. A yeast two-hybrid assay was used to test the binding activity of phthalates to CAR. This system is based on the ligand-dependent interaction of two proteins, a human CAR receptor and a coactivator, transcriptional intermediary factor 2 (TIF2). Briefly, two expression plasmids, pGBT9-CARLBD and pGAD424-TIF2, were introduced into yeast cells (Saccharomyces cerevisiae Y190) that carry a β-galactosidase reporter gene. When a chemical ligand binds to CAR, CAR interacts with the coactivator, resulting in production of β-galactosidase (Figure S1). The yeast cells were preincubated overnight at 30 °C (about 14−16 h) with vigorous shaking in 5 mL of SD medium (6.7 g/L Difco yeast nitrogen base without amino acids, 2% glucose, 300 mg/L L-isoleucine, 1500 mg/L L-valine, 200 mg/L L-adenine hemisulfate salt, 200 mg/L L-arginine HCl, 200 mg/L L-histidine HCl monohydrate, 300 mg/L L-lysine HCl, 200 mg/L L-methionine, 500 mg/L L-phenylalanine, 200 mg/L Lthreonine, 300 mg/L L-tyrosine, 200 mg/L L-uracil (Sigma, St. Louis, MO, USA) until the cell density reached an optical density (OD) level of 0.4, as measured by a microplate reader (Bio-Rad 550, Richmond, CA, USA) operating at a wavelength of 595 nm. Cell culture (50 μL) and 2.5 μL of phthalate standards dissolved in dimethyl sulfoxide (DMSO) solution diluted to the desired concentrations were then

U = 1000 × ([OD415] − [1.75 × OD570])/([v] × t × [OD595]) where t is the reaction time (min), v is the volume of culture used in the assay (mL), optical density (OD)595 is the cell density at the start of the assay, OD415 is the absorbance by o-nitrophenol at the end of the reaction, and OD570 is the light scattering at the end of the reaction. β-galactosidase activity (U) on the Y axis of the dose− response curve is expressed as the difference between the units of βgalactosidase activity at each concentration of a chemical tested and that of the control (DMSO). 6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), a human CAR-selective agonist, was used as a positive control. Stock solutions of test chemicals were subjected to a 2-fold serial dilution with DMSO to prepare 11 concentrations in the range of 97.7 nM to 100 μM. All results were from experiments performed in at least triplicate assays. It should be noted that the procedures were performed at 30 °C and that the binding activity of target chemicals to CAR would be different from that at body temperature considering C


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Chemical Research in Toxicology the potential effects of temperature on the binding activity. The CAR binding activity of a target compound was expressed as 10% relative effective concentration (REC10), which is the concentration of the test chemical corresponding to 10% of the maximum activity of CITCO, and was calculated based on the sigmoidal dose−response curve. 2.3. Molecular Docking. Molecular simulation software Scigress (ultra version 3.0.0, Fujitsu, USA) was used to dock flexible ligands into a rigid protein active site.19,20 This procedure included (1) ligand structure preparation, (2) protein structure preparation, and (3) docking calculations. In ligand structure preparation, the compounds were sketched and hydrogen atoms were added to each structure with the Scigress Explorer user interface and drawing tools, with the results stored in csf format. Then, their geometries were optimized and energy-minimized using the PM3 force-field method by Scigressintegrated procedures. This energy minimization procedure continued until the energy change was less than 0.001 kcal/mol; otherwise, the molecules were updated 300 times. In protein structure preparation, the X-ray crystal structure of the human CAR ligand-binding domain (CAR-LBD) (PDB ID code 1XVP) was derived from the RCSB Protein Data Bank website (PDB, http://www.rcsb.org/). The protein structure was cleaned up and reduced to a monomer of chain D, which contains the active site of the CAR-LBD. All water molecules were stripped from the crystal structure, and the original ligand was subsequently added to the target protein through the Scigress Workspace module. Hydrogen atoms were added to the target protein to achieve the correct valences of amino acid residues, which was downloaded from PDB (ID: 1xvp). The ionization and tautomeric states of ionizable groups were in accordance with the original crystallographic structure, e.g., histidine residues were protonated. Then, a radius of 6 Å around the bound ligand was used to obtain the active site, and, for simplification, calculations and energy minimization were done using PM3 force-field runs. In the docking calculations, the flexible target compounds were docked into the rigid human CAR ligand binding site to obtain a detailed analysis of the test chemical/ CAR protein interaction. Docking calculations were evaluated using a genetic algorithm with a 15 × 15 × 15 Å grid box with 0.3 Å grid spacing, which contained the active site for the original ligand. The procedure was set to run for 30 000 generations with an initial population size of 100, an elitism of 8, a crossover rate of 0.8, a mutation rate of 0.35, and a convergence of 1.0. Other parameters were set at their default values. The potential of mean force (PMF),21 a knowledge-based approach that extracts pairwise atomic potentials from the structure information on known protein−ligand complexes contained in the Protein Data Bank, was determined and used to score the binding activity of a chemical to the active site of human CAR. 2.4. Data Analysis. Data are expressed as the mean ± standard deviation (SD), unless otherwise stated. GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA) and Excel (Microsoft, NY, USA) were used to analyze the data. Sigmoidal dose−response curves were fitted with a least-squares (ordinary) fit method.

