Enantioselective Metabolism of Flufiprole in Rat and Human Liver

Mar 3, 2016 - The enantioselective metabolism of rac-flufiprole was dramatically different in rat and human liver microsomes in the presence of the ...
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Enantioselective Metabolism of Flufiprole in Rat and Human Liver Microsomes Chunmian Lin, Yelong Miao, Mingrong Qian, Qiang Wang, and Hu Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05853 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 12, 2016

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Journal of Agricultural and Food Chemistry

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Enantioselective Metabolism of Flufiprole in Rat and

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Human Liver Microsomes

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Chunmian Lin , Yelong Miao , Mingrong Qian , Qiang Wang , Hu Zhang





4 5







*,‡

College of Biological and Environmental Engineering, Zhejiang University of Technology,

Hangzhou 310014, China ‡

6

Institute of Quality and Standard for Agricultural Products, Zhejiang Academy of Agricultural

7

Sciences, Hangzhou 310021, China

8

ABSTRACT: The enantioselective metabolism of flufiprole in rat and human liver

9

microsomes in vitro was investigated in this study. The separation and determination

10

were performed using a liquid chromatography system equipped with a triple

11

quadrupole mass spectrometer and a Lux Cellulose-2 chiral column. The

12

enantioselective metabolism of rac-flufiprole was dramatically different in rat and

13

human liver microsomes in the presence of β-Nicotinamide adenine dinucleotide

14

phosphate regenerating system. The half-life (t1/2) of flufiprole in rat and human liver

15

microsomes were 7.22 and 21.00 min, respectively, for R-(+)-flufiprole, while the

16

value were 11.75 and 17.75 min, respectively, for S-(-)-flufiprole. In addition, the Vmax

17

of R-(+)-flufiprole was about 3-fold of S-(-)-flufiprole in rat liver microsomes, while

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its value in case of S-(-)-flufiprole was about 2-fold of R-(+)-flufiprole in human liver

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microsomes. The CLint of rac-flufiprole also showed opposite enantioselectivy in rat

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and human liver microsomes. The different compositions and contents of

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metabolizing enzyme in the two liver microsomes might be the reasons for the

22

difference in the metabolic behavior of the two enantiomers.

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KEYWORDS:

24

HPLC-MS/MS

flufiprole,

enantioselective

metabolism,

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liver

microsomes,

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INTRODUCTION

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Phenylpyrazole insecticides are widely used to protect a variety of crops, including

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rice, fruits and vegetables, from insects such as ants, beetles, ticks and cockroaches1-3.

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Another common phenylpyrazole insecticide, fipronil, has been banned in China in

29

2009, because of its high toxicity towards aquatic organisms, such as fish and

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shrimp4-6. Flufiprole (or butene-fipronil), (R,S)-1-[2,6-dichloro-4-(trifluoromethyl)

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phenyl]

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sulfinyl]-pyrazole-3-carbonitrile (Figure 1), is a new kind of phenylprazole insecticide

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introduced in 2002 by Dalian Raiser Pesticides Co., Ltd., China. It is a modified form

34

of fipronil with a similar effect against a broad spectrum of insect pests and has much

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lower toxicity7-9. Both flufiprole and fipronil have a chiral sulfur atom consisting of

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two enantiomers. In fact, although enantiomers have exactly the same physical and

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chemical properties, they behave differently in bioactivity10-11, toxicity10,12 or

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degradation10,13-14 when exposed to a chiral environment. In recent years, with the

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intensive study of chiral pesticides all over the world, many papers about the

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enantioselective degradation of chiral pesticides have been published15-18. For instance,

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S-fipronil has been shown to degrade faster than R-fipronil in earthworms and natural

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soil18. To our knowledge, there is no such report about the study on the metabolic

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behavior of flufiprole at the enantiomer level, which may lead to potential

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environmental risks.

-5-[N-(methallyl)

amino]-4-[(trifluoro-methyl)

45

Recently, it is popular to use in vitro models in pharmacology, toxicology and

46

toxicokinetic experiments, because there are fewer animals needed, fewer endogenous

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interfering substrates and much shorter experiment time compared with in vivo

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studies19-23. Liver is the major organ for the metabolism of many xenobiotics, where

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the majority of drug metabolic enzymes exist. In previous studies, many researchers

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have reported the stereoselective degradation of many chiral pesticides in rat, rabbit

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and human liver microsomes (HLMs), including triazole fungicides24-25, benalaxyl26

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and fluroxypyr methylheotyl ester27. Taking benalaxyl enantiomers as an example, the

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t1/2 of R-benalaxyl in rat liver microsomes (RLMs) was about 2-fold of S-benalaxyl.

