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Etoxazole is Metabolized Enantioselectively in Liver Microsomes of Rat and Human in Vitro Zhoulin Yao, Mingrong Qian, Hu Zhang, Jing Nie, Jingqing Ye, and Zu Guang Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02676 • Publication Date (Web): 01 Aug 2016 Downloaded from http://pubs.acs.org on August 13, 2016
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Etoxazole is Metabolized Enantioselectively in Liver Microsomes of Rat and Human in Vitro Zhoulin Yao†,‡,§, Mingrong Qian§, Hu Zhang§, Jing Nie†, Jingqing Ye†, Zuguang Li*,†
†
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou
310014, China ‡
Zhejiang Citrus Research Institute，Zhejiang Academy of Agricultural Sciences;
Taizhou 318020，China §
Institute of Quality and Standard for Agricultural Products, Zhejiang Academy of
Agricultural Sciences; Key Laboratory of Detection for Pesticide Residues and Control of Zhejiang Province; State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control; MOA Key Laboratory for Pesticide Residue Detection, Hangzhou 310021, China
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Acaricide etoxazole which belongs to
the ovicides/miticides
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ABSTRACT:
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diphenyloxazole class, affecting adults to lay sterile eggs by inhibiting chitin
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biosynthesis possibly. The reverse-phase HPLC-MS/MS method was used to
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determine the etoxazole enantiomers. The enantioselective degradation behavior
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of rac – etoxazole in liver microsomes of rat and human in vitro with NADPH was
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dramatically different. The t1/2 of (R) - etoxazole was 15.23 min in rat liver
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microsomes and 30.54 min in human liver microsomes, while 21.73 min and 23.50
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min were obtained for (S) - etoxazole, respectively. The Vmax of (R) - etoxazole was
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almost 5 folds of (S) - etoxazole in liver microsomes of rat in vitro. However, the
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Vmax of (S) - etoxazole was almost 2 folds of (R) - etoxazole in liver microsomes of
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human in vitro. The CLint of etoxazole was also shown the enantioselectivity on the
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contrary in liver microsomes of rat and human. These results indicated that the
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metabolism of two etoxazole enantiomers was selective in liver microsomes of rat
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and human in vitro, and enantioselectivity in the two kinds of liver microsomes was
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in difference degradation performance. The reason might be related to the
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composition and content involved in enzyme system.
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KEYWORDS: etoxazole, liver microsomes, enantioselective degradation, enzyme
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Kinetics
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INTRODUCTION
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Acaricide etoxazole which belongs to the ovicides/miticides diphenyloxazole class,
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affecting adults to lay sterile eggs by inhibiting chitin biosynthesis possibly. It is
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effective against the nymphs, eggs and larvae of spider mites but safety to mature.
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Tetranychid spider mites like Tetranychus ssp. and Panonychus ssp. are the main
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pests target of etoxazole. Etoxazole was launched in 1998, and registered on citrus
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since 2012 in China, for its high effectiveness. 2
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Figure 1 shows the chemical structure of etoxazole, which has two enantiomers
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for its asymmetrically substituted carbon. Enantiomers have the chemical and
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physical properties of the exactly same. When exposed to the chiral environment,
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optical isomers may behave different in activities,3, 4 toxicology5 or degradations.6, 7
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Then the one of the isomers might be toxic or inactive.8, 9 Metabolizing enzymes
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exhibiting enantioselectivity in the different biological models in the environment,
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because they often show the preference for one of the enantiomers.10,
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structural characteristics of the enzymes were related to the enantiomeric
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metabolism of chiral pesticides.12
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The
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The studies of the drugs in vitro were widely used in pharmacology, toxicology
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and toxicokinetic. Comparing to the studies in vivo, in vitro experiment studies need
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fewer animals, less experiment time, and have fewer endogenous interfering
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substrates.13-17 Etoxazole was tested for geno toxicity in vivo and in vitro in many
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studies. Some negative results were obtained from a range of researches. But a
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positive response was obtained at cytotoxic doses in a study with human