Table 2. Physicochemical Properties of Tested Chemicals (23 Phthalate Esters and 10 Phthalate Monoesters), Molecular Docking Scores, and Binding Activities (REC10) with Human Constitutive Androstane Receptor (CAR) by a Yeast Two-Hybrid Assay chemical (abbr.)

log Pa

L (Å)b

Vm (Å3)c

docking score (kcal/mol)

REC10 (μM)

CITCO BBP DnBP DAP DBzP DPRP DIBP DIPRP DIPP DnPP DEP DPHP DHXP BMPP DCHP DMP DEHP TBMEHP MEP MIBP DHP DnOP DIOP DnNP DINP DIDP TBPH MMP MnBP MBzP MOP MEHP MCHP MnHP

7.94 5.00 4.83 3.29 5.18 3.76 4.46 3.39 5.52 5.89 2.70 4.41 6.95 6.22 5.76 1.64 8.71 6.43 1.66 2.54 8.01 9.08 8.71 10.14 9.77 10.83 8.86 1.13 2.72 2.90 4.85 4.67 3.19 3.79

NDd 6.78 6.63 5.91 6.94 5.90 5.91 4.69 6.94 6.42 4.74 6.35 5.60 5.73 6.46 3.69 7.41 6.68 5.09 5.72 10.75 11.88 10.72 13.47 12.15 13.48 6.44 3.72 7.14 6.42 12.12 6.42 6.58 9.39

490.61 399.58 375.68 317.16 421.62 325.40 366.72 322.43 417.11 422.16 282.29 379.81 467.71 453.82 406.12 235.04 521.64 437.75 233.37 274.46 523.52 576.11 558.38 634.73 613.69 666.41 620.39 208.51 278.14 301.41 371.13 352.79 299.18 326.18

−132.15 −152.44 −132.11 −125.92 −169.85 −125.96 −131.68 −119.36 −126.46 −138.45 −113.40 −154.21 −128.41 −127.43 −136.49 −100.24 −123.50 −111.49 −103.89 −116.41 −127.99 −86.29 −103.80 −100.07 −117.36 −83.75 5.38 −97.70 −118.11 −143.88 −129.01 −122.07 −125.26 −122.07

0.49 0.28 0.35 0.44 0.47 0.72 0.83 0.98 1.12 1.41 1.45 1.74 2.75 9.77 10.23 11.75 29.51 0.66 4.27 14.13 NAe NA NA NA NA NA NA NA NA NA NA NA NA NA

a

log P: the logarithm of the octanol/water partition coefficient, calculated by ACD/logPow ver.1.0. bL: the length of ester side chain, calculated by Scigress, ultra version 2.2.0. cVm: molecular volume, calculated by Scigress, ultra version 2.2.0. dND: not determined. eNA: not activated.

3. RESULTS 3.1. CAR Recognizes a Broad Range of Phthalates. The human CAR binding activities of 33 target chemicals, including 23 phthalate esters and 10 phthalate monoesters (Table 1), were evaluated by a yeast two-hybrid assay. Phthalate esters, except for six high molecular weight compounds (DHP, DnOP, DIOP, DnNP, DINP, and DIDP), showed dose-dependent CAR binding activity (Figure 1B,C,D,E). The REC10 values are presented in Table 2. Of these phthalates, the binding activity of BBP with a benzene ring in its side chain was the highest, with an REC10 of 0.28 μM. It should be noted that phthalate esters with linear alkyl chain lengths less than 7 carbons usually elicited binding activity, and their binding activities generally increased with the number of carbons in the alkyl chain, up to 4 carbons, and then decreased with further increases in the number of carbons. For phthalate esters with an alkyl chain having less than 5 carbons, the binding activity for the phthalate

