Inhibition of UDP-glucuronosyltransferases (UGTs) by bromophenols (BPs)

Chemosphere 238 (2020) 124645

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Inhibition of UDP-glucuronosyltransferases (UGTs) by bromophenols (BPs) Feige Wang a, 1, Shang Wang a, 1, Kai Yang a, b, c, d, Yong-Zhe Liu a, b, c, d, Kun Yang a, b, c, d, Yao Chen e, **, Zhong-Ze Fang a, b, c, d, * a

Department of Toxicology and Sanitary Chemistry, School of Public Health, Tianjin Medical University, Tianjin, 300070, China Tianjin Key Laboratory of Environment, Nutrition and Public Health, Tianjin, China National Demonstration Center for Experimental Preventive Medicine Education, Tianjin Medical University, Tianjin, 300070, China d Tianjin Center for International Collaborative Research in Environment, Nutrition and Public Health, Tianjin, China e Shenyang Mental Health Center, Shenyang, Liaoning Province, 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


UGT1A7 was the most vulnerable UGT isoform for the inhibition of BPs.
Structure-activity relationship was obtained for the inhibition of BPs towards UGTs.
In vivo moderate possibility was found for the inhibition of 2,4,6-TBP towards UGT1A3 and UGT1A7.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 June 2019 Received in revised form 18 August 2019 Accepted 21 August 2019 Available online 24 August 2019

Bromophenols (BPs) are important organic compounds which have become dominant pollutants during these years. Our present study investigated the potential inhibition behaviour of BPs on the activity of one of the most important phase II drug-metabolizing enzymes (DMEs), UDP-glucuronosyltransferases (UGTs). Recombinant UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation of 4methylumbelliferone (4-MU) was utilized as the probe reaction. 100 mM of BPs was utilized as the inhibition screening concentrations, and the complete inhibition profile of UGT isoforms by BPs was obtained. UGT1A7 was the most vulnerable UGT isoform towards BPs. Some structure-activity relationship for the inhibition of UGTs by BPs was found, and this relationship can be furtherly explained by the hydrophobic contacts of BPs with the activity cavity of UGTs using in silico docking method. The inhibition kinetics determination showed that the inhibition kinetic parameter Ki value was calculated to be 2.85, 3.99 and 31.00 mM for the inhibition of UGT1A3, UGT1A7, and UGT2B7 by representative BPs, 2,4,6TBP. Combined with in vivo exposure concentration of 2,4,6-TBP, in vitro-in vivo extrapolation (IVIVE) was employed to demonstrate the moderate possibility for the inhibition of UGT1A3 and UGT1A7 by 2,4,6TBP. In conclusion, our study gave the full description towards the inhibition of BPs towards UGT

Handling Editor: A. Gies Keywords: Bromophenols (BPs) Human UDP-Glucuronosyltransferases (UGTs) Inhibition Bromine substituent In vitro-in vivo extrapolation (IVIVE)

* Corresponding author. Department of Toxicology and Sanitary Chemistry, School of Public Health, Tianjin Medical University, Tianjin, 300070, China. ** 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.2019.124645 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

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isoforms, which will provide a new perspective for elucidating the toxicity mechanism of bromophenols (BPs). © 2019 Elsevier Ltd. All rights reserved.

