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Inhibition of UDP-glucuronosyltransferases (UGTs) by phthalate monoesters
Chemosphere 197 (2018) 7e13
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Inhibition of UDP-glucuronosyltransferases (UGTs) by phthalate monoesters Zuo Du a, 1, Yun-Feng Cao b, 1, Sai-Nan Li a, Cui-Min Hu c, Zhi-Wei Fu a, Chun-Ting Huang d, Xiao-Yu Sun d, Yong-Zhe Liu a, Kun Yang a, Zhong-Ze Fang a, * a
Department of Toxicology, School of Public Health, Tianjin Medical University, 22 Qixiangtai Road, Heping District, Tianjin, China Key Laboratory of Liaoning Tumor Clinical Metabolomics (KLLTCM), Jinzhou, Liaoning, China Tianjin Life Science Research Center, Department of Microbiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China d RSKT Biopharma Inc., Liaoning, China b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Phthalate monoesters showed broad inhibition on UGT1A9. Threshold values for inducing in vivo inhibition of UGTs were calculated. Both hydrogen bonds and hydrophobic interaction contributed to the inhibition of UGT1A9 by phthalate monoestersi.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2017 Received in revised form 2 January 2018 Accepted 4 January 2018 Available online 5 January 2018
Phthalate monoesters are important metabolites of phthalate esters (PAEs) which have been extensively utilized in industry. This study aims to investigate the inhibition of phthalate monoesters on the activity of various isoforms of UDP-glucuronosyltransferases (UGTs), trying to elucidate the toxicity mechanism of environmental endocrine disruptors from the new perspectives. In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was employed to evaluate 8 kinds of phthalate monoesters on 11 sorts of main human UGT isoforms. 100 mM phthalate monoesters exhibited negligible inhibition towards the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A8, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17. The activity of UGT1A7 was strongly inhibited by monoethylhexyl phthalate (MEHP), but slightly inhibited by all the other phthalate monoesters. UGT1A9 was broadly inhibited by monobenzyl phthalate (MBZP), monocyclohexyl phthalate (MCHP), MEHP, monohexyl phthalate (MHP) and monooctyl phthalate (MOP), respectively. MEHP exhibited competitive inhibition towards UGT1A7, and MBZP, MCHP, MEHP, MHP and MOP showed competitive inhibition towards UGT1A9. The inhibition kinetic parameters (Ki) were calculated to be 11.25 mM for MEHP-UGT1A7, and 2.13, 0.09, 1.17, 7.47, 0.16 mM for MBZP-UGT1A9, MCHP-UGT1A9, MEHP-UGT1A9, MHP-UGT1A9, MOP-UGT1A9, respectively. Molecular docking indicated that both hydrogen bonds formation and hydrophobic interactions significantly contributed to the interaction between phthalate monoesters and UGT isoforms. All these information will be beneficial for understanding the adverse effects of PAEs. © 2018 Published by Elsevier Ltd.
Handling Editor:A. Gies Keywords: Phthalate esters (PAEs) Phthalate monoesters UDP-glucuronosyltransferases (UGTs) Enzyme inhibition
* Corresponding author. E-mail address: [email protected] (Z.-Z. Fang). 1 These two authors equally contributed to this work. https://doi.org/10.1016/j.chemosphere.2018.01.010 0045-6535/© 2018 Published by Elsevier Ltd.
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1. Introduction
Table 1 The abbreviations and side chains of phthalate monoesters.
Phthalate esters (PAEs), an important kind of chemical additives which have been extensively used in building materials, paints, insecticides, packaging, and cosmetics as plasticizers to reinforce the durability, elasticity and stretchability of plastics (Bui et al., 2016; Gao and Wen, 2016), can be released from plastics to contaminate air, water, sludge, food and medical instruments so as to cause human exposure via inhalation, dermal absorption, oral et al., 2013; Net et al., 2015). As uptake and medical injection (Berge shown in Fig. 1, PAEs undergo two-step metabolic elimination processes in human body. PAEs are initially converted to monoester metabolites by lipases via phase-I metabolism. And then, these monoesters react with uridine diphosphate glucuronic acid (UDPGA) to form glucuronide conjugates through the catalytic action of uridine diphosphate glucuronosyltransferases (UGTs). (Koch et al., 2006; Harris et al., 2016). As for simple short-branched PAEs, about 70% of the excreted monoesters was excreted unconjugated and a similar glucuronidation pattern was found in plasma (Silva et al., 2003). In vitro and in vivo studies of PAEs showed that phthalate monoesters had more biological activity and toxicity (Ito et al., 2006), so it is of important significance to study the toxic mechanism of phthalate monoesters. The abbreviations and side chains and of phthalate monoesters which are generally used in industry are shown in Table 1. As a kind of environmental endocrine disruptors, phthalate monoesters have a severe influence on human hormonal regulation system (e.g., thyroid hormone, steroid hormone, androgen, etc.). Some studies have reported significant mild negative correlations between phthalate monoesters and thyroid hormones, including thyroid-stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4) and free T4 (FT4), with the dimness of specific mechanism (Huang et al., 2007). In addition, phthalate monoesters in breast milk can reduce the activity of androgen and decrease the function of testis interstitial cells (Huang et al., 2007). Phthalate
Name
Abbreviation
Side chains
Monobutyl Phthalate Monobenzyl phthalate Monocyclohexyl phthalate Monoethylhexyl phthalate Monoethyl phthalate Monohexyl phthalate Monomethyl phthalate Monooctyl phthalate
MBP MBZP MCHP MEHP MEP MHP MMP MOP
Butyl Butyl Cyclohexyl Ethylhexyl Ethyl Hexyl Methyl Octyl
monoesters can also lead to premature breast development because of their estrogenic and anti-androgenic activity (Colon et al., 2000). UGTs, as the most important phase-II metabolizing enzymes, not only affect the metabolic behavior of many xenobiotics, but also metabolize many endogenous substances. Studies have report the associations between serum concentrations of phthalate monoesters (MBP and MEHP) and thyroid hormones including thyroidstimulating hormone (TSH), total thyroxine (TT4) and free thyroxine (FT4) (Colon et al., 2000). Estradiol levels are reduced by phthalate monoesters through a decrease in gene transcript levels of aromatase (Lovekamp and Davis, 2001). Bile acids are substrates of UGTs leading to more hydrophilic and less toxic glucuronides (Erichsen et al., 2010). Furthermore, metabolism of many other endogenous components such as bilirubin and serotonin (5hydroxytryptamine) are involved in the catalysis of UGTs (Krishnaswamy et al., 2003; Zheng et al., 2014). Therefore, the inhibition of UGT isoforms might seriously affect the metabolism of various endogenous substances. Our previous study has shown that PAEs showed limited inhibition on the activity of UGT1A6, -1A7 and -2B4, and UGT1A9 was broadly inhibited by PAEs, which indicated that there might be good interactions between phthalate
Fig. 1. Metabolic pathway of PAEs in human body.
