Evaluation of the Interaction between Pesticides and a Cell Membrane

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Evaluation of Interaction Between Pesticides and a Cell Membrane Model by Surface Plasmon Resonance Spectroscopy Analysis Hiroshi Moriwaki, Kotaro Yamada, and Hiromitsu Nakanishi J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 12, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Evaluation of Interaction Between Pesticides and a

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Cell Membrane Model by Surface Plasmon Resonance

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Spectroscopy Analysis

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Hiroshi Moriwaki*1,2, Kotaro Yamada1, Hiromitsu Nakanishi3

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Biology, 3-15-1, Tokida, Ueda 386-8567, Japan

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Supports to Advanced Science, 3-15-1, Tokida, Ueda 386-8567, Japan.

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Shinshu University, Faculty of Textile Science and Technology, Division of Applied

Shinshu University, Division of Instrumental Analysis (Ueda branch), Research Center for

Satellite Venture Business Laboratory, Shinshu University, 3-15-1 Tokida, Ueda,

Nagano 386-8567, Japan

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KEYWORDS: surface plasmon resonance, pesticide, interaction, cell membrane, liposome

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Abstract

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A surface plasmon resonance spectroscopy analysis (SPR) was used for the characterization of the

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interaction between pesticides and a cell membrane model. A liposome was immobilized onto the

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surface of the SPR sensor chip (L1), and the lipid bilayer membrane formed on the sensor chip was

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regarded as the cell membrane model. The solution containing a pesticide was flowed onto the sensor

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chip, and an SPR sensorgram, which reflected the interaction between the pesticide and the lipid

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bilayer membrane, was obtained. As the results, the pattern and strength of the interaction of the

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pesticides with the cell membrane model were visualized and quantified. Triflumizole, hexythiazox,

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and pentachlorophenol showed a strong interaction with the lipid bilayer. It is well known that

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triflumizole and pentachlorophenol interact with the membrane, and reveal toxicities for cell

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membranes. In addition, there was a tendency for higher residual ratios to be observed when the no

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observable adverse effect level (NOAEL) values for chronic toxicity (1-year toxicity study in dogs)

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were lower. We suggest that a novel parameter for the evaluation or presumption of the behaviors

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and chronic toxicities of pesticides is obtained by the presented method.

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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Pesticides have been widely used all over the world, and diffuse into the environment. In fact, they have

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been detected from various environmental media, such as river water,

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organisms, 3 and the human body. 4 The exposure of pesticides can induce adverse effects on living

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creatures. Therefore, it is very important to obtain information about their behavior in the body. Currently,

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there are many reports concerning the accumulation of pesticides in body organs and tissues. 5,6 However,

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there is little information about the behaviors of pesticides at the surface of cells on a biomolecular level.

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The log Kow values have been extensively used for the evaluation of the bioaccumulation of pesticides.7

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However, this parameter only reflects the hydrophobic nature of the compounds. The log Kow values are

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not suitable to consider the intravital behavior of the pesticides containing polar functional groups8,

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because they can interact with biomolecules in the body by hydrogen bonding or a Coulomb interaction.

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ground water,

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biological

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Surface plasmon resonance spectroscopy analysis (SPR) is a technique to visualize the interaction

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between a compound on a sensor chip and chemicals in the mobile phase in real time. SPR is a rapid and

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simple method to observe the interaction of the target compound with the surface of the sensor chip. The

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technique can sensitively detect the interactions, and have been applied to the analysis of pesticides or

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biomolecules using antigen-antibody complex reactions. 9, 10

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Miyano et al. reported the SPR method using sensor-chip-immobilized liposomes as a taste sensor.

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11,12

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sensor-chip-immobilized liposome was also applied to the characterization of the interactions between

The sensor-chip-immobilized liposome was the membrane model of the lingual cells. In addition, the

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liposomes and drugs, such as naptoxen, ketoprofen, pindolol, suprofen, etc., in the field of pharmaceutical

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research.13 Furthermore, the binding of air particles to phospholipid vesicles immobilized on an SPR

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sensor chip, which was a membrane model of epithelial cells in respiration organs, was measured in order

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to evaluate and characterize air particles.14

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In this study, we suggested a novel method for the evaluation of pesticides using SPR analysis. A

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liposome was immobilized onto the SPR L1 sensor chip, and a solution containing a pesticide was flowed

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onto the surface of the sensor chip (Fig. 1). The obtained SPR sensorgram was used for the evaluation of

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the interaction between the pesticide and the cell membrane model. It is expected that the patterns and

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strength of the interactions of the pesticide with the cell membrane could be obtained by this method. The

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obtained data would be useful for estimating the behavior and toxicities of pesticides. Seventeen pesticides

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were selected and analyzed for characterization of their interaction with the cell membrane model.