1. Introduction Bromophenols (BPs) are regarded as important phenolic contaminants, and the main sources of BPs are animal metabolism (e.g. polychaeta, algae, marine mammals, etc.) and artificial industrial production (e.g. aquaculture, pharmaceutical, chemical production, etc.) (Chang et al., 2010; Feng et al., 2016). BPs are extensively distributed in water bodies, indoor air, sediment, aquatic organisms and human biological systems (e.g., human milk, blood, etc.) (Chen et al., 2017; Sim et al., 2009). BPs have significant adverse effects on human bodies. For example, BPs have been reported to exhibit severe disruption effect towards thyroid hormone homeostasis and sex hormone steroidogenesis (Kerger et al., 1988; Nakagawa et al., 2007; Szabo et al., 2009). 2,4,6-TBP can disrupt the function of cellular Ca2þ ion channel in neuroendocrine cell and potentially disturb the endocrine system. It also has a negative impact on dehaloperoxidase-hemoglobin (Zhao and Franzen, 2013). UDP-glucuronosyltransferases (UGTs), an important member of phase II drug-metabolizing enzymes (DMEs), have been demonstrated to play an important role in the elimination of various substances through conjugating lipophilic substances with glucuronic acids. UGTs-catalyzed metabolic reaction can decrease the activity and increase the water solubility of compounds, which facilitates the excretion of xenobiotics into bile, urine and feces (Rowland et al., 2013). The elimination of many endogenous substances needs UGTs-catalyzed metabolic reaction. For example, UGT1A1 is responsible for the glucuronidation metabolism of bilirubin (Ai et al., 2014). UGT1A3 metabolizes some endogenous compounds such as estrone, 2-hydroxyestradiol and 2hydroxyestrone (Radominska-Pandya et al., 1999). UGT1A8 and UGT1A10 are involved in the glucuronidation of several steroids (Cheng et al., 1998, 1999). UGT1A9 glucuronidates the thyroid hormones, thyroxine and reverse triiodothyronine (Visser et al., 1993). UGT2B7, the most important UGT isoform, has been reported to catalyze approximately 35% of exogenous and endogenous substances (Bock, 2015; Oda et al., 2015). Our previous studies have reported the strong inhibition of UGTs by chlorophenols (CPs) (Yang et al., 2018). Bromophenols (BPs), as a kind of important halogenated phenols, have not been specifically studied on their inhibitory effect on UGT isoforms. Due to the structural similarity between BPs and CPs, the inhibition of BPs towards the activity of UGTs was speculated. In addition, the previous literatures have reported that some BPs are good substrates of UGTs. The experiment carried out by Ho et al., 2012, 2015 has demonstrated the glucuronidation of 2,4-dibromophenol (2,4-DBP) and 2,4,6tribromophenol (2,4,6-TBP). 2-Bromophenol (2-BP) and 4bromophenol (4-BP) have also been reported to be the substrates of UGTs (Koen et al., 2012; Adesina-Georgiadis et al., 2018). In this study, preliminary inhibition screening, inhibition kinetic determination, and in silico docking were utilized. In vitro-in vivo extrapolation (IVIVE) was carried out to investigate in vivo inhibition magnitude.

human UGT isoforms, expressed in baculoviral-infected cells, were obtained from BD Gentest Corp. (Woburn, MA, USA). 4methylumbelliferone (4-MU) and its glucuronide 4-MUG, UDPGA (trisodium salt), Tris-HCl, MgCl2 and 7-hydroxycoumarin were from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile was purchased from Tianjin Saifurui Technology Ltd. Millipore Elix 5 UV and Milli-Q Gradient Ultra-Pure Water System were used for preparation of ultra-pure water. The other reagents were of ultraperformance liquid chromatography (UPLC) grade or of the highest grade commercially available. 2.2. Preliminary screening of inhibition capability of BPs towards UGTs 4-MU was utilized as the nonselective probe substrate for recombinant UGTs to observe the inhibition of BPs towards UGT isoforms. Because UGT1A4 do not show catalytic activity towards 4MU, UGT1A4 is not included in this study. Incubation mixture (total volume ¼ 200 mL) contained BPs (100 mM), Tris-HCL buffer (50 mM, PH ¼ 7.4), MgCl2 (5 mM), UDPGA (5 mM), UGTs and 4-MU. The concentrations of 4-MU were 110, 1200, 110, 30, 750, 30, 30, 1000, 350, 250 and 2000 mM for UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, respectively. The concentrations of UGT isoforms were 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 for UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15 and UGT2B17, respectively. After 5-min pre-incubation at 37  C, the reactions were initiated through adding 5 mM of UDPGA. The incubation time has been described in the previous literatures (Li et al., 2018; Yang et al., 2018). The metabolic reaction was terminated through adding the equal volume of ice-cold acetonitrile with 100 mM of 7hydroxycoumarin as the internal standard. The incubation mixture without BPs was used as the negative control. The terminated mixture was centrifuged at 12,000 rpm for 10 min, and 10 mL of supernatants were analyzed using ultra-performance liquid chromatography (UPLC)-UV instrument according to the previous literatures (Li et al., 2018; Yang et al., 2018). All the experiments were carried out in triplicate. 2.3. Half inhibition concentration (IC50) and inhibition kinetics determination

2. Materials and methods

Half inhibition concentration (IC50) was determined through adding multiple concentrations of BPs ranging from 0 mM to 100 mM. Furthermore, the inhibition kinetics were determined with BPs and 4-MU covering the Km (for 4-MU) and IC50 values(for BPs). Lineweaver-Burk plot was drawn using 1/reaction velocity (v) versus 1/the concentration of 4-MU ([4-MU]), and used to determine the inhibition kinetic type. The second plot was drawn to determine the inhibition kinetic parameter (Ki), in which the slopes of the lines in the Lineweaver-Burk plots were calculated and drawn versus the concentrations of BPs.