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monoesters and UGTs (Krishnaswamy et al., 2003; Zheng et al., 2014). The aim of the present study is to investigate the inhibitory effects of phthalate monoesters towards the activities of human UGTs. To collect comprehensive information, the inhibition of 8 kinds of phthalate monoesters (MBP, MBZP, MCHP, MEHP, MEP, MHP, MMP and MOP) on 11 sorts of main human UGTs (UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17) were tested. Furthermore, inhibition kinetic type and parameters were determined, and in silico docking experiments were carried out to explain the kinetic relationship between phthalate monoesters and UGTs. 2. Materials and methods 2.1. Chemicals and reagents Phthalate monoesters (MBP, MBZP, MCHP, MEHP, MEP, MHP, MMP and MOP) were purchased from J&K Chemical (Beijing, China). Recombinant human UGT supersomes (UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17) expressed in baculovirus-infected insect cells were obtained form BD Gen test Corp (Woburn, MA, USA). 4-methylumbelliferone (4-MU), 4methylumbelliferone-b-D-glucuronide (4-MUG), Tris-HCl, 7hydroxycoumarin and uridine 50 -diphosphoglucuronic acid (UDPGA) (trisodium salt) were purchased from Sigma-Aldrich (St Louis,MO). Millipore Elix 5 UV and Milli-Q Gradient Ultra-Pure Water System was used to make ultra-pure water. The purity of these compounds was above 95%. All other reagents were of highperformance liquid chromatography (HPLC) grade or of the highest grade commercially available. 2.2. Initial screening of phthalate monoesters' inhibition towards recombinant UGTs-catalyzed 4-MU glucuronidation The evaluation of inhibition of phthalate monoesters on UGTs’ activity was performed according to the previous literature (Chen et al., 2017; Du et al., 2017). 4-MU was used as a nonselective probe substrate for recombinant UGTs to determine the inhibition of phthalate monoesters towards UGT isoforms. A classical incubation system (total volume ¼ 200 mL) containing 100 mM of phthalate monoesters, 50 mM of Tris-HCl buffer (pH ¼ 7.4) and 5 mM of MgCl2 was prepared, in which different concentrations of 4-MU and UGTs were involved. The used incubation time and
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protein concentration were previously determined, and the 4-MU concentration was equal to known Km values for each UGT isoform (110, 1200, 110, 30, 750, 30, 30, 1000, 350, 250 and 2000 mM 4MU for 0.125, 0.05, 0.025, 0.05, 0.025, 0.05, 0.05, 0.25, 0.05, 0.2 and 0.5 mg/ml UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, respectively) to ensure the reaction rate within the linear range. The absence of phthalate monoesters was set as control. The phthalate monoesters were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO was below 0.5% (v/v). After a 5 min preincubation at 37 C, the UDPGA was added in the mixture to initiate the reaction. The reactions were quenched by adding 200 mL acetonitrile with 100 mM 7-hydroxycoumarin as the internal standard after 30e120 min according to different UGTs (30 min for UGT1A6, UGT1A7, UGT1A8 and UGT1A9, and 120 min for UGT1A1, UGT1A3, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, respectively). The mixture was centrifuged at 10,625 g for 10 min to obtain the supernatant and an aliquot of supernatant was transferred to an auto-injector vial for UPLC analysis. Chromatographic separation was carried out using a C18 column (4.6 200 mm, 5 mm, Kromasil) at a flow rate of 0.2 mL/min and UV detector at 316 nm. The mobile phase consisted of H2O containing 0.5% (v/v) formic acid (A) and acetonitrile (B). The following gradient condition was applied: 0e3.50 min, 90% A and 10% B; 3.50e4.00 min, 35% A and 65% B; 4.01e7.00 min, 90% A and 10% B. The calculation curve was generated by peak area ratio (4-MUG/internal standard) and all experiments were performed in two independent experiments in duplicate. The phthalate monoesters whose inhibition ratios to UGTs were more than 80% were screened out to proceed with the subsequent experiment. 2.3. Inhibition kinetic evaluation and in vitro-in vivo extrapolation (IVIVE) To determine the inhibition kinetic behavior, concentrationdependent inhibition of phthalate monoesters on the activity of UGTs was determined. 0, 0.5, 1, 5, 10, 20, 40, 60, 80 and 100 mM phthalate monoesters selected from preliminary screening were prepared to determine the IC50 of these phthalate monoesters. The concentrations of phthalate monoesters whose inhibition ratios were 20%, 40%, 60% and 80% were chosen to undertake the inhibition kinetics assays. Various concentrations of 4-MU were used to determine the kinetic type and parameters in the presence or absence of phthalate monoesters based on each different UGT
Fig. 2. Inhibition potential of MBP, MBZP, MCHP, MEHP, MEP, MHP, MMP and MOP towards UGT1A7 and 1A9. The residual activity of recombinant UGTs was given, and calculated using the following equations: Residual activity (% CTRL) ¼ the activity at 100 mM of phthalate monoesters*100%/the activity at 0 mM of phthalate monoesters. Data were presented as the mean value plus S.D.*, p < .05.
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isoform. Lineweaver-Burk plots were employed to determine the inhibition kinetic type, and the second plot with the slopes from Lineweaver-Burk plot towards the concentrations of phthalate monoesters was utilized to determine the inhibition kinetic parameters (Ki). In vivo inhibition magnitude of UGTs was predicted using in vitro-in vivo extrapolation (IVIVE) to exhibit the area under the plasma concentration-time curve (AUC) alteration caused by exposure of phthalate monoesters. The following equation was used:
AUCi/AUC ¼ 1þ[I]/Ki In this equation, AUCi/AUC was the predicted ratio of in vivo exposure of xenobiotics or endogenous substances with or without the exposure of phthalate monoesters. [I] was the in vivo exposure concentration of phthalate monoesters, and the Ki was in vitro inhibition constant. The standard was as followed: [I]/Ki < 0.1, low possible; 0.1<[I]/Ki < 1, medium possible; [I]/Ki > 1, high possible.
Fig. 3. Inhibitory effects of MEHP on UGT1A7 and MBZP, MCHP, MEHP, MHP, MOP on UGT1A9 activity in human cDNA-expressed UGT1A7 and UGT1A9 supersomes. IC50 values of MEHP towards UGT1A7 was calculated to be 10.71 mM; IC50 values for the inhibition of MBZP, MCHP, MEHP, MHP and MOP towards UGT1A9 were calculated to be 9.36, 3.29, 1.57, 12.46 and 7.58 mM, respectively. Data were presented as the mean value plus S.D.