esters with a branched side chain tended to be lower than that of those with a linear side chain. For example, the REC10 value of DPRP (0.72 μM) was slightly lower than that (0.98 μM) of DIPRP, and the REC10 value of DnBP (0.35 μM) was 2.45-fold lower than that of DIBP (0.83 μM). However, DnPP with a linear alkyl chain (carbon number = 5) elicited relatively low activity (REC10: 1.41 μM) compared with that (1.12 μM) of DIPP with a branched alkyl chain. Of 10 phthalate monoesters, only MEP, MIBP, and TBMEHP elicited significant human CAR binding activity (Figure 1F), and their REC10 values were 4.27, 14.13, and 0.66 μM, respectively (Table 2). The binding activities of MEP and MIBP were both weaker than those of their corresponding phthalate esters (DEP and DIBP). TBMEHP, the brominated analogue of MEHP, elicited strong human CAR binding activity, with a REC10 of 0.66 μM, although we could not obtain D


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Figure 2. Molecular docking conformations of (A) the positive control (CITCO) and (B) the tested phthalate esters that elicited human CAR binding activities: DMP, DEP, DPRP, DAP, DIPRP, DnBP, DIBP, DIPP, DnPP, DHXP, DPHP, DBzP, BBP, DCHP, and BMPP. Phthalate esters are represented as balls and cylinders, and the amino acid residues in the active site are represented as sticks. Atoms are colored as follows: carbon (gray), oxygen (red), chlorine (green), sulfur (yellow), and nitrogen (blue).

Figure 3. Molecular docking conformations of the tested phthalates: (A) BBP, (B) DEHP, (C) MEP, and (D) MIBP. Phthalates are represented as balls and cylinders, and the amino acid residues in the active site are represented as sticks. Atoms are colored as follows: carbon (gray), oxygen (red), and nitrogen (blue).

E


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Chemical Research in Toxicology the whole dose−response curve due to the limited concentration of the commercially obtainable standard. No significant activation of human CAR was observed after incubation with TBMEHP’s corresponding phthalate ester, TBPH, even at the highest concentration tested (100 μM). 3.2. Structural Analysis of Molecular Docking. In order to understand the nature of interactions between phthalates and human CAR, automated molecular docking was performed. The PMF for CITCO, the original ligand in the complex (Figure 2A), when docked into the binding site was −132.15 kcal/mol, and the root-mean-square error (RMSE) between the predicted conformation and the actual conformation from the crystal structure was 1.21 Å, which was smaller than the X-ray crystallography resolution (2.60 Å),22 suggesting that the parameter set for the docking simulation was reasonable. The results of the molecular docking revealed that the interactions between the phthalates and the ligand binding domain of human CAR are primarily hydrophobic. All active phthalate esters except DEHP have almost the same binding mode with a U-shaped conformation: the phenyl rings of the phthalates interact with residues Phe238, Tyr326, Leu242, Phe234, and Phe161, and the alkyl chains interact with residues Phe132, Phe161, Ile164, Asn165, Leu206, Phe217, Tyr224, Asp228, Leu239, and Phe243 of human CAR (Figure 2B). BBP showed significant hydrophobic interactions with amino acid residues Phe161, Phe234, Phe238, Leu242, Phe243, and Tyr326 of human CAR; in particular, it showed π−π interactions between the benzene ring in the side chain of the compound and those of Tyr224 (face-to-face), Phe217 (edge-to-face), and Phe132 (edge-to-face) (Figure 3A). As for DEHP, a high molecular weight phthalate ester, the orientation in the pocket was slightly different from that of the other active phthalates (Figure 3B): the phenyl ring of DEHP appears to be oriented away from Phe238, Tyr326, Leu242, Phe234, and Phe161 and tends associate close to Phe217 and Phe243. The high molecular weight phthalates with alkyl side chains having more than six carbons (DHP, DnOP, DIOP, DnNP, DINP, and DIDP) adopt awkward conformations with their side chains folded in the pocket when docked into human CAR, as exemplified by the DINP−CAR complex (Figure S2). In the docking view of the two phthalate monoesters (MEP and MIBP) that elicit binding activity with human CAR, the side chains occupy only part of the pocket formed by residues Phe161, His203, Leu206, Phe217, Phe238, and Phe243 (Figure 3C,D). As for the interactions between TBMEHP and human CAR, the important features were the formation of weak hydrogen bonds [C−Br···H−C (Phe161) (Br1···H1 = 2.812 Å, Br1···H2 = 2.951 Å, Br2···H2 = 2.832 Å), C−Br···H−O (Tyr326) (Br1···H3 = 3.275 Å), C−Br1···H−N (Asn165) (Br1···H4 = 2.465 Å, Br1···H5 = 3.31 Å) and (Br2···H4 = 3.362 Å, Br2···H5 = 3.562 Å)] and weak halogen bonds [C−Br···N− CO (Asn165) (Br1···N1 = 3.113 Å, Br1···N2 = 3.316 Å)], in addition to the π−π interaction between the benzene ring of TBMEHP and Phe161 (edge-to-face) (Figure 4). The docking scores of all target chemicals are listed in Table 2. There was a correlation between the simulated and the experimental activities, with a correlation coefficient (r2) of 0.85 for low molecular weight phthalates and 0.61 for high molecular weight phthalates (Figure 5), showing the relatively high effectiveness of the molecular docking. From the molecular docking results, the interactions between phthalates and human CAR can be characterized by hydrophobic interactions and steric effects. Thus, the molecular structural parameters,