2.1. Chemicals and reagents

2.4. In vitro-in vivo extrapolation (IVIVE)

Eight BPs (the structures of BPs were given in Fig. 1) were purchased from J&K Chemical Ltd. (Beijing, China). Recombinant

The in vivo inhibition magnitude was determined through in vitro-in vivo extrapolation (IVIVE). The equation for IVIVE was

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Fig. 1. The structure of eight bromophenols (BPs).

given as followed:

2.5. In silico docking

AUCi =AUC ¼ 1  ½I=Ki

In silico docking was employed to elucidate the molecular interaction between BPs and UGTs. We constructed the structure of UGT isoforms by homology modeling method with MODELLER9v14 program. Autodock software (version 4.2) was utilized to dock BPs into the activity cavity of UGT isoforms, respectively. The non-polar hydrogen atoms of UGTs were merged. The gridbox was generated with 60  60  60 in X, Y and Z coordinate to cover the entire

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

Fig. 2. The preliminary inhibition screening of bromophenols (BPs) towards UGT1A1 (A), UGT1A3 (B), UGT1A7 (C). Data were given as mean value plus S.D. (n ¼ 3). *,p < 0.05; **, p < 0.01 compared with control (CTRL).

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Fig. 3. Concentration-dependent inhibition of 2,4,6-TBP towards UGTs. IC50 was determined by different concentrations of 2,4,6-TBP. Parallel samples were made, and the average values were used to draw the graph. Fig. 3AeC presents concentration-dependent inhibition of 2,4,6-TBP towards UGT1A3, UGT1A7, UGT2B7. Triplicate experiments were carried out. *,p < 0.05; **, p < 0.01 compared with control (CTRL).

Table 1 Half inhibition concentrations (IC50) of BPs towards UGT isoforms.

UGT1A1 UGT1A3 UGT1A6 UGT1A7 UGT1A8 UGT1A9 UGT2B7

2-BP

2,4-DBP

2,5-DBP

3,5-DBP

2,4,6-TBP

e e 1.90 mM e e e e

e e e 0.13 mM e 0.22 mM e

5.07 mM e e 0.62 mM e 0.15 mM 0.14 mM

e e 0.58 mM 0.10 mM 2.17 mM 0.18 mM 1.93 mM

e 7.47 mM e 0.64 mM e e 8.58 mM

ligand-binding site. Lamarckian Genetic Algorithm (LGA) method was employed to possess molecular docking study for the binding of BPs towards UGTs. The LGA runs were set to 50 runs for each BPs. The best conformation with the lowest docked energy was analyzed for the interactions between BPs and UGTs including hydrogen bonds and hydrophobic contacts. 2.6. Statistical analysis The experimental data were presented as the mean value plus standard deviation (S.D.). Statistical analysis was carried out using GraphPad Prism 5.0. Comparisons between two groups were performed using a two-tailed unpaired Student's t-test. Multiple groups were compared using the one-way ANOVA.

final volume of 200 mL 100 mM of BPs was utilized. Organic solvent was not more than 1% by volume. After 30-min incubation at 37  C, the reaction was terminated by the addition of 0.1 mL methanol, followed by centrifugation at 20,000g for 20 min. The supernatant was subjected to LC-MS/MS analysis. Targeted analysis of BPs was performed using Ekspert ultra LC 100 coupled to Triple TOF 5600 (AB SCIEX) with a C18 BEH column (2.1*100 mm, 3.5 mm; Waters) via MultipleReaction Monitoring in negative ionization modewith curtain gas, ion source gas 1 and ion source gas 2 at 30, 50, 50 psi, source temperature at 550  C and ion spray voltage floating at 4500 V, respectively. In auto MS/MS acquisition, the TOF MS scan and production of ion scan were using an m/z of 100e1200Da and 50e1200Da, respectively. The collision energy of the production ion scan was set at 35 ± 15 V and the declustering potential was set at 80 V. The analysis was achieved under a column temperature of 40  C using acetic acid amine (10 mM), formic acid (0.1%, v/v) and water (99.9%, v/v) as mobile phase A, acetic acid amine (10 mM), formic acid (0.1%, v/v), acetonitrile (20%, v/v) and methanol(80%, v/v) as mobile phase B. The gradient elution was set as follows: 0.01min, 35% (v/v) B; 0.5min, 35% (v/v) B; 3.01min, 60% (v/v) B; 10.01min, 80% (v/v) B; 16.01min, 90% (v/v) B; 20min, 35% (v/v) B.23min, 35% (v/v) B. 5 mL was injected for analysis and flow at 0.4 mL/min.