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2.4. In silico docking to explain the inhibition of phthalate monoesters on the activity of UGTs For better understanding the molecular interactions between phthalate monoesters and UGTs, in silico docking method was used to dock the chemical structure of phthalate monoesters into the activity cavity of UGTs. Comparative homology modeling with MODELLER9v14 program was carried out to elucidate the threedimensional structure of UGTs. Auto-dock version 4.2 program was employed to dock the flexible small molecule of phthalate monoesters into the rigid protein of UGTs. Polar hydrogen atoms were added to UGTs, and nonpolar hydrogen atoms were merged.
Table 2 The inhibition kinetics of phthalate monoesters to UGTs, including IC50, inhibition kinetic type and parameters. Phthalate monoesters-UGTs
IC50(mM)
inhibition type
Ki (mM)
MEHP-UGT1A7 MBZP-UGT1A9 MCHP-UGT1A9 MEHP-UGT1A9 MHP-UGT1A9 MOP-UGT1A9
10.71 9.36 3.29 1.57 12.46 7.58
competitive competitive competitive competitive competitive competitive
11.25 2.13 0.09 1.17 7.47 0.16
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The grid box was generated with a dimension of 50 50 50 in X, Y and Z coordinate, and the grid point spacing was set to 0.375 Å. Lamarckian Genetic Algorithm (LGA) method was selected to deal with the protein-fixed ligand-flexible docking calculations for the binding of phthalate monoesters towards UGTs. The best conformation with the lowest docked energy was selected to analyze the interactions between phthalate monoesters and UGTs, including hydrogen bonds and hydrophobic contacts.
3. Results 3.1. Inhibition potential of phthalate monoesters towards UGT isoforms 100 mM phthalate monoesters exhibited weak inhibition towards the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A8, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17 by reducing the glucuronidation less than 80% (Supplemental Fig. 1AeI). The inhibition ratios of phthalate monoesters towards UGT1A7 and UGT1A9 were shown in Fig. 2. The activity of UGT1A7 was inhibited by 85.26% at 100 mM of MEHP (p < .05), but slightly inhibited by all the other phthalate monoesters. Unlike the other UGTs, UGT1A9 was broadly inhibited by MBZP, MCHP, MEHP, MHP and MOP, with
Fig. 4. Inhibition kinetics of MEHP on UGT1A9. (A) Lineweaver-Burk plot of the inhibition of MEHP on the activity of UGT1A9. Each data point represents the mean value of duplicate experiments. (B) Determination of inhibition kinetic parameter (Ki) of MEHP on the activity of UGT1A9. The verticle axis represents the slopes of the lines from Lineweaver-Burk plot, and the horizontal axis represents the concentrations of MEHP.
Fig. 5. Activity pocket of UGT1A9 binding with MEHP. The active site of UGT1A9 binding with MEHP was composed of amino acids residues SER-11, PHE-14, CYS-252, LEU-258, PRO259, PHE-277, ARG-308, TRP-326, LEU-327, PRO-328, GLN-329, ASN-330, HIS-348 and GLU-352.
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the reductions of 4-MU glucuronidation activity by 92.49%, 95.90%, 97.61%, 85.83% and 91.21%, respectively. 3.2. Inhibition kinetic analysis The concentration-dependent inhibition of phthalate monoesters towards UGT isoforms was exhibited. The IC50 value for the inhibition MEHP towards UGT1A7 was calculated to be 10.71 mM; IC50 values for the inhibition of MBZP, MCHP, MEHP, MHP and MOP towards UGT1A9 were calculated to be 9.36, 3.29, 1.57, 12.46 and 7.58 mM, respectively (Fig. 3, Table 2). Furthermore, the inhibition kinetics was determined, including kinetic type and parameters (Ki). MEHP exhibited competitive inhibition towards UGT1A7, and MBZP, MCHP, MEHP, MHP and MOP showed competitive inhibition towards UGT1A9. The inhibition kinetic parameters (Ki) were calculated to be 11.25 mM for MEHPUGT1A7, and 2.13, 0.09, 1.17, 7.47, 0.16 mM for MBZP-UGT1A9, MCHP-UGT1A9, MEHP-UGT1A9, MHP-UGT1A9, MOP-UGT1A9, respectively. The representative figures were given in Fig. 4 in which the Lineweaver-Burk plot (Fig. 4A) and the second plot (Fig. 4B) were given for the inhibition of MEHP towards UGT1A9. The Lineweaver-Burk and second plot for the inhibition of other phthalate monoesters towards other UGT isoforms were given in Supplemental Fig. 2AeE. 3.3. Molecular docking of phthalate monoesters towards UGT1A9
Fig. 3AeD. The contribution of hydrogen bonds and hydrophobic interaction towards the binding of other phthalate monoesters with UGT1A9 was given in Supplemental Fig. 4AeD and 5A-D. The binding free energy of MBZP, MCHP, MEHP, MHP and MOP towards UGT1A9 was 6.46, 6.98, 5.75, 6.27 and 5.81 kcal/mol, respectively. 4. Discussion This study used in vitro determination system to investigate the inhibition behavior of phthalate monoesters on the activity of various isoforms of UGTs, and the results showed that some kinds of phthalate monoesters exhibited strong inhibition on UGT1A7 and UGT1A9. UGT1A7 and UGT1A9 were turned out to be the main metabolic enzymes of thyroxine (Yamanaka et al., 2007), indicating that phthalate monoesters could cause thyroid diseases by disturbing the metabolism of thyroxine. Likewise, UGT1A9 was involved in glucuronidation of estradiol (E2) and estrone (E1), their hydroxyls (OH), and their methoxy derivatives (MeO) (Lepine et al., 2004), so there could be diseases of reproductive system induced by phthalate monoesters through reproductive hormone abnormalities. Therefore, there could be an intense disturbance to the metabolic homeostasis of these endogenous substances caused by the inhibition effect of phthalate monoesters on the activity of these UGT isoforms. As for the structure specificity of phthalate monoesters, the
Because phthalate monoesters showed broad inhibition on UGT1A9, the mechanism analysis for the inhibition of phthalate monoesters towards UGT1A9 was performed using in silico docking methods. Homology modeling was carried out to construct the crystal structure of UGT1A9, and the chemical structures of phthalate monoesters were docked into the activity cavities of UGT1A9 using in silico docking method. The representative docking results were given for MEHP. The active site of UGT1A9 binding with MEHP was composed of amino acids residues SER-11, PHE-14, CYS-252, LEU-258, PRO-259, PHE-277, ARG-308, TRP-326, LEU-327, PRO-328, GLN-329, ASN-330, HIS-348 and GLU-352, which were shown in Fig. 5. In the binding pocket of UGT1A9, MEHP formed five hydrogen bonds to ARG-308, LEU-327 and GLN-329 (Fig. 6). MEHP formed hydrophobic contacts with amino acids residues PHE-14, CYS-252, PRO-328, ASN-330, HIS-348 and GLU-352 in the active pocket of UGT1A9, which were shown in Fig. 7. The binding behavior of other phthalate monoesters was given in Supplemental
Fig. 6. Hydrogen bonds interaction between MEHP with the activity cavity of UGT1A9. In the binding pocket of UGT1A9, MEHP formed five hydrogen bonds to ARG-308, LEU327 and GLN-329.
