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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
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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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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(