3. Results

2.7. Determination of glucuronidation metabolism of BPs

3.1. Preliminary inhibition screening

To determine whether tested BPs are the substrates of UGTs, the glucuronidation of BPs was investigated using human liver microsomes (HLMs) incubation system. The incubation system for the UGT reaction included HLM (5 mg/mL) (or 0.5 mg/mL of UGT1A7), UDPGA (40 mM), Tris-HCl buffer (PH 7.4), MgCl2 (50 mM), 25 mg/mL alamethicin, 10 mM D-saccharic acid 1,4-lactone, and substrates in a

The inhibition of BPs towards representative UGT isoforms was given in Fig. 2, including UGT1A1, UGT1A3, and UGT1A7. As shown in Fig. 2C, UGT1A7 was determined to be the most vulnerable UGT isoform for BPs, and negligible inhibition has been found for the inhibition of BPs towards UGT1A10, UGT2B4 and UGT2B17 (Supplemental Figure 1). 2-BP strongly inhibited the activity of UGT1A6,

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Fig. 4. Inhibition kinetics determination of 2,4,6-TBP on UGTs. Fig. 4AeC represents the Lineweaver-Burk plot of 2,4,6-TBP towards UGT1A3, -1A7, and -2B7. The horizontal axis represents the value of 1/[4 -MU], and the vertical axis represents 1/v. V is the velocity of the reaction. Fig. 4DeF represents the second plots of 2,4,6-TBP towards UGT1A3, -1A7, and -2B7. The horizontal axis represents the concentrations of 2,4,6-TBP, and the verticle axis represents the slopes of the lines from Lineweaver-Burk plots.

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Table 2 The inhibition kinetics of BPs towards UGT isoforms.

UGT1A1 UGT1A3 UGT1A6 UGT1A7 UGT1A8 UGT1A9 UGT2B7

2-BP

2,4-DBP

2,5-DBP

3,5-DBP

2,4,6-TBP

e e C,1.74 mM e e e e

e e e C,1.46 mM e C,3.72 mM e

C,0.94 mM e e C,1.56 mM e C, 0.95 mM C, 0.02 mM

e e C, 2.46 mM e C, 2.26 mM e C, 0.48 mM

e C, 2.85 mM e C, 3.99 mM e e NC, 31.00 mM

Note: C represents competitive, and NC represents noncompetitive.

isoforms, the addition of brominated substituent in the fifth site of BPs significantly increased the inhibition capability of BPs towards UGT1A1, UGT1A3, UGT2B4, UGT2B7, and UGT2B15. The addition of brominated substituent in the sixth site of BPs exhibited complex influence on the inhibition potential of BPs towards UGTs. The addition of brominated substituent in the sixth site of BPs increased the inhibition potential towards UGT1A1 and UGT1A3. In the contrast, the introduction of brominated substituent in the sixth site of BPs decreased the inhibition capability of BPs towards some UGT isoforms, such as UGT1A6, UGT1A7, and UGT1A9.

Fig. 5. Hydrophobic interaction between 2-BP (A), 2,6-DBP (B), 2,4-DBP (C), 2,4,6-TBP (D) and the activity cavity of UGT1A1. The involved amino acids in hydrophobic contacts are Leu 16, Tyr 194, Phe 211 (A), Leu 16, Tyr 194, Phe 211, Leu 19 (B), Leu 16, Tyr 194, Phe 211 (C), Leu 16, Tyr 194, Phe 211, Leu 19 (D), respectively.