Fig. 7. Hydrophobic interaction between MEHP and the activity cavity of UGT1A9. MEHP formed hydrophobic contacts with amino acids residues PHE-14, CYS-252, PRO328, ASN-330, HIS-348 and GLU-352 in the active pocket of UGT1A9.
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results showed that there was a wide inhibition on UGT1A9 by MBZP, MCHP, MEHP, MHP and MOP, which was mainly eliminated from body by UGTs to form glucuronic acid conjugate (Silva et al., 2003). In contrast, phthalate monoesters with short chains like MEP and MMP, showed a slight inhibition to all UGT isoforms. Therefore, the preliminary structure-activity relationship for the inhibition of phthalate monoesters towards UGT isoforms was indicated to be that phthalate monoesters with long chains exhibited stronger inhibition towards UGTs, and UGT1A9 was the most vulnerable to the inhibition of phthalate monoesters. The in vivo inhibition magnitude can be extrapolated using the combination of in vitro parameters (Ki) and in vivo exposure of phthalate monoesters. According to [I]/Ki ratio ([I]/Ki > 0.1) evaluation standard, the threshold values for inducing in vivo inhibition was calculated to be 1.125 mM for MEHP-UGT1A7 and 0.213, 0.009, 0.117, 0.747, 0.016 mM for MBZP-UGT1A9, MCHP-UGT1A9, MEHPUGT1A9, MHP-UGT1A9, MOP-UGT1A9, respectively. Plasma concentration of MEHP was demonstrated to reach 0.38 mg/ml in human body (Silva et al., 2003). Based on this value, the in vivo exposure concentration was calculated to be approximately 1.37 mM, which exceed the threshold value of MEHP to UGT1A7 and UGT1A9. Therefore, the exposure of phthalate monoesters might disturb the metabolic elimination of endogenous substances through inhibiting the activity of UGTs in vivo. It should be noted that the biotransformation process for PAEs to their phthalate monoesters will significantly alter the inhibition potential on UGT isoforms. For example, the biotransformation of BBZP to MBZP significantly weakens the inhibition potential towards UGT1A6. In addition, the biotransformation of DBP to MBP, BBZP to MBZP significantly weakens the inhibition potential towards UGT2B4. In conclusion, the complete inhibition profile of phthalate monoesters towards UGT isoforms was given, and high specific inhibition of phthalate monoesters towards UGT1A7 and UGT1A9 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the possible concentration threshold of phthalate monoesters to effect endogenous hormone signaling and drug therapy was obtained. All these results of the present study indicating that phthalate monoesters could possibly disturb endogenous metabolism by inhibit UGTs, so close monitoring the exposure of phthalate monoesters should be paid much attention to decrease the disease risk caused by phthalate monoesters. Conflicts of interest The authors have declared that there are no conflicts of interest. Acknowledgement This work was supported by the project for the National Key Research and Development Program (2016YFC0903100, 2016YFC0903102), The 13th five year plan and TMU talent project (11601501/2016KJ0313), individualized diagnosis and treatment of colorectal cancer (No. LNCCC-B05-2015), Foundation of Committee on Science and Technology of Tianjin (Grant No. 15JCYBJC54700, 14JCQNJC11300), the China Postdoctoral Science Foundation (2016M590210, 2017T100164), Tianjin Health Bureau Science Foundation Key Project (16KG154) and Tianjin Project of Thousand Youth Talents, and Key Laboratory Open Project Fund from State
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Key Laboratory of Environment Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (KF2017-02), and National Natural Science Fund (81602850). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.chemosphere.2018.01.010. References , A., Cladie re, M., Gasperi, J., Coursimault, A., Tassin, B., Moilleron, R., 2013. Berge Meta-analysis of environmental contamination by phthalates. Environ. Sci. Pollut. Res. 20, 8057e8076. Bui, T.T., Giovanoulis, G., Cousins, A.P., Magner, J., Cousins, I.T., de Wit, C.A., 2016. Human exposure, hazard and risk of alternative plasticizers to phthalate esters. Sci. Total Environ. 541, 451e467. Chen, D.W., Du, Z., Zhang, C.Z., Zhang, W.H., Cao, Y.F., Sun, H.Z., Zhu, Z.T., Yang, K., Liu, Y.Z., Zhao, Z.W., Fu, Z.W., Gu, W.Q., Yu, Y., Fang, Z.Z., 2017. The inhibition of UDP-glucuronosyltransferases (UGTs) by tetraiodothyronine (T4) and triiodothyronine (T3). Xenobiotica 1e8. Colon, I., Caro, D., Bourdony, C.J., Rosario, O., 2000. Identification of phthalate esters in the serum of young Puerto Rican girls with premature breast development. Environ. Health Perspect. 108, 895e900. Du, Z., Wang, G., Cao, Y.F., Hu, C.M., Yang, K., Liu, Y.Z., Zhang, C.Z., Zhang, W.H., Zhu, Z.T., Sun, H.Z., Sun, X.Y., Hong, M., Fang, Z.Z., 2017. Everolimus-inhibited multiple isoforms of UDP-glucuronosyltransferases (UGTs). Xenobiotica 1e7. Erichsen, T.J., Aehlen, A., Ehmer, U., Kalthoff, S., Manns, M.P., Strassburg, C.P., 2010. Regulation of the human bile acid UDP-glucuronosyltransferase 1A3 by the farnesoid X receptor and bile acids. J. Hepatol. 52, 570e578. Gao, D.W., Wen, Z.D., 2016. Phthalate esters in the environment: a critical review of their occurrence, biodegradation, and removal during wastewater treatment processes. Sci. Total Environ. 541, 986e1001. Harris, S., Wegner, S., Hong, S.W., Faustman, E.M., 2016. Phthalate metabolism and kinetics in an in vitro model of testis development. Toxicol. Vitro 32, 123e131. Huang, P.C., Kuo, P.L., Guo, Y.L., Liao, P.C., Lee, C.C., 2007. Associations between urinary phthalate monoesters and thyroid hormones in pregnant women. Hum. Reprod. 