Assessment of the inhibitory effects of pyrethroids against human carboxylesterases

Article abstract of DOI:10.1016/j.taap.2017.02.018

Pyrethroids are broad-spectrum insecticides that widely used in many countries, while humans may be exposed to these toxins by drinking or eating pesticide-contaminated foods. This study aimed to investigate the inhibitory effects of six commonly used pyrethroids against two major human carboxylesterases (CES) including CES1 and CES2. Three optical probe substrates for CES1 (DME, BMBT and DMCB) and a fluorescent probe substrate for CES2 (DDAB) were used to characterize the inhibitory effects of these pyrethroids. The results demonstrated that most of the tested pyrethroids showed moderate to weak inhibitory effects against both CES1 and CES2, but deltamethrin displayed strong inhibition towards CES1. The IC₅₀ values of deltamethrin against CES1-mediated BMBT, DME, and DMCB hydrolysis were determined as 1.58?μM, 2.39?μM, and 3.3?μM, respectively. Moreover, deltamethrin was cell membrane permeable and capable of inhibition endogenous CES1 in living cells. Further investigation revealed that deltamethrin inhibited CES1-mediated BMBT hydrolysis via competitive manner but noncompetitively inhibited DME or DMCB hydrolysis. The inhibition behaviors of deltamethrin against CES1 were also studied by molecular docking simulation. The results demonstrated that CES1 had at least two different ligand-binding sites, one was the DME site and another was the BMBT site which was identical to the binding site of deltamethrin. In summary, deltamethrin was a strong reversible inhibitor against CES1 and it could tightly bind on CES1 at the same ligand-binding site as BMBT. These findings are helpful for the deep understanding of the interactions between xenobiotics and CES1.

Full text of DOI:10.1016/j.taap.2017.02.018

Accepted Manuscript
Assessment of the inhibitory effects of pyrethroids against human
carboxylesterases
Wei Lei, Dan-Dan Wang, Tong-Yi Dou, Jie Hou, Liang Feng,
Heng Yin, Qun Luo, Jie Sun, Guang-Bo Ge, Ling Yang
PII:
S0041-008X(17)30085-6
doi: 10.1016/j.taap.2017.02.018
YTAAP 13878
DOI:
Reference:
To appear in:
Toxicology and Applied Pharmacology
Received date:
Revised date:
Accepted date:
6 January 2017
12 February 2017
22 February 2017
Please cite this article as: Wei Lei, Dan-Dan Wang, Tong-Yi Dou, Jie Hou, Liang Feng,
Heng Yin, Qun Luo, Jie Sun, Guang-Bo Ge, Ling Yang , Assessment of the inhibitory
effects of pyrethroids against human carboxylesterases. The address for the corresponding
author was captured as affiliation for all authors. Please check if appropriate. Ytaap(2017),
doi: 10.1016/j.taap.2017.02.018
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ACCEPTED MANUSCRIPT
Research paper
Assessment of the inhibitory effects of pyrethroids against human
carboxylesterases
a,b,1
b,1
b
c
b
b
Wei Lei
, Dan-Dan Wang , Tong-Yi Dou , Jie Hou , Liang Feng , Heng Yin , Qun
d
a
b,*
b
Luo , Jie Sun , Guang-Bo Ge , Ling Yang
a
The Second Affiliated Hospital of Dalian Medical University, Dalian110623, China.
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Dalian Medical University, Dalian 116044, China.
b
c
d
Beijing National Laboratory for Molecular Science, Institute of Chemistry, Chinese
Academy of Sciences, Beijing, 100080, China.
1
These authors contributed equally to this work.
∗Corresponding authors
Tel.: +86 411 84379317; fax: +86 411 84676961
E-mail addresses: geguangbo@dicp.ac.cn (G-B. Ge)

ACCEPTED MANUSCRIPT
Abstract
Pyrethroids are broad-spectrum insecticides that widely used in many countries, while
humans may be exposed to these toxins by drinking or eating pesticide-contaminated foods.
This study aimed to investigate the inhibitory effects of six commonly used pyrethroids
against two major human carboxylesterases (CES) including CES1 and CES2. Three optical
probe substrates for CES1 (DME, BMBT and DMCB) and a fluorescent probe substrate for
CES2 (DDAB) were used to characterize the inhibitory effects of these pyrethroids. The
results demonstrated that most of the tested pyrethroids showed moderate to weak inhibitory
effects against both CES1 and CES2, but deltamethrin displayed strong inhibition towards
CES1. The IC values of deltamethrin against CES1-mediated BMBT, DME, and DMCB
5
0
hydrolysis were determined as 1.58 μM, 2.39 μM, and 3.3 μM, respectively. Moreover,
deltamethrin was cell membrane permeable and capable of inhibition endogenous CES1 in
living cells. Further investigation revealed that deltamethrin inhibited CES1-mediated BMBT
hydrolysis via competitive manner but noncompetitively inhibited DME or DMCB hydrolysis.