UGT1A7, UGT1A8, and UGT1A9. 3-BP strongly inhibited UGT1A6, UGT1A7, and UGT1A9. 4-BP strongly inhibited UGT1A7 and UGT1A9. 2,4-DBP strongly inhibited the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT2B7, and UGT2B15.2,5-DBP strongly inhibited the activity of UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT2B7, and UGT2B15.2,6-DBP strongly inhibited UGT1A1, UGT1A3, and UGT1A8. 3,5-DBP strongly inhibited the activity of UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT2B7, and UGT2B15.2,4,6TBP inhibited UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT2B7, and UGT2B15. From the above preliminary screening results, we can find some structure-activity relationship for the inhibition of UGTs by BPs. Through comparison of the inhibitory effect of 2-BP and 2,4DBP, and 2,6-DBP and 2,4,6-TBP, the addition of brominated substituent in the fourth site of BPs significantly increased the inhibition capability of BPs towards UGT1A1, UGT1A3, UGT1A7, UGT1A9, and UGT2B7. Through comparison of the inhibitory effect of 2-BP and 2,5-DBP, and 3-BP and 3,5-DBP on the activity of UGT

3.2. Inhibition kinetics determination 2,4,6-TBP was chosen as the representative BP to determine the inhibition kinetics since 2,4,6-TBP was one of the most common used BPs in industry and could accumulate in human bile, blood, brain and liver. 2,4,6-TBP showed concentration-dependent inhibition towards UGT1A3, UGT1A7, and UGT2B7, and the IC50 values for UGT1A3, UGT1A7, and UGT2B7 were calculated to be 7.48, 0.64, and 8.58 mM, respectively (Fig. 3). The concentration-dependent inhibition curve and calculated IC50 values for other UGT isoforms were given in Supplemental Figure 2 and Table 1. As shown in Fig. 4A and Fig. 4B, the intersection point was located in the vertical axis for the inhibition of UGT1A3 and UGT1A7 by 2,4,6-TBP, indicating the competitive inhibition of 2,4,6-TBP towards UGT1A3 and UGT1A7. The intersection point was located in the horizontal axis for the inhibition of 2,4,6-TBP towards UGT2B7 (Fig. 4C), showing the noncompetitive inhibition of 2,4,6-TBP towards UGT2B7. The slopes of the lines in the Lineweaver-Burk plot were drawn versus

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Fig. 6. Hydrophobic interaction between 2-BP (A), 2,5-DBP (B), 3-BP (C), 3,5-DBP (D) and the activity cavity of UGT1A3. The involved amino acids in hydrophobic contacts are Leu 192, Tyr 194, Phe 211 (A), Leu 89, Val 126, Phe 367, Phe 211 (B), Leu 14, Leu 328, Trp 327 (C), Leu 89, Leu 192, Tyr 194, Phe 367 (D), respectively.

Table 3 In vitro-in vivo extrapolation for BPs.[I] presents the average concentration of BPs in human urine or serum. Compound

Sources

Concentration

[I] (mM)

[I]/Ki

Possibility

2-BP 2,4-DBP

Urine, Hongkong Serum, USA

2.04 ± 4.33 mg$L1 a 27 ng$g-1 lipid b

0.037 0.178

2,4,6-TBP

Urine, Hongkong

5.57 ± 4.05 mg L1 a

0.029

Serum, Norway

81 ng/g lipidb

0.612

Serum, Finland

53.8 ng/g lipidb

0.406

Serum, USA

27 ng$g-1 lipidb

0.204

0.02(UGT1A6) 0.001(UGT1A9) 0.08(UGT1A7) 0.05(UGT1A9) 0.01(UGT1A3) 0.007(UGT1A7) 0.001(UGT2B7) 0.19(UGT1A3) 0.13(UGT1A7) 0.02(UGT2B7) 0.12(UGT1A3) 0.09(UGT1A7) 0.01(UGT2B7) 0.06(UGT1A3) 0.04(UGT1A7) 0.01(UGT2B7)

Low possibility Low possibility Low possibility Low possibility Low possibility Low possibility Low possibility Moderate possibility Moderate possibility Low possibility Moderate possibility Low possibility Low possibility Low possibility Low possibility Low possibility

Note:It is known that the total blood volume of human body is about 8% of body weight and the fat content of human body is about 20% of body weight. For an adult of 50 kg, the total blood volume is about 4000 ml and the fat content is about 10 kg. The relative molecular mass (M) of 2-BP, 2,4-DBP,2,4,6-TBP are 173, 252 and 331. [I] ¼ Concentration*10 kg lipid/M/4000 ml blood (for serum) or Concentration/M (for urine). a (Wang et al., 2012). b (Gao et al., 2015).

the concentrations of 2,4,6-TBP (also named the second plot), and the results were given in Fig. 4DeF. According to these second plots, the inhibition kinetic parameters (Ki) were calculated to be 2.9, 4.0, and 31.0 mM for the inhibition of 2,4,6-TBP towards UGT1A3, UGT1A7, and UGT2B7, respectively. We also determined the inhibition kinetics of some other BPs towards UGTs, and the results were given in Table 2 and Supplemental Figure 3-12.