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However, R-benalaxyl degraded faster than S-benalaxyl in rabbit liver microsomes, at

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the same time there was no chiral inversion between R- and S-benalaxyl in both liver

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microsomes26. To evaluate the environmental risk of flufiprole comprehensively, it is

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necessary to evaluate the enantioselective metabolism of flufiprole in different liver

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microsomes.

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In this study, the metabolic behaviors of rac-flufiprole in RLMs and HLMs were

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investigated to determine that if similar enantioselectivity occurred. The

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determination of chiral flufiprole was achieved in high-performance liquid

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chromatography-tandem mass spectrometry (HPLC-MS/MS) equipped with a Lux

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Cellulose-2 chiral column. The significant kinetic parameters of the enantiomers, such

64

as half-life (t1/2), maximum velocity of metabolism (Vmax) and Michaelis constant

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(CLint), were also calculated to explore the enantioselective behaviors of flufiprole.

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The results might be helpful to understand the risks associated with flufiprole to

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humans, animals and the environment.

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MATERIALS AND METHODS

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Chemicals and Materials. Flufiprole with a purity of 96.6% was obtained from

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Dalian Raiser Pesticides Co., Ltd (Dalian, China). Two enantiomers of flufiproles

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with purity ≥98.0% were obtained from Daicel (Shanghai, China). HPLC-grade

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methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). A stock

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solution of racemic standard was prepared in methanol and stored at 4 °C. Working

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standard solutions were obtained by dilutions of the stock solution in methanol.

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Purified water was produced by a Milli-Q water purification system (Millipore

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Bedford,

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regenerating system, rat and human liver microsomes (20 mg/mL, 0.5 mL) were

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purchased from XenoTech (Lenexa, KS). Acetic acid (99.7% purity) was purchased

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from Sigma-Aldrich. All other chemicals and solvents were of analytical grade and

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purchased from commercial sources. The chiral analytical column, cellulose tris

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(3-chloro-4methylphenycarbamate)

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Phenomenex (Torrance, CA), and the column was sized 150 mm × 2.0 mm i.d. and

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packed with 3 µm particles.

MA).

β-Nicotinamide

adenine

(Lux

dinucleotide

Cellulose-2),

phosphate

was

(NADPH)

purchased

from

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Incubation in Liver Microsomes. Substrate-depletion studies in vitro were

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performed by incubation of rac-flufiprole (10 µM) for rat liver microsomes and

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rac-flufiprole (5 µM) for human liver microsomes with 0.5 mg of microsomal protein

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in 50 mM Tris-HCl buffer (pH 7.4) with 5.0 mM MgCl2. The concentrations of

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flufiprole were chosen based on pre-experiments. Flufiprole was prepared in methanol

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and added to the incubation media with the final methanol concentration not

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exceeding 1.0% v/v. All reaction mixtures were preincubated in a heated water bath at

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37 °C for 5 min before initiation of the reaction by addition of NADPH at a final

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reaction concentration of 1.0 mM. Incubation mixtures without NADPH served as

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controls (with deactivated microsomes). The final total reaction volume was 0.5 mL.

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After incubation in a water bath (37 °C) for 5-90 min, the reactions were terminated

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by adding 0.5 mL of ice-cold methanol and the sample was vortexed for 5 min. After

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centrifugating at 4000 rpm for 5 min, the supernatant was filtered through the

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membrane (PTFE, 0.22 µm) for enatioselective HPLC-MS/MS analysis. All

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experiments were performed as three independent trials.

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Kinetic metabolism Assays. The in vitro metabolic kinetics of flufiprole was

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studied by adding variable concentrations of flufiprole stock solution to the incubation

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mixtures. The final substrate concentrations were from 2 to 100 µM and the

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incubation time was 10 min with liver microsomes (0.5 mg protein/mL). Then, the

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sample preparation was performed according to the section above. The Vmax and Km

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values were calculated from nonlinear regression analysis of experimental data

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according to the Michaelis-Menten equation. Intrinsic clearance (CLint) was calculated

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as a ratio of the Vmax and to the Km. Nonlinear regression analysis was performed with

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Origin pro 8.5.