The inhibition behaviors of deltamethrin against CES1 were also studied by molecular
docking simulation. The results demonstrated that CES1 had at least two different
ligand-binding sites, one was the DME site and another was the BMBT site which was
identical to the binding site of deltamethrin. In summary, deltamethrin was a strong reversible
inhibitor against CES1 and it could tightly bind on CES1 at the same ligand-binding site as
BMBT. These findings are helpful for the deep understanding of the interactions between
xenobiotics and CES1.
Key words: Pyrethroids; Human carboxylesterase 1; Deltamethrin; Ligand-binding sites

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1
. Introduction
Pyrethroids are a class of broad-spectrum insecticides which have been widely used in
many countries for decades (Anadon et al., 2009). Owing to their relatively low toxicity to
mammals in contrast to organophosphorus insecticides, synthetic pyrethroids are among the
most frequently used pesticides and account for more than one-third of the insecticides
currently marketed in the world (Soderlund, 2012a; Power et al., 2007; Morgan, 2012;
Walters et al., 2009; Walters et al., 2007; Quirós-Alcalá et al., 2014). Now pyrethroids are not
only used for crop and forestry protection but also used in indoor environments for pest
control (Colume et al., 2001; Saillenfait et al., 2015; Li et al., 2016a). Because of the
numerous applications and increasing worldwide use of pyrethroids, the exposure of
pyrethroids in the environment is a major risk factor for chronic toxicity to animals and
humans (Moretto, 1991; Zawiyah et al., 2007; Lu et al., 2012). The reported toxic effects of
pyrethroids include neurotoxicity, skin contact toxicity, respiratory toxicity and reproductive
system toxicity (Pauluhn, 1996; Soderlund et al., 2002; Jurewicz et al., 2015; Li et al.,
2
016b). Several previous studies have demonstrated that some pyrethroids can inhibit the
catalytic activity of acetylcholinesterase (AchE), a member of the serine hydrolase
superfamily (Szegletes et al., 1995; Hossain et al., 2005). It is more likely that pyrethroids
may also display inhibitory effects towards other important serine hydrolases, which may
bring potential toxic consequences or undesirable effects.
Mammalian carboxylesterases (CES) are important members of the serine hydrolase
superfamily, which are localized within the lumen of the endoplasmic reticulum in many
tissues (Xiao et al., 2013). CEs catalyze the esters cleavage of a vast number of structurally
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diverse ester- or amide-containing compounds, including endogenous and exogenous
molecules (Redinbo et al., 2003). In human, the majority of CES have been identified belong
to the CES1 or CES2 gene family, two major isoenzymes distributed in human. CES1 and
CES2 share 47% amino acid sequence identity, but exhibit extremely differential tissue
distribution and distinct substrate specificity (Schwer et al., 1997; Sanghani et al., 2009).
Generally, CES1 is predominantly distributed in the liver and prefers to hydrolyze the ester
substrates with small alcoholic groups and large acyl groups. In contrast, CES2 is abundant in
small intestine and prefers to hydrolyze the esters with large alcohol and small acyl group
(Hosokawa, 2008; Sato et al., 2012). The main biological functions of CES1 include the
hydrolysis of cholesterol esters in both adipocyte and the liver (Jernas et al., 2009; Satoh and
Hosokawa, 2009; Marrades et al., 2010; Quiroga et al., 2012), as well as the detoxification or
metabolic activation of various ester xenobiotics including clinical drugs (such as clopidogrel
and oseltamivir) and environmental toxins (such as permethrin and deltamethrin) (Ross et al.,
2
006; Hosokawa, 2008; Yang et al., 2009; Sato et al., 2012; Zhu and Markowitz, 2013; Zhu
et al., 2013; Laizure et al., 2014; Tarkiainen et al., 2015). Although the endogenous substrates
of CES2 have not been reported, CES2 plays key roles in the metabolic activation of
anticancer prodrugs including irinotecan, capecitabine and LY2334737 (the prodrug of
gemcitabine) (Imai, 2006; Satoh and Hosokawa, 2006; Hatfield et al., 2011). Strong
inhibition of these two major CES in human may slow down the metabolism of endogenous
esters or ester drugs, and thereby affect the endogenous metabolism and the clinical efficacy
and outcomes of various CEs-substrate drugs (Khanna et al., 2000; Wu et al., 2002). Notably,
CES1 and CES2 played key roles in the detoxification of pyrethroids, these ester insecticides
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could be hydrolyzed to non-toxic or low toxic metabolites by CES (Nishi et al., 2006; Ross et
al., 2006; Crow et al., 2007). However, the inhibition effects of these pyrethroids towards
human CEs have not been investigated yet. Given that pyrethroids are substrates of CES1 and
CES2, these compounds are more likely to affect the catalytic activities of these human CES,
thus bring undesirable or harmful effects via modulation the metabolism of therapeutic drugs
or endogenous esters.