3.3. In silico docking to elucidate the inhibition mechanism In silico docking was employed to elucidate the mechanism for the influence of introduction of substituted bromine on the inhibition capability. Through comparison, we found that the binding free energy decreased with the introduction of 6-substitued bromine towards UGT1A1. The binding free energy of 2-BP and

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2,6-DBP were 5.10 kcal/mol and 5.69 kcal/mol, respectively. The binding free energy of 2,4-DBP and 2,4,6-TBP were 5.77 kcal/mol and 6.41 kcal/mol, respectively. 2-BP and 2,4-DBP formed 4 hydrophobic contacts with UGT1A1. 2,6-DBP and 2,4,6-TBP formed 5 hydrophobic contacts with UGT1A1 (Fig. 5AeD). Therefore, stronger hydrophobic interaction contributed to the result that 2,6-DBP (2,4,6-TBP) showed stronger inhibition towards UGT1A1 than 2-BP (2,4-DBP). Also, the same conclusion was obtained through the in silico docking of 2-BP (3-BP) and 2,5-DBP (3,5-DBP) towards UGT1A3. The binding free energy of 2-BP, 2,5-DBP, 3-BP and 3,5DBP towards UGT1A3 were 4.84, 5.44, 5.10 and 5.52 kcal/ mol, respectively. 3 hydrophobic contacts were formed between 2BP (3-BP) and the activity cavity of UGT1A3. 4 hydrophobic contacts were formed between 2,5-DBP (3,5-DBP) and the activity cavity of UGT1A3 (Fig. 6AeD). The introduction of 5-substitued bromine induced stronger inhibition of 2,5-DBP and 3,5-DBP towards UGT1A3 through stronger hydrophobic contacts. 3.4. Substrate properties of BPs for UGTs The detection of glucuronide was shown in Supplemental Figure 11. The results showed that the incubation of all the tested BPs with HLMs system can form their glucuronides. The MS spectrum of BPs and their glucuronides were given in Supplemental Figure 12-15, showing that the molecular weight (MW) of BP glucuronides have 176 more than BPs which indicating the addition of glucuronic acid of BPs. Because UGT1A7 is not expressed (or very low level) in liver, we determined whether UGT1A7 can metabolize BPs using a recombinant UGT1A7 incubation system. Indeed, all the BPs can be metabolized by recombinant UGT1A7 (Supplemental Figure 16). 4. Discussion Significant inhibition of BPs towards multiple UGTs was demonstrated in this study. UGT1A7 was regarded as the most fragile subtype inhibited by BPs. In our recent study, we also reported some structure-activity relationship for the inhibition of UGTs by BPs, which can be well explained using in silico docking methods in this study. In vitro-in vivo extrapolation (IVIVE) was carried out to calculate in vivo inhibition magnitude of BPs towards UGT isoforms, and 2,4,6-TBP was chosen as the representative BP because it is the most abundant BP for the human exposure. The exposure resources of 2,4,6-TBP have been reported to be dietary intake of seafood and bioconversion of industrial pollutants. The concentrations of 2,4,6TBP in different body fluid from different races were listed in Table 3. From these reports, we can make a conclusion that the highest concentration of 2,4,6-TBP in vivo was 0.612 mM in serum. The determined Ki values were 2.85, 3.99, 31.00 mM for UGT1A3, UGT1A7, and UGT2B7, respectively. Detailed IVIVE results were also summarized in Table 3. According to evaluation standards using [I]/ Ki ratio ([I]/Ki > 0.1), the threshold values were calculated to be 0.285, 0.399, 3.10 mM for the inhibition of 2,4,6-TBP towards UGT1A3, UGT1A7, UGT2B7, respectively. Therefore, moderate inhibition magnitude of 2,4,6-TBP towards UGT1A3 and UGT1A7 was found in vivo. Decreased activity of UGT1A7 has been reported to have close relationship with the susceptibility of some diseases, especially for cancers. For example, the individuals with decreased activity of UGT1A7 due to the UGT1A7mutant-type (especially *3 and *4) gene polymorphisms have higher risk of cancer susceptibility (Zhang et al., 2017). The decreased activity of UGT1A7 has high correlation with the increased risk of Gilbert's syndrome (Teng et al., 2007). Therefore, the exposure of 2,4,6-TBP might increase the susceptibility of some diseases through the inhibition of