22, 2715e2722. Ito, R., Seshimo, F., Miura, N., Kawaguchi, M., Saito, K., Nakazawa, H., 2006. Effect of sterilization process on the formation of mono(2-ethylhexyl)phthalate from di(2-ethylhexyl)phthalate. J. Pharm. Biomed. Anal. 41, 455e460. Koch, H.M., Preuss, R., Angerer, J., 2006. Di(2-ethylhexyl)phthalate (DEHP): human metabolism and internal exposuree an update and latest results. Int. J. Androl. 29 (155e165), 181e185. Krishnaswamy, S., Duan, S.X., Von Moltke, L.L., Greenblatt, D.J., Sudmeier, J.L., Bachovchin, W.W., Court, M.H., 2003. Serotonin (5-hydroxytryptamine) glucuronidation in vitro: assay development, human liver microsome activities and species differences. Xenobiotica 33, 169e180. Lepine, J., Bernard, O., Plante, M., Tetu, B., Pelletier, G., Labrie, F., Belanger, A., Guillemette, C., 2004. Specificity and regioselectivity of the conjugation of estradiol, estrone, and their catecholestrogen and methoxyestrogen metabolites by human uridine diphospho-glucuronosyltransferases expressed in endometrium. J. Clin. Endocrinol. Metabol. 89, 5222e5232. Lovekamp, T.N., Davis, B.J., 2001. Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells. Toxicol. Appl. Pharmacol. 172, 217e224. re , R., Paluselli, A., Ouddane, B., 2015. Reliable quantiNet, S., Delmont, A., Sempe fication of phthalates in environmental matrices (air, water, sludge, sediment and soil): a review. Sci. Total Environ. 515, 162e180. Silva, M.J., Barr, D.B., Reidy, J.A., Kato, K., Malek, N.A., Hodge, C.C., Hurtz, D., Calafat, A.M., Needham, L.L., Brock, J.W., 2003. Glucuronidation patterns of common urinary and serum monoester phthalate metabolites. Arch. Toxicol. 77, 561e567. Yamanaka, H., Nakajima, M., Katoh, M., Yokoi, T., 2007. Glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes. Drug Metabol. Dispos. 35, 1642e1648. Zheng, B., Hu, G., Yu, J., Liu, Z., 2014. Crigler-Najjar syndrome type II in a Chinese boy resulting from three mutations in the bilirubin uridine 5'-diphosphate-glucuronosyltransferase (UGT1A1) gene and a family genetic analysis. BMC Pediatr. 14, 267.
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Inhibition of UDP-glucuronosyltransferases (UGTs) by phthalate monoesters Zuo Du a, 1, Yun-Feng Cao b, 1, Sai-Nan Li a, Cui-Min Hu c, Zhi-Wei Fu a, Chun-Ting Huang d, Xiao-Yu Sun d, Yong-Zhe Liu a, Kun Yang a, Zhong-Ze Fang a, * a
Department of Toxicology, School of Public Health, Tianjin Medical University, 22 Qixiangtai Road, Heping District, Tianjin, China Key Laboratory of Liaoning Tumor Clinical Metabolomics (KLLTCM), Jinzhou, Liaoning, China Tianjin Life Science Research Center, Department of Microbiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China d RSKT Biopharma Inc., Liaoning, China b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Phthalate monoesters showed broad inhibition on UGT1A9. Threshold values for inducing in vivo inhibition of UGTs were calculated. Both hydrogen bonds and hydrophobic interaction contributed to the inhibition of UGT1A9 by phthalate monoestersi.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2017 Received in revised form 2 January 2018 Accepted 4 January 2018 Available online 5 January 2018
Phthalate monoesters are important metabolites of phthalate esters (PAEs) which have been extensively utilized in industry. This study aims to investigate the inhibition of phthalate monoesters on the activity of various isoforms of UDP-glucuronosyltransferases (UGTs), trying to elucidate the toxicity mechanism of environmental endocrine disruptors from the new perspectives. In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was employed to evaluate 8 kinds of phthalate monoesters on 11 sorts of main human UGT isoforms. 100 mM phthalate monoesters exhibited negligible inhibition towards the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A8, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17. The activity of UGT1A7 was strongly inhibited by monoethylhexyl phthalate (MEHP), but slightly inhibited by all the other phthalate monoesters. UGT1A9 was broadly inhibited by monobenzyl phthalate (MBZP), monocyclohexyl phthalate (MCHP), MEHP, monohexyl phthalate (MHP) and monooctyl phthalate (MOP), respectively. MEHP exhibited competitive inhibition towards UGT1A7, and MBZP, MCHP, MEHP, MHP and MOP showed competitive inhibition towards UGT1A9. The inhibition kinetic parameters (Ki) were calculated to be 11.25 mM for MEHP-UGT1A7, and 2.13, 0.09, 1.17, 7.47, 0.16 mM for MBZP-UGT1A9, MCHP-UGT1A9, MEHP-UGT1A9, MHP-UGT1A9, MOP-UGT1A9, respectively. Molecular docking indicated that both hydrogen bonds formation and hydrophobic interactions significantly contributed to the interaction between phthalate monoesters and UGT isoforms. All these information will be beneficial for understanding the adverse effects of PAEs. © 2018 Published by Elsevier Ltd.
Handling Editor:A. Gies Keywords: Phthalate esters (PAEs) Phthalate monoesters UDP-glucuronosyltransferases (UGTs) Enzyme inhibition
* Corresponding author. E-mail address: [email protected] (Z.-Z. Fang). 1 These two authors equally contributed to this work. https://doi.org/10.1016/j.chemosphere.2018.01.010 0045-6535/© 2018 Published by Elsevier Ltd.
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1. Introduction
Table 1 The abbreviations and side chains of phthalate monoesters.