In the present study, the inhibitory effects of six commonly used pyrethroids including
fenpropathrin, cyhalothrin, deltamethrin, fenvalerate, permethrin and tau-fluvalinate, against
both CES1 and CES2 were investigated for the first time. The highly selective optical probe
substrates for CES1 and CES2 were used to rapid screen and characterize the inhibition
potency of these pyrethroids against CES1 and CES2, by using human liver microsomes
(HLM) as enzyme source. The results demonstrated that most of tested pyrethroids showed
moderate to weak inhibitory effects against both CES1 and CES2, but deltamethrin displayed
strong inhibition against CES1. In this case, three optical probe substrates for CES1 including
DME, BMBT and DMCB (Table 1) were used to further investigate the inhibition behaviors
of this pyrethroid against CES1-mediated hydrolysis, while docking simulations were utilized
to provide deep insights into the interactions between deltamethrin and CES1. All these
findings are very helpful for the deep understanding of the interactions between pyrethroids
and CES1, as well as for the risk assessment of these environmental toxins.
2
. Materials and methods
2
.1 Chemicals and reagents
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Fenpropathrin (cis and trans isomers) was acquired from Fluka (USA), cyhalothrin,
deltamethrin, fenvalerate (cis and trans isomers), tau-fluvalinate and permethrin (cis and
trans isomers) were purchased from Aladdin Industrial Corporation. (Shanghai, China). The
purities of these six pyrethroids were > 98%. Bis-p-nitrophenyl phosphate (BNPP) was
purchased from TCI (Tokyo, Japan). Stock solutions of all pyrethroids were prepared in
DMSO and stored at 4 °C until use. Phosphate buffer (100 mM, pH 7.4 and pH 6.5) was
prepared by using Millipore water and stored at 4 °C until use. Human liver microsomes
(BioreclamationIVT In Vitro CYP™ M-class™ Human Liver Microsomes, Product Number:
X008067) were obtained from RILD Co. Ltd (Shanghai, China). The specific probe
substrates including (S)-methyl 2-(6- hydroxybenzo[d]thiazol -2-yl) -4,5- dihydrothiazole -4-
carboxylate (DME), 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl benzoate
(DDAB),
3,5-dimethyl-8-methoxycarbonyl-BODIPY
(DMCB),
and
2
-(2-benzoyloxy-3-methoxyphenyl) benzothiazole (BMBT) (the chemical structures were
depicted in Table 1) were synthesized by the authors as described previously (Liu et al., 2014;
Jin et al., 2016; Wang et al., 2016b; Ding et al., 2017). The Luciferin Detection Reagent
(
LDR) was obtained from Promega Corporation (USA). Millipore water (Millipore, USA)
was used throughout. All other reagents, fine chemicals and LC solvents with the highest
grade commercially available were obtained from J&K Chemical Ltd. (China) and Tedia
(USA).
2
.2 Analytical instrument and methods
1
The Synergy H Hybrid Multi-Mode Microplate (BioTek) was applied for analysis the
probe reactions of DME, DDAB, and DMCB. The analytical methods for DME and its
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hydrolytic metabolite were set as follow, the gain value of luminescence detection was set as
1
35, while the integration time was set as 1 s. The fluorescence detection conditions for the
hydrolytic metabolite of DDAB were set as follow, the excitation wavelength was set at 600
nm, the emission wavelength was set at 662 nm, and the gain value was 100. The
fluorescence detection conditions for the hydrolytic metabolite of DMCB were set as follow,
the excitation wavelength was set at 505 nm, the emission wavelength was set at 560 nm, and
the gain value was 80.