Figure 4. Molecular docking conformation of TBMEHP. Carbon is in gray, hydrogen, white, nitrogen, blue, oxygen, red, and bromine, orange. Weak hydrogen bonds and weak halogen bonds are expressed by red dashed lines between the bromine atom and residues Phe161, Tyr326, and Asn165.

Figure 5. Relationships between 10% relative effective concentration (REC10) of phthalates and docking score. Low molecular weight phthalates (DMP, DEP, DPRP, DIPRP, DAP, DnBP, and DIBP): log REC10 = 0.041, docking score +8.0 (n = 7, r2 = 0.85, p < 0.05). High molecular weight phthalates (DIPP, DnPP, DHXP, DEHP, DCHP, BMPP, BBP, DBzP, and DPHP): log REC10 = 0.035, docking score +8.2 (n = 9, r2 = 0.61, p < 0.05).

including the logarithm of the octanol/water partition coefficient (log P, ACD/logPow ver.1.0, Advanced Chemistry Development Inc.), molecular volume (Vm, Scigress, ultra version 2.2.0, Fujitsu, USA), and the length of ester side chain (L, Scigress, ultra version 2.2.0, Fujitsu, USA), were calculated in this study (Table 2). For lower molecular weight phthalates (DMP, DEP, DPRP, DIPRP, DAP, DnBP, and DIBP), their binding activities linearly correlated with log P (r2 = 0.72, n = 7, p < 0.05) (Figure 6A), and better correlation between their REC10 values with the side chain length L (r2 = 0.82, n = 7, p < 0.01) was achieved (Figure 6B). However, for high molecular weight phthalates (DIPP, DnPP, DHXP, DEHP, DCHP, BMPP, BBP, DBzP, and DPHP), their binding activities were less correlated with log P (r2 = 0.54, n = 9, p < 0.05) and L (r2 = 0.20, n = 9, p > 0.05). F


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Figure 6. Relationships between 10% relative effective concentration (REC10) of low molecular weight phthalates (DMP, DEP, DPRP, DIPRP, DAP, DnBP, and DIBP) and (A) log P or (B) length of side chain (L).