Phthalate esters (PAEs), an important kind of chemical additives which have been extensively used in building materials, paints, insecticides, packaging, and cosmetics as plasticizers to reinforce the durability, elasticity and stretchability of plastics (Bui et al., 2016; Gao and Wen, 2016), can be released from plastics to contaminate air, water, sludge, food and medical instruments so as to cause human exposure via inhalation, dermal absorption, oral et al., 2013; Net et al., 2015). As uptake and medical injection (Berge shown in Fig. 1, PAEs undergo two-step metabolic elimination processes in human body. PAEs are initially converted to monoester metabolites by lipases via phase-I metabolism. And then, these monoesters react with uridine diphosphate glucuronic acid (UDPGA) to form glucuronide conjugates through the catalytic action of uridine diphosphate glucuronosyltransferases (UGTs). (Koch et al., 2006; Harris et al., 2016). As for simple short-branched PAEs, about 70% of the excreted monoesters was excreted unconjugated and a similar glucuronidation pattern was found in plasma (Silva et al., 2003). In vitro and in vivo studies of PAEs showed that phthalate monoesters had more biological activity and toxicity (Ito et al., 2006), so it is of important significance to study the toxic mechanism of phthalate monoesters. The abbreviations and side chains and of phthalate monoesters which are generally used in industry are shown in Table 1. As a kind of environmental endocrine disruptors, phthalate monoesters have a severe influence on human hormonal regulation system (e.g., thyroid hormone, steroid hormone, androgen, etc.). Some studies have reported significant mild negative correlations between phthalate monoesters and thyroid hormones, including thyroid-stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4) and free T4 (FT4), with the dimness of specific mechanism (Huang et al., 2007). In addition, phthalate monoesters in breast milk can reduce the activity of androgen and decrease the function of testis interstitial cells (Huang et al., 2007). Phthalate
Name
Abbreviation
Side chains
Monobutyl Phthalate Monobenzyl phthalate Monocyclohexyl phthalate Monoethylhexyl phthalate Monoethyl phthalate Monohexyl phthalate Monomethyl phthalate Monooctyl phthalate
MBP MBZP MCHP MEHP MEP MHP MMP MOP
Butyl Butyl Cyclohexyl Ethylhexyl Ethyl Hexyl Methyl Octyl
monoesters can also lead to premature breast development because of their estrogenic and anti-androgenic activity (Colon et al., 2000). UGTs, as the most important phase-II metabolizing enzymes, not only affect the metabolic behavior of many xenobiotics, but also metabolize many endogenous substances. Studies have report the associations between serum concentrations of phthalate monoesters (MBP and MEHP) and thyroid hormones including thyroidstimulating hormone (TSH), total thyroxine (TT4) and free thyroxine (FT4) (Colon et al., 2000). Estradiol levels are reduced by phthalate monoesters through a decrease in gene transcript levels of aromatase (Lovekamp and Davis, 2001). Bile acids are substrates of UGTs leading to more hydrophilic and less toxic glucuronides (Erichsen et al., 2010). Furthermore, metabolism of many other endogenous components such as bilirubin and serotonin (5hydroxytryptamine) are involved in the catalysis of UGTs (Krishnaswamy et al., 2003; Zheng et al., 2014). Therefore, the inhibition of UGT isoforms might seriously affect the metabolism of various endogenous substances. Our previous study has shown that PAEs showed limited inhibition on the activity of UGT1A6, -1A7 and -2B4, and UGT1A9 was broadly inhibited by PAEs, which indicated that there might be good interactions between phthalate
Fig. 1. Metabolic pathway of PAEs in human body.
Z. Du et al. / Chemosphere 197 (2018) 7e13
monoesters and UGTs (Krishnaswamy et al., 2003; Zheng et al., 2014). The aim of the present study is to investigate the inhibitory effects of phthalate monoesters towards the activities of human UGTs. To collect comprehensive information, the inhibition of 8 kinds of phthalate monoesters (MBP, MBZP, MCHP, MEHP, MEP, MHP, MMP and MOP) on 11 sorts of main human UGTs (UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17) were tested. Furthermore, inhibition kinetic type and parameters were determined, and in silico docking experiments were carried out to explain the kinetic relationship between phthalate monoesters and UGTs. 2. Materials and methods 2.1. Chemicals and reagents Phthalate monoesters (MBP, MBZP, MCHP, MEHP, MEP, MHP, MMP and MOP) were purchased from J&K Chemical (Beijing, China). Recombinant human UGT supersomes (UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17) expressed in baculovirus-infected insect cells were obtained form BD Gen test Corp (Woburn, MA, USA). 4-methylumbelliferone (4-MU), 4methylumbelliferone-b-D-glucuronide (4-MUG), Tris-HCl, 7hydroxycoumarin and uridine 50 -diphosphoglucuronic acid (UDPGA) (trisodium salt) were purchased from Sigma-Aldrich (St Louis,MO). Millipore Elix 5 UV and Milli-Q Gradient Ultra-Pure Water System was used to make ultra-pure water. The purity of these compounds was above 95%. All other reagents were of highperformance liquid chromatography (HPLC) grade or of the highest grade commercially available. 2.2. Initial screening of phthalate monoesters' inhibition towards recombinant UGTs-catalyzed 4-MU glucuronidation The evaluation of inhibition of phthalate monoesters on UGTs’ activity was performed according to the previous literature (Chen et al., 2017; Du et al., 2017). 4-MU was used as a nonselective probe substrate for recombinant UGTs to determine the inhibition of phthalate monoesters towards UGT isoforms. A classical incubation system (total volume ¼ 200 mL) containing 100 mM of phthalate monoesters, 50 mM of Tris-HCl buffer (pH ¼ 7.4) and 5 mM of MgCl2 was prepared, in which different concentrations of 4-MU and UGTs were involved. The used incubation time and
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protein concentration were previously determined, and the 4-MU concentration was equal to known Km values for each UGT isoform (110, 1200, 110, 30, 750, 30, 30, 1000, 350, 250 and 2000 mM 4MU for 0.125, 0.05, 0.025, 0.05, 0.025, 0.05, 0.05, 0.25, 0.05, 0.2 and 0.5 mg/ml UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, respectively) to ensure the reaction rate within the linear range. The absence of phthalate monoesters was set as control. The phthalate monoesters were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO was below 0.5% (v/v). After a 5 min preincubation at 37 C, the UDPGA was added in the mixture to initiate the reaction. The reactions were quenched by adding 200 mL acetonitrile with 100 mM 7-hydroxycoumarin as the internal standard after 30e120 min according to different UGTs (30 min for UGT1A6, UGT1A7, UGT1A8 and UGT1A9, and 120 min for UGT1A1, UGT1A3, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, respectively). The mixture was centrifuged at 10,625 g for 10 min to obtain the supernatant and an aliquot of supernatant was transferred to an auto-injector vial for UPLC analysis. Chromatographic separation was carried out using a C18 column (4.6 200 mm, 5 mm, Kromasil) at a flow rate of 0.2 mL/min and UV detector at 316 nm. The mobile phase consisted of H2O containing 0.5% (v/v) formic acid (A) and acetonitrile (B). The following gradient condition was applied: 0e3.50 min, 90% A and 10% B; 3.50e4.00 min, 35% A and 65% B; 4.01e7.00 min, 90% A and 10% B. The calculation curve was generated by peak area ratio (4-MUG/internal standard) and all experiments were performed in two independent experiments in duplicate. The phthalate monoesters whose inhibition ratios to UGTs were more than 80% were screened out to proceed with the subsequent experiment. 2.3. Inhibition kinetic evaluation and in vitro-in vivo extrapolation (IVIVE) To determine the inhibition kinetic behavior, concentrationdependent inhibition of phthalate monoesters on the activity of UGTs was determined. 0, 0.5, 1, 5, 10, 20, 40, 60, 80 and 100 mM phthalate monoesters selected from preliminary screening were prepared to determine the IC50 of these phthalate monoesters. The concentrations of phthalate monoesters whose inhibition ratios were 20%, 40%, 60% and 80% were chosen to undertake the inhibition kinetics assays. Various concentrations of 4-MU were used to determine the kinetic type and parameters in the presence or absence of phthalate monoesters based on each different UGT
Fig. 2. Inhibition potential of MBP, MBZP, MCHP, MEHP, MEP, MHP, MMP and MOP towards UGT1A7 and 1A9. The residual activity of recombinant UGTs was given, and calculated using the following equations: Residual activity (% CTRL) ¼ the activity at 100 mM of phthalate monoesters*100%/the activity at 0 mM of phthalate monoesters. Data were presented as the mean value plus S.D.*, p < .05.