As for probe BMBT, a liquid chromatography-fluorescence detection (LC-FD) based
method was used as our previously reported (Wang et al., 2016a). Briefly, liquid
chromatography separation was performed with a ultra-fast liquid chromatography (UFLC)
spectrometry system equipped with a DGU-20A vacuum degasser, two LC-30AD pumps, an
SIL-30AC autosampler, an RF-20A fluorescence detector, a CTO-30A column oven, and a
chromatography workstation (SHIMADZU, Kyoto, Japan). The chromatographic separation
was achieved using a Shim-pack XR-ODS column (75 mm × 2.0 mm, 2 µm, Shimadzu). The
mobile phase consisted of CH CN (A) and 0.2% formic acid in water (B) with the following
3
gradient: 0–3 min, 60–90 % A; 3–4 min, 90 % A; 4–5 min, 60 % A. The system was operated
at a flow rate of 0.4 mL/min. The column temperature was kept at 40 ℃ and the injection
volume was 10 µL. The fluorescence detection for BMBT and HMBT was achieved with
excitation wavelength at 304 nm, and the emission wavelength was set at 488 nm.
2
.3 Enzyme inhibition assays
In this study, chemical inhibition assays were performed as described previously (Wang et
al., 2016b). In brief, for inhibition assays with probes BMBT, DDAB, and DMCB, the
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incubation mixtures included PBS (100 mM, pH 7.4), potential inhibitors, and HLMs. After
o
pre-incubation at 37 C for 5 min, the reaction was initiated by the addition of the stock
solution of probe. After 20 min incubation, the reactions were terminated by adding 200 µL
ice-cold acetonitrile (equal volume to incubation mixture) followed by centrifugation at
2
0,000×g for 20 min by using Allegra 64R centrifuge. It is noteworthy that all the probe
reactions were conducted within the linear response. As for BMBT, the substrate
concentration was 10 μM and the HLMs concentration was 0.5 ug/ml; For DDAB, the
substrate concentration was 10 μM and the HLMs concentration was 5 ug/ml; Fo DMCB the
substrate concentration was 5 μM and the HLMs concentration was 0.2 ug/ml. Control
incubations without inhibitors were also performed. For all experiments, the final
concentration of DMSO was less than 2% (V/V). Aliquots of the supernatant were then taken
for further LC-FD or microplate reader analysis.
As for inhibition assays with probe DME, the incubation mixture (50 μL total volume)
consisted of PBS (100 mM, pH 6.5), enzyme sources (10 μg/mL，HLM), inhibitors (one of
six tested pyrethroids or bavachinin) and DME (3 μM, final concentration). First, each of the
potential esterase inhibitors was pre-incubated with HLM at 37 °C for 10 min with shaking in
the microplate reader. Then DME was added to start the reaction. After incubation at 37 °C
for 10 min in a shaking bath, LDR (equal volume of incubation mixture, 50 uL) was added to
terminate the reaction, luminescence measurements were conducted as described above. The
residual enzyme activity (%) was determined by the percent of D-fluorescein production in
the presence of tested compounds to the control (in the absence of inhibitors).
2
.4 Inhibition kinetic analysis
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The method for inhibition kinetic analyses was performed by using different inhibitor
concentrations as mentioned previously (Sun et al., 2016). The half inhibition concentration
(
IC ) was determined by using various inhibitor concentrations under the incubation
50
conditions described above. Inhibition constants (K ), the affinity of inhibitors towards target
i
enzyme, were determined by using various concentrations of substrates in the presence or
absence of inhibitors (Fang et al., 2013; He et al., 2015; Wang et al., 2015; Zhu et al., 2015;
Lv et al., 2016; Xin et al., 2016). Three kinetic models were used to calculate the K values
i
by nonlinear regression using the equations in the program Prism (Version 5.0.1, GraphPad,
San Diego, CA) (He et al., 2015). The type of inhibition was determined by fitting the data to
the enzyme inhibition models.
2
.5 Cell culture and fluorescence imaging
HepG2 cells were grown in MEM/EBSS medium supplemented with 10% fetal bovine
serum (FBS). HepG2 cells were regularly grown in an atmosphere of 5% CO and 95% air at
2
5
3
7 °C. For fluorescence imaging, cells were seeding at a density of 10 cells per confocal dish
(Φ 20 mm) and incubated for 24 hours. After washed twice with FBS-free culture medium,
the adherent cells were incubated with 0, 10, 25, 50, 100 µM of deltamethrin (preparing in
FBS-free) for 60 min at incubator. For evaluating the intracellular CE1 function, the living
cells were then co-incubated with DMCB (5 μM) for another 30 min. After washed with PBS
(pH 7.4) for three times, cells were imaged on confocal microscope (Olympus, FV1000,
Japan).