4. DISCUSSION To our knowledge, this is the first study to examine the binding activities of a broad range of phthalate esters and monoesters to human CAR using a fast and sensitive yeast two-hybrid assay. Recently, the strong binding activities of DnBP and DEHP to splice forms of the human CAR receptor have been demonstrated.23 In the present study, 16 phthalates (DMP, DEP, DPRP, DIPRP, DAP, DnBP, DIBP, DnPP, DIPP, DHXP, BMPP, DBzP, BBP, DPHP, DCHP, and DEHP) elicited human CAR binding activities with the REC10 values of 0.28− 29.51 μM (Table 2). In particular, BBP, DnBP, DIBP, and DEP elicited much higher human CAR binding activities, with REC10 values of 0.28−1.45 μM, and are ubiquitous in the environment (food, air, water, and dust) and human body fluids and matrices.3−10 For example, high levels of phthalates have been detected in semen samples, with mean concentrations of 3.11 μg/mL for DEP and 1.65 μg/mL for DnBP,8 which are sufficient to activate CAR; therefore, these phthalate esters may pose a health risk by interfering with the metabolism of endogenous and exogenous substrates through activation of CAR. Given the important role of CAR in bioactivating chemicals to induce toxicity, further studies are warranted to clarify the potential toxicity of phthalate esters. The present study, for the first time, reported that phthalate monoesters (MIBP, MEP, and TBMEHP) also elicit binding activity to human CAR. As the major metabolites of phthalates, phthalate monoesters are continuously introduced into the aqueous environment via municipal wastewater discharge. Monoesters can also be generated from phthalate esters by microorganisms in sewage treatment plants and in surface waters, soils, and sediments.24 Therefore, the environmental occurrence of phthalate monoesters is of great concern. MMP, MnBP, and MEHP have been detected in river water from Tokyo at concentrations of 0.030−0.0340, 0.010−0.480, and 0.010−1.30 μg/L, respectively.25 Phthalate monoesters were also detected in biota, with concentrations of MMP (4.43-21.82 ng/g wet wt), MEP (5.63−25.54 ng/g wet wt), and MnBP (75.0−585 ng/g wet wt) being significantly higher (p < 0.05) than their corresponding parent product concentrations in mussels.26 Given that phthalate monoesters are ubiquitous in the environment, the high human CAR binding activities of MEP and MiBP observed in the present study suggest that phthalate monoesters should be given more attention in addition to phthalate esters. In particular, TBPH is a novel brominated flame retardant with an annual production volume of 450 to 4500 tons from 1990 to 2006 by Chemtura Chemical Corporation,27 and it has been recently developed as a substitute for banned PBDEs.28 It has been reported that the

median concentration of TBPH in dust from indoor environments is up to 410 ng/g,28 and TBPH has also been detected in marine mammals from the Pearl River Delta with mean concentrations of 342 ± 883 ng/g lipid−1.29 Since TBPH can be metabolized to TBMEHP,28 TBMEHP would be discharged into the aqueous environment and would accumulate in aquatic organisms and humans. Considering the high human CAR binding activity of TBMEHP (REC10 = 0.66 μM), more toxicological studies on its endocrine disrupting effects are needed in order to evaluate whether TBPH is suitable for use as a substitute for common brominated flame retardants. Molecular docking indicated that the hydrophobicity and steric effects of phthalates play important roles in their binding to human CAR, as the 22 important amino acid residues (Phe129, Phe161, Ile164, Asn165, Met168, Val199, Cys202, His203, Leu206, Thr209, Phe217, Tyr224, Phe234, His246, Phe238, Tyr326, Ile330, Gln331, Ile333, Met339, Leu343, and Ile346) present in the inner surface of the ligand binding domain of human CAR form the hydrophobic agonist pocket with a volume of 675 Å3.22 Structure−activity relationships were also demonstrated by linear correlation between the binding activities and the log P values for phthalate esters with alkyl chain carbon numbers less than 5 (Figure 6A). The binding activities of phthalates were found to increase with increasing length of the linear alkyl chain from DMP to DnBP and to decrease with the extension of the alkyl chain of DnBP (Figure 6B). Binding activities for DHP, DNOP, DIOP, DnNP, DINP, and DIDP were not observed, possibly due to their larger volume and longer side chains. Additionally, all active phthalate esters except DEHP were extend into the most hydrophobic region of the pocket (Figure 2B), and the chemicals are found to be away from the region of Leu343, which is thought to have a negative effect on the ability of ligands to bind human CAR.30 Furthermore, they come to a similar position in the pocket, as the distances between amino acid residues are similar (Figure S3). The U-shaped conformations of the phthalates upon binding are in good agreement with the conformation of CITCO in the human CAR crystal structure.22 The molecular docking result demonstrated that BBP fitted well into the active site of human CAR via significant hydrophobic interactions, especially π−π interactions (Figure 3A). The simulated binding energy of BBP is −152.44 kcal/ mol, much more negative than that of the known human CAR agonist CITCO (−132.15 kcal/mol), which supports the finding that BBP demonstrated the highest CAR binding among the phthalates. On the other hand, the phenyl ring of DEHP was placed in the deep of the hydrophobic pocket formed by Phe217 and Phae243, and its alkyl chains were G