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Enantioselective HPLC-MS/MS Analysis. The determination of flufiprole was

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achieved on a Surveyor liquid chromatography (Thermo Fisher Scientific, Waltham,

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MA, USA) equipped with a triple-quadrupole mass spectrometer. Xcalibur 2.0.7

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(Thermo Fisher Scientific) software was used to process the quantitative data obtained

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from calibration standards and samples. Complete chiral separation of flufiprole was

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achieved on a Lux Cellulose-2 chiral column28. The mobile phase consisted of 65%

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(v/v) (A) acetonitrile and 35% (v/v) (B) 2 mM ammonium acetate aqueous solution

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containing 0.1% formic acid. The flow rate was set to 0.3 mL/min, and the injection

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volume was 5 µL. The ESI-MS/MS interface (electrospray ionization coupled with

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tandem mass spectrometry) was operated in the negative ion mode. ESI source

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conditions were as follows: source temperature, 110 °C; desolvation temperature,

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500 °C; desolvation gas (N2) flow rate, 12 mL/min; cone gas (N2) flow rate, 40 mL/Hr;

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and collision gas (Ar) flow rate, 0.15 mL/min. Multiple reaction monitoring (MRM)

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was applied for flufiprole determination; transition m/z 488.9→288 was used for

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quantification, and m/z 488.9→249.9 was used for confirmation.

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Method validation. Calibration standards were prepared by adding a series of

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working standard solution of rac-flufiprole into the blank matrix (BM, the extracts

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from the bovine serum protein). The preparing and extracting processes were the same

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as incubation samples. The calibration curves were generated by plotting peak areas

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of quantification ion transition against the concentration of each enantiomer from 0.1

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to 50 µM with regression analysis. Linear regression analysis was performed with

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Microsoft Excel 2007. The precision and accuracy of the method were calculated by

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analyzing calibration standards at three concentration levels (0.5, 10 and 50 µM for

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each flufiprole enantiomer) and comparing the predicted concentration (obtained from

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the calibration curve) to the actual concentration of each enantiomer spiked in the BM.

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Both intraday and interday precisions are presented as relative standard deviation

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(RSD [SD/mean] × 100%). Limit of detection LOD (signal-to-noise ratio [S/N]=3)

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and limit of quantification LOQ (S/N=10) were determined with the method using the

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matrix-matched standards.

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RESULTS AND DISCUSSION

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Assay validation. Using m/z 488.9→288 as the quantification transition, linear

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calibration curves were obtained over the concentration range of 0.1-50 µM in blank

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matrix for R-flufiprole (y = 6.9227x × 10^4 - 29.9224, R2 = 0.9994) and S-flufiprole (y

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= 6.9938x × 10^4 – 14.3144, R2 = 0.9996). The RSD was less than 7% at all

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concentration.

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The precision and accuracy of intraday and interday, expressed with the RSD and

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the ratios of predicted concentration to acctural concentration, was shown in Table 1.

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For both enantiomers, the accuracies obtained were in the acceptable range of 90.3%-

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98.7% and the RSD ranged from 1.8% to 5.7%. The main recoveries ranged from

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97.5% to 100.8% with 2.4%-6.9% RSD (Table 2). The LODs for both enantiomers

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were estimated to be 0.005 µM, while the LOQs were 0.015 µM. In addition, the

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extraction procedure did not cause epimerize of flufiprole enantiomers as shown in

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Figure 2 F-G.

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Data Ananlysis. Under the HPLC-MS/MS condition, the enantiomers of flufiprole

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were separated completely on a Lux Cellulose-2 column (as shown in Figure 2A), and

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no endogenous interference peaks eluted at retention times in blank samples (without

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insecticides). The order elution was confirmed to be S-(-)-flufiprole for peak 1 and

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R-(+)-flufiprole for peak 2 based on published data28.

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The enantiomer fraction (EF) was used to measure the enantioselectivity of the

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flufiprole enantiomers during incubation in the liver microsomes. EF was calculated

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from the peak areas for the signals, defined by eq. 1. The EF values ranged from 0 to

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1 with EF=0.5 representing the racemic mixture according to the equation.

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EF= (S)-(-)-enantiomer / [(R)-(+)-enantiomer + (S)-(-)-enantiomer]

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The metabolism of rac-flufiprole and its enantiomers was assumed to follow the

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first-order kinetics model, and the degradation constants (k) were calculated by eq. 2

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using regression analysis. The half-life (t1/2, min) was estimated from eq. 3. C = C0e-kt

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t1/2 = ln2/k = 0.693/k

165 166 167

(1)

(2) (3)

C and C0 are the concentrations of the R-(+)- or S-(-)-enantiomer at time t and time 0, respectively.