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isoform. Lineweaver-Burk plots were employed to determine the inhibition kinetic type, and the second plot with the slopes from Lineweaver-Burk plot towards the concentrations of phthalate monoesters was utilized to determine the inhibition kinetic parameters (Ki). In vivo inhibition magnitude of UGTs was predicted using in vitro-in vivo extrapolation (IVIVE) to exhibit the area under the plasma concentration-time curve (AUC) alteration caused by exposure of phthalate monoesters. The following equation was used:
AUCi/AUC ¼ 1þ[I]/Ki In this equation, AUCi/AUC was the predicted ratio of in vivo exposure of xenobiotics or endogenous substances with or without the exposure of phthalate monoesters. [I] was the in vivo exposure concentration of phthalate monoesters, and the Ki was in vitro inhibition constant. The standard was as followed: [I]/Ki < 0.1, low possible; 0.1<[I]/Ki < 1, medium possible; [I]/Ki > 1, high possible.
Fig. 3. Inhibitory effects of MEHP on UGT1A7 and MBZP, MCHP, MEHP, MHP, MOP on UGT1A9 activity in human cDNA-expressed UGT1A7 and UGT1A9 supersomes. IC50 values of MEHP towards UGT1A7 was calculated to be 10.71 mM; IC50 values for the inhibition of MBZP, MCHP, MEHP, MHP and MOP towards UGT1A9 were calculated to be 9.36, 3.29, 1.57, 12.46 and 7.58 mM, respectively. Data were presented as the mean value plus S.D.
Z. Du et al. / Chemosphere 197 (2018) 7e13
2.4. In silico docking to explain the inhibition of phthalate monoesters on the activity of UGTs For better understanding the molecular interactions between phthalate monoesters and UGTs, in silico docking method was used to dock the chemical structure of phthalate monoesters into the activity cavity of UGTs. Comparative homology modeling with MODELLER9v14 program was carried out to elucidate the threedimensional structure of UGTs. Auto-dock version 4.2 program was employed to dock the flexible small molecule of phthalate monoesters into the rigid protein of UGTs. Polar hydrogen atoms were added to UGTs, and nonpolar hydrogen atoms were merged.
Table 2 The inhibition kinetics of phthalate monoesters to UGTs, including IC50, inhibition kinetic type and parameters. Phthalate monoesters-UGTs
IC50(mM)
inhibition type
Ki (mM)
MEHP-UGT1A7 MBZP-UGT1A9 MCHP-UGT1A9 MEHP-UGT1A9 MHP-UGT1A9 MOP-UGT1A9
10.71 9.36 3.29 1.57 12.46 7.58
competitive competitive competitive competitive competitive competitive
11.25 2.13 0.09 1.17 7.47 0.16
11
The grid box was generated with a dimension of 50 50 50 in X, Y and Z coordinate, and the grid point spacing was set to 0.375 Å. Lamarckian Genetic Algorithm (LGA) method was selected to deal with the protein-fixed ligand-flexible docking calculations for the binding of phthalate monoesters towards UGTs. The best conformation with the lowest docked energy was selected to analyze the interactions between phthalate monoesters and UGTs, including hydrogen bonds and hydrophobic contacts.
3. Results 3.1. Inhibition potential of phthalate monoesters towards UGT isoforms 100 mM phthalate monoesters exhibited weak inhibition towards the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A8, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17 by reducing the glucuronidation less than 80% (Supplemental Fig. 1AeI). The inhibition ratios of phthalate monoesters towards UGT1A7 and UGT1A9 were shown in Fig. 2. The activity of UGT1A7 was inhibited by 85.26% at 100 mM of MEHP (p < .05), but slightly inhibited by all the other phthalate monoesters. Unlike the other UGTs, UGT1A9 was broadly inhibited by MBZP, MCHP, MEHP, MHP and MOP, with
Fig. 4. Inhibition kinetics of MEHP on UGT1A9. (A) Lineweaver-Burk plot of the inhibition of MEHP on the activity of UGT1A9. Each data point represents the mean value of duplicate experiments. (B) Determination of inhibition kinetic parameter (Ki) of MEHP on the activity of UGT1A9. The verticle axis represents the slopes of the lines from Lineweaver-Burk plot, and the horizontal axis represents the concentrations of MEHP.
Fig. 5. Activity pocket of UGT1A9 binding with MEHP. The active site of UGT1A9 binding with MEHP was composed of amino acids residues SER-11, PHE-14, CYS-252, LEU-258, PRO259, PHE-277, ARG-308, TRP-326, LEU-327, PRO-328, GLN-329, ASN-330, HIS-348 and GLU-352.