2
.6 Molecular docking simulations
The protein structures of CES1 were obtained from the Protein Data Bank (PDB ID: 1MX5)
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(Bencharit et al., 2003b). The whole molecular docking process in this work was performed
using Discovery Studio (BIOVIA Discovery Studio 2016, Dassault Systèmes, San Diego,
USA). The ‘Prepare Protein’ procedure was used to prepare the input protein structures for
docking. Tasks including inserting missing atoms in incomplete residues, modelling missing
loop regions, deleting alternate conformations (disorder), removing waters, standardizing
atom names, and protonating titratable residues using predicted pKs were performed.
Meanwhile, the ‘Prepare Ligand’ procedure was used to prepare the input ligands for docking.
Tasks including removing duplicates, enumerating isomers and tautomers, and generating 3D
conformations were performed. The CHARMM 40.1 force field was used to represent the
protein and ligand structures. Docking simulations were performed by a standard LibDock
protocol, where protein features are referred to as hotspots. After a final energy-minimization
step (allowing the ligand poses to be flexible), the top scoring ligand poses are saved. The
rigid poses are placed into the active site of CES1 and the hotspots are matched as triplets.
For docking process, all ligands including DME, BMBT, DCMB and Deltramethrin were
inputted, the protein-ligand complexes with the highest LibDock score were taken from the
docking results and depicted in full text.
3
Results
3
.1 The IC of six pyrethroids against hCEs
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0
The inhibitory effects of the six pyrethroids against two major CES (including CES1 and
CES2) were investigated using DME and DDAB as the corresponding probe substrates. The
dose-dependent inhibition curves of these pyrethroids were depicted in Fig. 1 and Fig. 2, and
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the corresponding IC values were listed in Table 2. It is evident from Fig. 1, Fig. 2, and
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0
Table 2 that all tested pesticides can inhibit the catalytic activities of CES1 and CES2 in dose
dependent manners, but most of them showed moderate to weak inhibitory effects on these
two hCEs (>10 μM). Notably, among all test pyrethroids, deltamethrin exhibited strong
inhibition against CES1-mediated DME hydrolysis. The IC value of deltamethrin against
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0
DME hydrolysis in HLMs were determined as 2.39 μM, which is more potent than that of
bavachinin (a known reversible inhibitor of CES1) determined under the same conditions
(Fig. S1). In addition, the time-dependent inhibition of deltamethrin demonstrated that
pre-incubation of deltamethrin could not lead to the change in inhibition potency (Fig. S2),
suggesting that deltamethrin is a reversible inhibitor against CES1 which is different from the
organophosphorus insecticides (Sogorb and Vilanova, 2002; Mikata et al., 2012).
3
.2 Inhibition behaviors of deltamethrin against DME and BMBT hydrolysis
Given that deltamethrin displayed strong inhibitory effects towards CES1 in the HLM and
the multiple ligand-binding sites of this key enzyme, three different substrates (DME, BMBT,
and DMCB) of CES1 were used for further investigation of the inhibition behaviors of
deltamethrin. (Bencharit et al., 2002; Bencharit et al., 2003a; Bencharit et al., 2003b;
Fleming et al., 2007) As shown in Table 3, deltamethrin could inhibit CES1-mediated BMBT,
DME, and DMCB hydrolysis in HLMs, with the IC values of 1.58 μM, 2.39 μM, and 3.3
5
0
μM, respectively (Fig. S2 & S3). Furthermore, both Dixon and Lineweaver-Burk plots
demonstrated that deltamethrin displayed noncompetitive inhibition behavior against
CES1-mediated DME and DMCB hydrolysis, while it acted as a competitive inhibitor against
CES1-mediated BMBT hydrolysis (Fig. 3). The inhibition parameters (K ) of deltamethrin
i
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against DME hydrolysis, BMBT, and DMCB hydrolysis in HLMs were also calculated as
4
.58 μM, 2.21 μM, and 5.94 μM (Table 3), respectively. These findings demonstrated that
deltamethrin displayed different inhibition types against CES1-mediated DME, DMCB, and
BMBT hydrolysis, and this pyrethroid exhibited more potent inhibition against
CES1-mediated BMBT hydrolysis.