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EXPERIMENTAL SECTION

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Materials. Sodium chloride, sodium hydroxide, chloroform, methanol, acephate, acetamiprid, atrazine,

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carbofuran, clofentezine, 2,4-D, difolatan, hexythiazox, imidacloprid, methamidophos, methomyl, paraquat,

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pentachlorophenol sodium salt, 2,4,5-T, thiamethoxam, triclopyr, triflumizole, perfluoroocanoic acid

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(PFOA), and perfluorooctanesulfonic acid sodium salt (PFOS) were purchased from Wako Pure Chemicals

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(Osaka,

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n-octyl-β-D-glucoside were obtained from Dojin Molecular Technologies, Inc. (Kumamoto, Japan). DOPC

Japan).

HEPES

(2-[4-(2-hydroxyethyl)-1-piperazinyl]

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ethanesulfonic

acid)

and

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(1,2-dioleoyl-sn-glycero-3-phosphocholine) was from Sigma-Aldrich (St. Louis, MO, USA). The SPR

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reagents for washing the SPR system, BIA desorb solution1and BIA desorb solution 2, were purchased

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from GE Healthcare (Buckingham shire, UK). The 0.1% bovine serum albumin (BSA) solution was from

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Takara Bio, Inc. (Shiga, Japan). Pure water was prepared by an automatic water distillation apparatus (MQ

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academic A10, Millipore, Billerica, MA, USA). HBS-N was used as the running buffer.

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Immobilization of DOPC onto the SPR sensor chip. The immobilization method of DOPC onto

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the sensor chip L1 was similar to that in references 10. DOPC was suspended in the HBS-N running buffer.

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The DOPC suspension (10 mM) was frozen, thawed, and vortexed five times to completely agitate it. The

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obtained DOPC suspension was then extruded 25 times through a 50-nm polycarbonate filter using a

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Mini-Extruder (Avanti Polar Lipid, Alabaster, AL, USA), and the liposomes were formed.

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The L1 sensor chip (GE Healthcare) was placed in the Biacore X system (GE Healthcare) and washed

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with 40 mM n-octyl-β-D-glucopyranoside at the flow rate of 10 µL min-1 for 10 min. The liposome was

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diluted in the HBS-N running buffer to a concentration of 0.5 mM. The suspension was added to the sensor

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chip at a flow rate of 2.0 µL min-1 to generate a lipid bilayer on the chip until achieving the resonance unit

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value (RU) at 6500. Erb et al. reported that a stable lipid bilayer was generated on the surfaces of the L1

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sensor chip by adding the liposome suspension.15 The liposome was fused, and homogeneously covered

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the surface of the L1 sensor chip. The phenomenon was confirmed by atomic force microscopy and

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fluorescence microscopy.15 The surface of the sensor chip was washed with 50 mM NaOH at 2.0 µL min-1

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for 5 min, then 100 µg mL-1 of BSA was flowed over the sensor chip at 2.0 µL min-1. It was reported that

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BSA binds strongly to the dextran matrix of the L1 sensor chip, but binds weakly to the liposome, and the

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increased signal by an injection of BSA onto the sensor chip immobilized the liposome was low (37± 25

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RU).15 Therefore, it was regarded that the stabilization of the liposome onto the sensor chip was achieved

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when the increased RU by the addition of BSA was < 100. The analysis methods mentioned below were

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used when the increased RU was below 100.

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SPR analysis. The methanol/ HEPES buffer (1/99; v/v) solutions containing a pesticide (62.5, 125, 250,

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500, or 1000 µM) were prepared. The SPR signals of the pesticides at 62.5 µM were enough high to detect

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(signal / noise (at 50-100 sec) > 10). In addition, higher the concentration of the solution (62.5-1000 µM)

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were, the higher the RU values were. The result indicated that the adsorption sites on the sensor chip were

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not fully saturated at these concentrations. Based on these results, the concentration of the solution was set

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at 62.5, 125, 250, 500, or 1000 µM. It was reported that the addition of methanol to the analyte solution

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(