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the reductions of 4-MU glucuronidation activity by 92.49%, 95.90%, 97.61%, 85.83% and 91.21%, respectively. 3.2. Inhibition kinetic analysis The concentration-dependent inhibition of phthalate monoesters towards UGT isoforms was exhibited. The IC50 value for the inhibition MEHP towards UGT1A7 was calculated to be 10.71 mM; IC50 values for the inhibition of MBZP, MCHP, MEHP, MHP and MOP towards UGT1A9 were calculated to be 9.36, 3.29, 1.57, 12.46 and 7.58 mM, respectively (Fig. 3, Table 2). Furthermore, the inhibition kinetics was determined, including kinetic type and parameters (Ki). MEHP exhibited competitive inhibition towards UGT1A7, and MBZP, MCHP, MEHP, MHP and MOP showed competitive inhibition towards UGT1A9. The inhibition kinetic parameters (Ki) were calculated to be 11.25 mM for MEHPUGT1A7, and 2.13, 0.09, 1.17, 7.47, 0.16 mM for MBZP-UGT1A9, MCHP-UGT1A9, MEHP-UGT1A9, MHP-UGT1A9, MOP-UGT1A9, respectively. The representative figures were given in Fig. 4 in which the Lineweaver-Burk plot (Fig. 4A) and the second plot (Fig. 4B) were given for the inhibition of MEHP towards UGT1A9. The Lineweaver-Burk and second plot for the inhibition of other phthalate monoesters towards other UGT isoforms were given in Supplemental Fig. 2AeE. 3.3. Molecular docking of phthalate monoesters towards UGT1A9
Fig. 3AeD. The contribution of hydrogen bonds and hydrophobic interaction towards the binding of other phthalate monoesters with UGT1A9 was given in Supplemental Fig. 4AeD and 5A-D. The binding free energy of MBZP, MCHP, MEHP, MHP and MOP towards UGT1A9 was 6.46, 6.98, 5.75, 6.27 and 5.81 kcal/mol, respectively. 4. Discussion This study used in vitro determination system to investigate the inhibition behavior of phthalate monoesters on the activity of various isoforms of UGTs, and the results showed that some kinds of phthalate monoesters exhibited strong inhibition on UGT1A7 and UGT1A9. UGT1A7 and UGT1A9 were turned out to be the main metabolic enzymes of thyroxine (Yamanaka et al., 2007), indicating that phthalate monoesters could cause thyroid diseases by disturbing the metabolism of thyroxine. Likewise, UGT1A9 was involved in glucuronidation of estradiol (E2) and estrone (E1), their hydroxyls (OH), and their methoxy derivatives (MeO) (Lepine et al., 2004), so there could be diseases of reproductive system induced by phthalate monoesters through reproductive hormone abnormalities. Therefore, there could be an intense disturbance to the metabolic homeostasis of these endogenous substances caused by the inhibition effect of phthalate monoesters on the activity of these UGT isoforms. As for the structure specificity of phthalate monoesters, the
Because phthalate monoesters showed broad inhibition on UGT1A9, the mechanism analysis for the inhibition of phthalate monoesters towards UGT1A9 was performed using in silico docking methods. Homology modeling was carried out to construct the crystal structure of UGT1A9, and the chemical structures of phthalate monoesters were docked into the activity cavities of UGT1A9 using in silico docking method. The representative docking results were given for MEHP. The active site of UGT1A9 binding with MEHP was composed of amino acids residues SER-11, PHE-14, CYS-252, LEU-258, PRO-259, PHE-277, ARG-308, TRP-326, LEU-327, PRO-328, GLN-329, ASN-330, HIS-348 and GLU-352, which were shown in Fig. 5. In the binding pocket of UGT1A9, MEHP formed five hydrogen bonds to ARG-308, LEU-327 and GLN-329 (Fig. 6). MEHP formed hydrophobic contacts with amino acids residues PHE-14, CYS-252, PRO-328, ASN-330, HIS-348 and GLU-352 in the active pocket of UGT1A9, which were shown in Fig. 7. The binding behavior of other phthalate monoesters was given in Supplemental
Fig. 6. Hydrogen bonds interaction between MEHP with the activity cavity of UGT1A9. In the binding pocket of UGT1A9, MEHP formed five hydrogen bonds to ARG-308, LEU327 and GLN-329.
Fig. 7. Hydrophobic interaction between MEHP and the activity cavity of UGT1A9. MEHP formed hydrophobic contacts with amino acids residues PHE-14, CYS-252, PRO328, ASN-330, HIS-348 and GLU-352 in the active pocket of UGT1A9.
Z. Du et al. / Chemosphere 197 (2018) 7e13
results showed that there was a wide inhibition on UGT1A9 by MBZP, MCHP, MEHP, MHP and MOP, which was mainly eliminated from body by UGTs to form glucuronic acid conjugate (Silva et al., 2003). In contrast, phthalate monoesters with short chains like MEP and MMP, showed a slight inhibition to all UGT isoforms. Therefore, the preliminary structure-activity relationship for the inhibition of phthalate monoesters towards UGT isoforms was indicated to be that phthalate monoesters with long chains exhibited stronger inhibition towards UGTs, and UGT1A9 was the most vulnerable to the inhibition of phthalate monoesters. The in vivo inhibition magnitude can be extrapolated using the combination of in vitro parameters (Ki) and in vivo exposure of phthalate monoesters. According to [I]/Ki ratio ([I]/Ki > 0.1) evaluation standard, the threshold values for inducing in vivo inhibition was calculated to be 1.125 mM for MEHP-UGT1A7 and 0.213, 0.009, 0.117, 0.747, 0.016 mM for MBZP-UGT1A9, MCHP-UGT1A9, MEHPUGT1A9, MHP-UGT1A9, MOP-UGT1A9, respectively. Plasma concentration of MEHP was demonstrated to reach 0.38 mg/ml in human body (Silva et al., 2003). Based on this value, the in vivo exposure concentration was calculated to be approximately 1.37 mM, which exceed the threshold value of MEHP to UGT1A7 and UGT1A9. Therefore, the exposure of phthalate monoesters might disturb the metabolic elimination of endogenous substances through inhibiting the activity of UGTs in vivo. It should be noted that the biotransformation process for PAEs to their phthalate monoesters will significantly alter the inhibition potential on UGT isoforms. For example, the biotransformation of BBZP to MBZP significantly weakens the inhibition potential towards UGT1A6. In addition, the biotransformation of DBP to MBP, BBZP to MBZP significantly weakens the inhibition potential towards UGT2B4. In conclusion, the complete inhibition profile of phthalate monoesters towards UGT isoforms was given, and high specific inhibition of phthalate monoesters towards UGT1A7 and UGT1A9 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the possible concentration threshold of phthalate monoesters to effect endogenous hormone signaling and drug therapy was obtained. All these results of the present study indicating that phthalate monoesters could possibly disturb endogenous metabolism by inhibit UGTs, so close monitoring the exposure of phthalate monoesters should be paid much attention to decrease the disease risk caused by phthalate monoesters. Conflicts of interest The authors have declared that there are no conflicts of interest. Acknowledgement This work was supported by the project for the National Key Research and Development Program (2016YFC0903100, 2016YFC0903102), The 13th five year plan and TMU talent project (11601501/2016KJ0313), individualized diagnosis and treatment of colorectal cancer (No. LNCCC-B05-2015), Foundation of Committee on Science and Technology of Tianjin (Grant No. 15JCYBJC54700, 14JCQNJC11300), the China Postdoctoral Science Foundation (2016M590210, 2017T100164), Tianjin Health Bureau Science Foundation Key Project (16KG154) and Tianjin Project of Thousand Youth Talents, and Key Laboratory Open Project Fund from State
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