3
.3 Inhibition of intracellular CES1 activities by deltamethrin
Taking into account that CES1 is mainly localized within the lumen of the endoplasmic
reticulum in human tissues, we next to explore whether deltamethrin could inhibit the
function of intracellular CES1 in living cells. It is well-known that many reported inhibitors
against a target intracellular enzyme(s) cannot work in living systems, due to the poor cell
membrane permeability or extensive metabolism (Li et al.,2013). In this study, the real
activities of intracellular CES1 with or without deltamethrin were assayed using DMCB (a
newly developed OFF-ON fluorescence probe), due to the excellent specificity and good
performance of this probe (Ding et al., 2017). As show in Fig. 4, following co-incubation of
HepG2 cells with DMCB (5 μM) at 37 °C for 30 min, the cells started to show strong green
fluorescence. In sharp contrast, when the cells were pretreated with deltamethrin, the cells
showed dim green fluorescence, suggesting the catalytic activities of intracellular CES1 was
strongly inhibited by deltamethrin. The fluorescence intensities of HepG2 cells with or
without deltamethrin were obtained by image processing and analysis Software ImageJ. As
depicted in Fig. 5, the decrease in fluorescence intensity of the hydrolytic product of DMCB
via a dose-dependent manner, and more than half of intracellular CES1 could be inhibited
when HepG2 cells were incubated with a 10 µM of deltamethrin. This finding suggested that
1
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deltamethrin was cell membrane permeable and capable of inhibition endogenous CES1 in
living cells. To the best of our knowledge, this is the first report to characterize the inhibitory
effects of a given xenobiotic on intracellular CES1 in living cells by a practical
fluorescence-based assay.
3
.4 Molecular docking simulations
In order to gain deep insights into the interactions between deltamethrin and CES1, as well
as to explore the binding site of deltamethrin on CES1, molecular docking simulations were
carried out. As shown in Fig. 6A, Fig. 6B and Fig. 6C, three optical substrates of CES1
including DME, DCBM, and BMBT could be well-docked into the active cavity of CES1,
but these three substrates bind on CES1 at two different binding sites. As shown in Fig. 6,
Fig.S4, Fig.S5, and Fig.S6, DME and DCBM bind on CES1 at the same site (also called
ligand-bind site I) which is surrounded by Gly356, Leu368, Met361 and Pro461, while the
BMBT (also called ligand-bind site II) bind on CES1 at another site which is surrounded by
Ser221, Ile359, Leu388 and His468, etc. As expected, deltamethrin could also be well-docked
into the active cavity of CES1, and the optimal binding mode was depicted in Fig. 6D. It was
evident from Fig. 6 and Fig.S4, that the binding area of deltamethrin is highly overlapped
with that of BMBT, but this area is different form the binding area of DME or DCBM. These
findings agreed well with the experimental results from inhibition kinetic analyses, in which
deltamethrin functioned as a competitive inhibitor against CES1-mediated BMBT hydrolysis
but was a non-competitive inhibitor against CES1-mediated DME/DCBM hydrolysis. Thus,
both experimental and docking simulations demonstrated that the preferred binding site of
deltamethrin on CES1 was identical to the BMBT site (the ligand-binding site II), which was
1
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different from the binding site of DME/DCBM. These findings demonstrated that CES1 had
at least two different ligand-binding sites, while DME/DCBM and BMBT could serve as
typical site markers for further investigations on the interactions between xenobiotics and
CES1.
4
．Discussion
The safety of pesticides is drawing more and more attentions, since growing cases of
illness and injuries have been reported following exposure to pyrethroids (Williams et al.,
008). It is well established that many pyrethroids could directly target on sodium channels,
2
chloride channels, and other channel and receptor systems in neuronal tissues, and thus bring
toxic and undesirable effects (Anadon et al., 2009). Notably, several previously studies have
demonstrated that some pyrethroids could also inhibit the catalytic activity of
acetylcholinesterase (AchE), an important member of the serine hydrolase superfamily
(Szegletes et al., 1995; Hossain et al., 2005). Furthermore, several studies regarding the
detoxification and metabolism of pyrethroids have found that CES1 and CES2 are key
enzymes responsible for the hydrolysis of many pyrethroids (Nishi et al., 2006; Ross et al.,
2
006; Crow et al., 2007). Taking into account the importance of CES in both endobiotic and
xenobiotic metabolism, as well as the ignorable roles in the process of pesticides
detoxification, it is necessary to explore the potential pesticides-CEs interactions which may
bring potential toxic consequences or undesirable effects.