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Nonlinear regression of substrate concentration versus reaction velocity curves

169

were analyzed using Origin 8.5 software by fitting the experimental data to the

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Michaelis-Menten equation. The in vitro kinetic parameters were determined by

171

fitting the data to eq. 4, and the intrinsic metabolic clearance (CLint) was calculated by

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eq. 5.

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V=Vmax×S/(Km+S)

(4)

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CLint=Vmax/Km

(5)

175 176

V, S, Vmax and Km represent the velocity of metabolism, substrate concentration, maximum velocity of metabolism and Michaelis constant, respectively.

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Metabolism of Racemic Flufiprole in RLMs and HLMs. The rac-flufiprole was

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dissipated rapidly in RLMs and HLMs with NADPH, and did not degradate obviously

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in the absence of NADPH. Typical chromatograms of extracts from the samples after

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10 min incubation are displayed in Figures 2B-E. The concentrations of two

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enantiomers were different from each other after incubation in RLMs and HLMs. The

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R-(+)-flufiprole degraded faster than its antipode in RLMs, in contrast, the

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S-(-)-flufiprole degraded faster in HLMs.

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Concentration-time curves of R-(+)- and S-(-)-flufiprole after incubation in RLMs

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with rac-flufiprole at 10 µM and HLMs with rac-flufiprole at 5 µM were shown in

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Figure 3. The results demonstrated that the metabolisms of each enantiomer in RLMs

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and HLMs were in accordance with the first-order kinetics decay model. Rate

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constants (k), correlation coefficients (R2) and half-lives (t1/2) were calculated (Table

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3). It was shown that the half-life t1/2 of R-(+)-flufiprole was 7.22 and 21 min in

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RLMs and HLMs, while the half-life t1/2 of S-(-)-flufiprole was 11.75 and 15.75 min,

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respectively. Thus, metabolism of R-(+)- and S-(-)-flufiprole was much faster in

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RLMs, and indicated opposite enantioselectivity in RLMs and HLMs.

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The enantioselective metabolic behavior could also be conducted by EFs. In the

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experiment, the EF value of flufiprole increased from the initial 0.50 to 0.71 rapidly in

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RLMs because R-(+)-flufiprole degradated much faster than S-(-)-flufiprole under the

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same concentration. Then with the concentration of R-(+)-flufiprole decreasing

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quickly, the degradation rate of R-(+)-flufiprole also decreased until it became smaller

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than its value of S-(-)-flufiprole. As a result, the EF value decreased to 0.62

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(concentration RS) gradually in HLMs, indicating that there

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was significant enantioselectivity in the two kinds of liver microsomes.

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Enzyme Kinetics of Flufiprole Enantiomers. The Michaelis-Menten model was

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applied to different initial concentrations (5-100 µM) of rac-flufiprole using RLMs

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and HLMs as examples to elucidate the mechanism of stereoselective toxicokinetics

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of flufiprole. Metabolic rate constants (apparent Km and Vmax) were determined after a

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10 min incubation period in liver microsomes. The Michalis-Menten plots are shown

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in Figure 4. By nonlinear regression analysis, the values of Km, Vmax and CLint were

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calculated and are shown in table 4. The Vmax of R-(+)-flufiprole (3906.3±80.6

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µM/min/mg) was about 3-fold of S-(-)-flufiprole (1430.6±52.9 µM/min/mg) after

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incubation for 10 min in RLMs, however, the opposite trendency was found after

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incubation in HLMs. For the CLint, the ability of an organism to eliminate a particular

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chemical demonstrated diversity, the CLint of rac-flufiprole also showed opposite

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enantioselectivy in RLMs and HLMs. Besides, the CLint of R-(+)-flufiprole and

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S-(-)-flufiprole in RLMs was higher than that in HLMs. These enzyme kinetic results

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suggested that the rat liver microsomes in vitro had a stronger potency to eliminate

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flufiprole than the human liver microsomes. Significant differences were found for the

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stereoselective metabolism of rac-flufiprole in RLMs and HLMs.