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lymphocytes. Metabolism activation on the mouse lymphoma, a weak positive
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reaction was obtained at closing to the cytotoxic dose of etoxazole. Without any
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metabolic activation, the result was inconclusive.18 But the difference between two
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etoxazole isomers on degradation activities and toxicology has not been reported up
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to present. The investigations into the pattern of metabolite, kinetics, intermediate
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formation, and pesticide metabolic pathways, formed into the main studies of
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metabolism in liver microsomes of rat and human in vitro. 19-21Recently, papers have
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been published on the stereoselective degradation in liver microsomes of rabbit,
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human and rat in vitro of many chiral pesticides, including metalaxyl,22
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flucetosulfuron,23 paclobutrazol24 and so on. Taking two enantiomers of (2R, 3R) –
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paclobutrazol and (2S, 3S) – paclobutrazol as an example, the t1/2 of paclobutrazol in
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liver microsomes of rat were 18.60 min and 10.93 min, respectively.24 The results
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displayed that stereoselective degradation occurred in liver microsomes of rat in
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vitro, and the rate of (2R, 3R) – paclobutrazol was slower than the other
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paclobutrazol isomer. In this study, enantioselective metabolization and degradation
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of etoxazole were investigated into liver microsomes of rat and human in vitro.
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EXPERIMENTAL SECTION
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Chemicals and Reagents. The ≥98.7% purity of racemic etoxazole (CAS:
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153233-91-1) standard, which was bought from Sigma-Aldrich (St. Louis, USA).
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Chromatographic purity acetonitrile (CH3CN) and methanol (CH3OH), Merck
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(Darmstadt, Germany). The ≥96% purity of formic acid, TEDIA (Fairfield, USA). The
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regenerating system of β-nicotinamide adenine dinucleotide phosphate (NADPH),
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liver microsomes of rat (0.5 mL, 20 mg/mL) and liver microsomes of human (0.5 mL,
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20 mg/mL), were bought from XenoTech (Lenexa, KS). Ultrapure water, preparation
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of Milli-Q water purification system (Billerica, USA). The other analytical reagents,
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which were obtained from commercial sources.
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Dissolving the etoxazole standard in acetonitrile to prepare the approximately
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100 mg/L stock standard. Serial dissolving the etoxazole stock standard to obtain
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the lower concentrations standards. All standard solutions in dark-brown glass
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bottles were stored at -20°C. There was no degradation for 3 months of the
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standard solutions.
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Apparatus and Chromatographic Conditions. The ESI-MS/MS experiments of
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etoxazole were carried out on a Bruker Daltonics micrOTOF QII (Q-TOF) mass
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spectrometer (Billerica, MA, USA), operated in the positive-ion mode. Diluted
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solutions (about 10 μg/mL) were prepared by dissolving the samples in a mixed
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solvent of methanol. The diluted sample was directly infused into the electrospray
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ionization source of the mass spectrometer with a syringe pump at a flow rate of
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180 μL/h. The parameters of Q-TOF mass spectrometers were set as follows: drying
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gas flow in the ion source was set up to 2 L/h and nebulizer gas pressure was set up
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to 0.4×105 Pa (both were N2), the temperature of ion source was 200 °C, and voltage
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was −4.5 kV. Tandem mass spectra were obtained by collision-induced dissociation
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(CID) of the ion of interest, with argon gas as the collision gas at the mass width of 2
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Da. The collision energy was set at 20 eV for all precursor ions. The instrument was
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operated at a resolution higher than 15,000 full widths at half maximum using the
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micrOTOF-Q control program ver. 2.3. Data processing was performed using the
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Data Analysis ver. 4 software package delivered by Bruker Daltonics. The molecular
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fragments do the transitions m/z 360>141 and m/z 360>177 refer to were shown in
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Figure 2.