Our previous studies have developed several specific activity-based optical probes for both
CES1 and CES2 which have been successfully used for rapid screening and characterization
1
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of inhibitors against CES (Liu et al., 2014; Jin et al., 2016; Wang et al., 2016b; Wang et al.,
2
017). DME is a bioluminescent probe substrate for CES1, and the principle for monitoring
of CES1 activity is as follows, DME could be rapidly converted to D-luciferin upon addition
of CES1, which then initiated the firefly luciferase–luciferin system to generate a
bioluminescent signal (Wang et al., 2016b). Probe BMBT and DCBM are fluorescent probes
for CES1, the CES1-mediated hydrolysis of which could lead to a remarkable change in
fluorescence spectrum upon addition of CES1, which provide the basis for the sensitive
detection of CES1 activity under physiological conditions (Liu et al., 2014). DDAB is a
near-infrared fluorescent probe for CES2 and the fluorescence signal of its metabolite in real
samples is modulated via CES2-mediated amide bond cleavage (Jin et al., 2016). All these
optical probe substrates display excellent selectivity, highly sensitivity, fast-response, and
anti-interference capability. With these tools in hands, the rapid screening and
characterization of inhibitors against CES can be achieved using human tissue preparations as
enzyme sources rather than recombinant enzymes. Moreover, the fluorescent property of the
present probes makes it possible to directly evaluate the inhibitory effects in live cells.
Actually, it is able to acquired further information of small molecules-hCEs interactions that
much more closely mimics the in vivo biological system using human tissue preparations or
in vitro cell systems. Notably, the present study demonstrated that these optical probes for
CES1 bind on this enzyme at two different ligand-binding sites including binding site I and
binding site II, thus these probes provide indispensable tools to explore the ligand-binding
sites of potential CES1 inhibitors. Our findings demonstrated that deltamethrin displayed
noncompetitive inhibition against CES1-mediated DME and DMCB hydrolysis, while it
1
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acted as a competitive inhibitor against CES1-mediated BMBT hydrolysis. Furthermore,
molecular docking simulations also give similar results to the experimental results. All these
results suggested that CES1 had at least two ligand-binding sites, while deltamethrin bind on
the same ligand-binding site as BMBT.
Although deltamethrin could be metabolized by CES, the metabolic clearance rate of
deltamethrin is far lower than other pyrethroids, such as bioresmethrin, trans-permethrin,
cis-permethrin (Godin et al., 2006; Ross et al., 2006), implying that deltamethrin is a poor
substrate of CES1. Meanwhile, deltamethrin displays the most potent inhibition against CES1
in contrast to other pyrethroids, suggesting that deltamethrin is a strong inhibitor against
CES1. On the basis of these findings, it is easily conceivable that the detoxification of
deltamethrin is relatively slow in human body after exposure when it is compared to other
pyrethroids. It is well-known that deltamethrin are mainly absorbed through the skin and
respiratory tract, the accumulation process may easily occur in these tissues in case of
long-term or high-dosage exposure. Base on the fact that the important roles of CES1 in
several physiological processes and xenobiotic-metabolic clearance (Xu et al., 2014), the
inhibition of CES1 activity by deltamethrin may affect the clinical efficacy and outcomes of
various CE1-substrate drugs or disturb CES1-associated lipid metabolism. Taking into
account of the widespread use of deltamethrin, humans may be exposed to this toxin from
deltamethrin-contaminated foods or environmental matrices. Hence, the potential
pesticide-drug interactions risks of deltamethrin via CES1 inhibition should be fully
considered, especially for those farmers or chemists taking the opportunity to long-term
exposure to deltamethrin.
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6

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It should be noted that the inhibitory type of deltamethrin against CES1 is totally different
from that of organophosphorus insecticides, which react covalently with the serine residue in
the active site of serine hydrolases, thereby inactivating these enzymes including CES
(Szegletes et al., 1995; Cacciatore et al., 2013). Unlike organophosphorus pesticides,
deltamethrin is a reversible inhibitor against CES1 which has been confirmed by a
time-dependent inhibition assay (also named IC shift assay, Fig. S7). In other hands, the K
i
5
0
values of organophosphorus insecticides against CES1 have been determined as low as nM
level, which is much potent than that of deltamethrin. It is easily conceivable that exposure to
organophosphorus insecticides may bring the inactivation of CES1 or other serine hydrolases
in human, and then cause acute poisoning if a large amount of organophosphate insecticides
were taken into the body. Comparing with organophosphorus insecticide (IC at low nM
5
0
level for CES), the inhibitory effects of pyrethroids (such as deltamethrin with the IC value
5
0
of 2.39 uM) against CES1 is relatively weak. Taking into account that the allowed residue
levels for pyrethroids are at low or sub ppm level, the potential exposure risk of a single
pyrethroids compound is minimal. However, in most cases, various pyrethroids are used
simultaneously in agriculture and other aspects. Considering the total exposure amount of
pyrethroids and the similarity of their structures, it is worth for attention to the undesirable
effects associated with the total pyrethroids. The long-term exposure to the class of
pyrethroids may bring cumulative effects in human body and then affect CES1-mediated
toxin clearances and disturb CES1-mediated endogenous metabolism. Thus, further
investigations on long-term exposure to deltamethrin should be performed to explore whether
this pesticide could trigger undesirable effects on human health.