UGT1A7. UGT1A3 plays a key role in the metabolism of some important endogenous substances, such as bile acids, androgens and estrogens (Erichsen et al., 2010; Trottier et al., 2010). The inhibition of UGT1A3 by 2,4,6-TBP might strongly interfere with the metabolism of these endogenous substances. It should be noted that the in vivo concentrations of 2,4,6-TBP were obtained from normal people. Occupational population will be exposed to higher concentration of mixed BPs, which might result in more severe inhibition magnitude in vivo. The results from this study can be discussed with our previous paper describing the inhibition of chlorophenols (CPs) towards UGT isoforms (Yang et al., 2018). This inhibition behaviour results from the substrates properties of BPs for UGTs. Some different inhibition situation can be found between CPs and BPs for the same UGT isoforms. For example, 2-CP showed negligible inhibitory effect towards UGT1A8, however, 2-BP exerted strong inhibition effect towards UGT1A8 (with more than 80% activity inhibited). In the contrast, 2,4-DCP exerted strong inhibition towards UGT1A10 (with nearly 80% activity inhibited), but 2,4-DBP showed no inhibition towards UGT1A10. Therefore, much attention should be given when explaining these results for the introduction of different halogenated substituents. In conclusion, our study gave the full description towards the inhibition of BPs towards UGTs isoforms. These results will provide a new perspective for the toxicity of bromophenols (BPs). Acknowledgement This work was supported by the project for the National Key Research and Development Program of China (2017YFC0211600, 2016YFC0211605), he State Key Project on Infectious Diseases of China (2018ZX10723204), Liaoning Natural Science Foundation (20170540364), The 13th five year plan and TMU talent project (11601501/2016KJ0313), National Natural Science Foundation of China (81602850), the China Postdoctoral Science Foundation (2016M590210, 2017T100164), Tianjin Health Bureau Science Foundation Key Project (16KG154), Tianjin Project of Thousand Youth Talents, and Key Laboratory Open Project Fund from State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (KF2017), Postgraduate Innovation Fund of ‘13th FiveYear comprehensive investment’, Tianjin Medical University (YJSCX201816), and The Open Project of the Key Laboratory of Modern Toxicology of Ministry of Education, Nanjing Medical University (NMUMT201809). References Adesina-Georgiadis, K.N., Gray, N., Plumb, R.S., Thompson, D.F., Holmes, E., Nicholson, J.K., Wilson, I.D., 2018. The metabolic fate and effects of 2Bromophenol in male Sprague-Dawley rats. Xenobiotica 17, 1e8. Ai, L., Zhu, L., Yang, L., Ge, G., Cao, Y., Liu, Y., Fang, Z., Zhang, Y., 2014. Selectivity for inhibition of nilotinib on the catalytic activity of human UDP-glucuronosyltransferases. Xenobiotica 44, 320e325 the fate of foreign compounds in biological systems. Bock, K.W., 2015. Roles of human UDP-glucuronosyltransferases in clearance and homeostasis of endogenous substrates, and functional implications. Biochem. Pharmacol. 96 (2), 77e82. Chang, H., Wan, Y., Naile, J., Zhang, X., Wiseman, S., Hecker, M., Lam, M.H., Giesy, J.P., Jones, P.D., 2010. Simultaneous quantification of multiple classes of phenolic compounds in blood plasma by liquid chromatography-electrospray tandem mass spectrometry. J. Chromatogr. A 1217 (4), 506e513. Chen, C.L., Yang, P.H., Kao, Y.C., Chen, P.Y., Chung, C.L., Wang, S.W., 2017. Pentabromophenol suppresses TGF-b signaling by accelerating degradation of type II TGF-b receptors via caveolae-mediated endocytosis. Sci. Rep. 7, 43206. Cheng, Z., Radominska-Pandya, A., Tephly, T.R., 1998. Cloning and expression of human UDP-glucuronosyltransferase (UGT) 1A8. Arch. Biochem. Biophys. 356 (2), 301e305. Cheng, Z., Radominska-Pandya, A., Tephly, T.R., 1999. Studies on the substrate specificity of human intestinal UDP- lucuronosyltransferases 1A8 and 1A10.

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