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The conditions of chromatography and mass spectrometry of etoxazole, referring
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to our previous research work.25 The enantioseparation of etoxazole was performed
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on a Chiralpak AD-3R column at 30°C using acetonitrile with 0.1 % formic acid
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solution (80/20; v/v) on a reverse-phase liquid chromatography–tandem mass
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spectrometry system. The first eluted enantiomer was (S)-etoxazole. The injection
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volume was 5 μl. The flow rate was set at 0.3 mL/min.
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Method Validation. The validation procedure was carried out through the
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following parameters: linearity, matrix effect, accuracy and precision, limit of
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quantification (LOQ), and limit of detection (LOD). The linearity was evaluated on
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etoxazole standard in range of 0.1–20 μM, using the matrixes of solvent acetonitrile,
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blank liver microsomes of rat and human. The least-squares linear regression
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analysis was used to construct the (R)-and (S)-etoxazole calibration curves
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(0.05–10μM) of nine points. The regression analysis was performed on the analyte
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concentrations of the quantification ion peak areas. The method accuracy was
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evaluated on each enantiomer comparing the actual concentration to the calculated
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concentrain from matrix-matched calibration curve. The accuracy is generally
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expressed by the recovery rate. Three etoxazole enantiomers concentration levels of
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0.05, 1, and 10 μM with six spiked samples were evaluated of the intraday accuracy.
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Three earlier concentration levels with six spiked samples on three different days
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were tested of the interday accuracy. RSD (relative standard deviation) was used to
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assess the precisions of intraday and interday. LOD (limit of detection) was defined
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as 3 times of the signal-to-noise (S/N). LOQ (limit of quantification) was S/N = 10.
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Both LOD and LOQ used matrix-matched standards.
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Metabolism of Etoxazole in Liver Microsomes of Rat and Human in Vitro.
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Substrate-depletion studies were performed on incubation the racemate etoxazole
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(5 μM) in liver microsomes of rat and human in vitro. The experimental condition
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was 0.5 mg microsomal protein in the buffer (pH 7.4) of 5.0 mM MgCl2-50 mM
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Tris-HCl. Etoxazole in acetonitrile added into the incubation media, and the
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acetonitrile in final system concentration was less than 1.0% v/v. The mixtures of
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the reaction were pre-incubated in 37°C water, bathing for 5 minutes. Then NADPH
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was added in the pre-incubate system to initiate the reaction, with the volume of
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0.5 mL and concentration of 1.0 mM in the final reaction system. The reaction was
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ended by adding ice-cold methanol of 0.5 mL, after incubating for 5–90 minutes.
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Samples were centrifuged for 5 minutes at 4000 rpm, after vortex of 5 minutes. The
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supernatant of centrifugation was filtered into the 0.22 μm membrane filter prior to
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the analysis of HPLC-MS/MS.
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Enzyme Kinetic Assays. Different concentrations of etoxazole were added into
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the incubation system with liver microsomes (1 mg protein / mL) for 10 minutes to
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estimate the kinetic parameters. The final concentrations of etoxazole were 1–100
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μM. Michaelis constant Km is the characteristic constant of the enzyme. It is equal to
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the substrate concentration at half of the maximum metabolic reaction velocity. The
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values of Vmax and Km were base on the nonlinear regression analysis of
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Michaelis–Menten equation, V= Vmax×S / (Km + S). Kinetic parameters of V, Vmax and
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S were respectively represented the velocity of metabolism, maximum velocity of
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metabolism and substrate concentration. CLint (the intrinsic clearance) was defined
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as Vmax /Km. It shows the different ability of eliminating the particular chemical of
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the organism.
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Assay Validation. Figure 3 shows the typical chromatograms of (S) - and (R) -
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etoxazole extracted from liver microsomes of rat and human samples. (S) - etoxazole
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and (R) - etoxazole were baseline separated from each other, with the retention
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time of 2.53 min and 3.41 min respectively in blank liver microsomes samples.
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There was not any endogenous interference peak eluted.
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The stock solution stability test. The standard solution (approximately 100 mg/L)
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was stored at -20°C in dark, and was observed at 7, 14 days, 1, 2, 3 months which
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were determined in parallel. The results show that the RSD