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In conclusion, we investigated the enantioselective metabolism of rac-flufiprole in

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RLMs and HLMs. The degradation velocity of flufiprole in HLMs was slower than

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that in RLMs. Moreover, there was significant opposite enantioselective behavior for

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the metabolism of flufiprole enantiomers in the two liver microsomes. Some

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studies24-27 verified that other fungicides, such as (2RS,3RS)-paclobutrazol,

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tebuconazole, benalaxyl and fluroxypyr methylheotyl ester, exhibited stereoselectivity

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of metabolism in liver microsomes. The reason for the difference in the metabolism

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behavior of the two enantiomers might be the different compositions and contents of

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metabolizing enzyme (especially cytochrome P450) in various species liver

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microsomes26, 29-30. Future studies should be focused on the isoforms of cytochrome

228

P-450 (CYP) that contribute to its stereoselectivy. These data should be important to

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elucidate the mechanism of stereoselective toxicokinetics of flufiprole in RLMs and

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HLMs, and contribute to environmental and public health.

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AUTHOR INFORMATION

232

Corresponding Author *

233

E-mail [email protected]

234

Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGEMENT

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We acknowledge financial support from the National Natural Science Foundation of

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China (21207118), the Project of Science and Technology Plan of Zhejiang Province,

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China (2014C37103), and the Natural Science Foundation of Zhejiang Province,

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China (Y15B070017).

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FIGURE CAPTIONS NC

CN Cl

F 3C

N N HN Cl H3C

O

O S CF3 CH2

S F3C

H2C

N N NH Cl

Cl

CF3

CH3

mirror Figure 1. Chemical structure of flufiprole enantiomers (Asterisk indicated the chiral center).

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Figure 2. Representative HPLC-MS/MS chromatograms of (A) standard flufiprole, extract from (B) RLMs with 10 µM rac-flufiprole and (C) HLMs with 5 µM rac-flufiprole after incubation for 10min in the absence of NADPH, extract from (D) RLMs with 10 µM rac-flufiprole and (E) HLMs with 5 µM rac-flufiprole after incubation for 10min with NADPH, extract from RLMs with (F-G) individual flufiprole enantiomer.

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Figure 3. Degradation of (A) rac-flufiprole (10 µM) in RLMs, (C) rac-flufiprole (5 µM) in HLMs for 90 min. The changes of EF values in (B) RLMs and (D) HLMs.

Figure 4. Michaelis-Menten kinetic analyses of rac-flufiprole (A) in RLMs and (B) in HLMs after 10min incubation.

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TABLE CAPTIONS Table 1. . Accuracy and Precision of Assay Method for Measurement of Flufiprole Enantiomers (n = 6) Intraday Concentration (µM) R-flufiprole 0.1 10 50 S-flufiprole 0.1 10 50

Interday

Accuracy (%)

RSD (%)

Accuracy (%)

RSD (%)

91.9 96.7 97.5

1.8 5.0 5.7

90.3 93.0 95.5

1.4 4.5 4.7

91.9 95.2 97.7

2.7 4.7 3.5

92.6 94.9 98.7

2.3 5.4 4.9

Table 2. . Method Recovery for Meassurement of Flufiprole Enantiomers (n = 3) R-flufiprole

S-flufiprole

Concentration (µM)

Accuracy (%)

RSD (%)

Accuracy (%)

RSD (%)

0.1 10 50

100.4 97.5 100.8

2.4 2.6 3.1

99.1 97.7 100.7

2.7 6.9 2.5

Table 3 . Degradation Rate Constant (k), Half-Life (t1/2) and Correlation Coefficients (R2) Values for the Metabolism of rac-Flufiprole in Liver Microsomes Liver microsomes

spiked compd

RLMs

rac-flufiprole

HLMs

rac-flufiprole

detected compd

k (min-1)

t1/2(min)

R2

R-(+)-flufiprole S-(-)-flufiprole R-(+)-flufiprole S-(-)-flufiprole

0.096 0.059 0.033 0.044

7.22 11.75 21.00 15.75

0.885 0.889 0.991 0.915

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Table 4. . Metabolic Parameters of rac-Flufiprole Incubated in RLMs and HLMs Sample RLMs R-(+)-flufiprole S-(-)-flufiprole HLMs R-(+)-flufiprole S-(-)-flufiprole

Vmax (µM/min/mg)

Km (µM)

CLint (mL/min/mg protein)

R2

3906.3±80.6 1430.6±52.9

43.12±1.53 17.22±0.84

90.58 83.06

0.99 0.99

961.5±52.7 1785.7±86.3

27.12±2.49 35.18±2.34

35.45 50.76

0.98 0.99

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ABSTRACT GRAPHICS

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