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In summary, this study investigated the inhibitory effects of six commonly used
pyrethroids against CES1 and CES2 in human tissue preparations for the first time. The
results demonstrated that most of tested pyrethroids showed moderate to weak inhibitory
effects against both CES1 and CES2, but deltamethrin displayed strong inhibition against
CES1. Further investigation revealed that deltamethrin was a strong reversible inhibitor
against CES1, which could inhibit CES1-mediated BMBT hydrolysis via competitive manner
but it acted as a noncompetitive inhibitor against DME hydrolysis. Additionally, both
experimental results and docking simulations suggested that CES1 had at least two
ligand-binding sites, while deltamethrin bind on the same ligand-binding site as BMBT. All
these findings will be very helpful for the deep understanding of the interactions between
xenobiotics and CES1.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by the National Basic Research Program of China (2013CB531805),
NSF of China (81473181, 81573501, 81302793, 21602219 & 31600641) and Beijing
National Laboratory for Molecular Sciences (BNLMS20150124).
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Figure / table legends
Table 1 The chemical structures of three specific probes towards CES1 and CES2.
Table 2 The IC values of six pyrethroids compounds and positive control towards CES1
5
0
and CES2 using DME and DDAB as probe substrates, respectively.
Table 3 The apparent K values and the type of inhibition of deltamethrin against CES1
i
meidated DME, BMBT, and DMCB hydrolysis.
Fig. 1 The dose-response inhibition curves of six pyrethroids towards the activity of CES1 by
using DME as probe substrate.
Fig. 2 The Dose-response inhibition curves of six pyrethroids towards the activity of CES2
by using DDAB as probe substrate.
Fig. 3 The inhibition kinetics of deltamethrin towards the activity of CES1. A, C, E:
Lineweaver–Burk plots for the inhibition of deltamethrin towards the activity of CES1 using
BMBT, DME, DMCB as probe substrates, respectively. B, D, F: The corresponding Dionx
plots.
Fig. 4 Confocal fluorescence images of HepG2 cells stained with DMCB. (A) Cells treated
with DMSO (1%, V/V); (B) Cells treated with DMCB (5 μM) at 37 °C for 30 min; (C-F)
Cells pretreated with DMT (10, 25, 50, 100 μM) for1 hour and further stained with DMCB (5
μM) at 37 °C for 30 min. Scar bars= 20 μm.
Fig. 5 Relative fluorescence intensity measurements from the corresponding confocal
fluorescence images. The fluorescence intensity from images of 0 μM DMT is defined as
1
00%.
Fig. 6 A stereo view of the crystal structure of CES1 and the stereodiagram of each ligand
aligned in its active site. (A) DME can be well docked into the cavity of CES1 on site I; (B)
BMBT can be well docked into the cavity of CES1 on site II; (C) DMCB can be well docked
into the cavity of CES1 on site I; (D) DMT can be well docked into the cavity of CES1 on
site II.
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Tables
Table 1
Probe
Probe substrate
Target enzyme
Hydrolytic product
DME
CES1
BMBT
CES1
CES2
CES1
DDAB
DMCB
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Table 2
Compound
name
Chemical
structure
Molecular
weight
IC50 (μM)
CES1
CES2
Fenpropathrin
3
4
47.4
49.8
26.09±4.92
13.62±2.70
39.64±4.93
(FPT)
Cyhalothrin
CLT)
17.13±3.23
25.16±3.93
28.06±1.70
19.68±1.81
(
Deltamethrin
DMT)
5
4
5
3
05.2
19.9
02.9
91.2
2.39±0.23
35.98±7.29
25.33±2.19
(
Fenvalerate
FVT)
(
Tau-fluvalinate
TFVT)
(
Permethrin
PMT)
37.24±4.32
5.49±0.66
41.15±5.07
18.54±1.63
(
Bavachinin
338.4
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8

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Table 3
Probe
substrate
DME
IC50
(μM)
2.39
1.58
3.30
K
i
2
The type of inhibition
R
(μM)
4.58
2.21
5.94
Noncompetitive inhibition
Competitive inhibition
0.99
0.99
0.99
BMBT
DMCB
Noncompetitive inhibition
2
9