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folded due to its large volume and branched side chains. Such fitting made DEHP less flexible for interaction with hydrophobic residues around the pocket (Figure 3B), therefore leading to its weak binding activity to human CAR. In comparison to the docking of DEHP, when other phthalates with a higher molecular weight than DEHP were docked into the human CAR pocket, their side chains twisted much more because the length of the side chains of DHP (10.75 Å) and DIDP (13.48 Å) is greater than that of DEHP (7.41 Å). Also, the length of the binding cavity calculated in CAR is around 10 Å, spanning from Phe234 to Tyr224 and Phe161 to Phe217. Therefore, these chemicals are too large to be confined inside the binding cavity without any strong torsion, and the docked complexes are expected to be unstable. Such a binding mode is different from that described for active chemicals, which may lead to the result that docking scores cannot separate the active and inactive chemicals shown in Table 2. As for MEP and MIBP, because these two phthalate monoesters have only one side chain to interact with the active pocket, their binding activities are lower than those of their corresponding phthalate esters. In the interaction mode of other phthalate monoesters, we also considered the relationship between CAR binding cavity and the extent of hydrophobic contact in the ligand−receptor complex based on size. When the number of carbons in the side chain is greater than four, their side chains folded in the pocket and the benzene ring deviated from Phe238 and Phe234, as exemplified by the docking conformation of MHXP (Figure S4). The hydrophobic interactions with MMP are fewer due to its shorter carbon chains (carbon number = 1). Such a binding mode may explain why these phthalate monoesters did not elicit binding activity. While the steric hindrance of TBMEHP was expected to be similar to that of TBHP, weak hydrogen bonds and weak halogen bonds in addition to hydrophobic and π−π interactions greatly contributed to the relatively high binding activity of TBMEHP (Figure 4).

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: 86-10-62765520. E-mail: [email protected]. Funding

This study was supported by the National Natural Science Foundation of China (41330637), the National Basic Research Program of China (2012BAD20B05), and the 111 Project (B14001). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS PXR, pregnane X receptor; PPAR, peroxisome proliferatoractivated receptor; AhR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; CYP, cytochrome P450; DEN, diethylnitrosamine; DEHP, di(2-ethylhexyl) phthalate; TIF2, transcriptional intermediary factor 2; OD, optical density; DMSO, dimethyl sulfoxide; ONPG, 2-nitrophenyl-βD-galactoside; REC10, 10% relative effective concentrations; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime; SD, standard deviation; LBD, ligand-binding domain; PDB, Protein Data Bank; RMSE, root-mean-square error; PMF, potential of mean force; DnBP, di-n-butyl phthalate; DIBP, diisobutyl phthalate; DnPP, di-n-pentyl phthalate; BBP, benzyl butyl phthalate; MEHP, mono-2-ethylhexyl phthalate; MnBP, mono-n-butyl phthalate; DHP, diheptyl phthalate; DnOP, di-n-octyl phthalate; DIOP, di-iso-octyl phthalate; DINP, diisononyl phthalate; DnNP, di-nnonyl phthalate; DIDP, diisodecyl phthalate; DPRP, di-npropyl phthalate; DIPRP, diisopropyl phthalate; MEP, monoethyl phthalate; MIBP, monoisobutyl phthalate; TBMEHP, mono-(2-ethyhexyl) tetrabromophthalate; DEP, diethyl phthalate; TBPH, bis(2-ethylhexyl) tetrabromophthalate; DMP, dimethyl phthalate; DAP, diallyl phthalate; DIPP, diisopentyl phthalate; DHXP, di-n-hexyl phthalate; DCHP, dicyclohexyl phthalate; BMPP, bis-4-methyl-2-pentyl ester; DBzP, dibenzyl phthalate; DPHP, diphenyl 1,2-phthalate; MMP, monomethyl phthalate; MHXP, monohexyl phthalate; MCHP, monocyclohexyl phthalate; MOP, monooctyl phthalate; MBZP, monobenzyl phthalate

5. CONCLUSIONS Overall, the present study provides a qualitative and quantitative analysis of the binding activities of 23 phthalate esters and 10 phthalate monoesters toward human CAR, and molecular docking explains the binding of phthalates to human CAR via hydrophobic interactions and steric effects. As a result, 16 phthalate esters and 3 phthalate monoesters are identified as being activators of human CAR, which will be helpful for understanding the potential toxicological implications of human exposure to phthalates in light of the regulatory role of CAR in metabolism.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information on chemicals and reagents, measurement of atom−atom distances, the mechanism of the yeast twohybrid assay to determine the human CAR binding activities of phthalate esters, distances between activated phthalates (DMP, DEP, DPRP, DAP, DIPRP, DnBP, DIBP, DIPP, DnPP, DHXP, DPHP, DBzP, BBP, DCHP, and BMPP) and the neighbored aromatic amino acid residues, and molecular docking conformations of DINP and MHXP in human CAR’s pocket. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00028. H


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