Comparison of the Metabolic Behaviors of Six Systemic Insecticides in

Jul 22, 2018 - in a Newly Established Cell Suspension Culture Derived from Tea ... State Key Laboratory of Tea Plant Biology and Utilization, School o...
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Comparison of the Metabolic Behaviors of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia sinensis L.) Leaves Weiting Jiao,†,⊥,‡,# Yizheng Hu,†,⊥,# Guoqin Ge,†,⊥ Jianchao Li,†,⊥ Yu Xiao,†,⊥,§ Huimei Cai,†,⊥ Lili He,∥ Rimao Hua,‡ Jun Sun,*,†,⊥ and Ruyan Hou*,†,⊥

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State Key Laboratory of Tea Plant Biology and Utilization, School of Tea and Food Science & Technology, Anhui Agricultural University, Hefei 230036, P. R. China ⊥ Anhui Province Key Lab of Analysis and Detection for Food Safety, Hefei 230022, P. R. China ‡ School of Resource & Environment of Anhui Agricultural University, Key Laboratory of Agri-food Safety of Anhui Province, Hefei 230036, P. R. China § Anhui Entry-Exit Inspection and Quarantine Bureau of the P. R. China, Hefei 230022, P. R. China ∥ Department of Food Science, University of Massachusetts, Amherst, Massachusetts 01003, United States S Supporting Information *

ABSTRACT: The use of an in vitro cell suspension to study insecticide metabolism is a simpler strategy compared to using intact plants, especially for a difficult matrix such as tea. In this study, a sterile tea leaf callus was inoculated into B5 liquid media with 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L−1) and Kinetin (KT, 0.1 mg L−1). After 3−4 subcultures (28 days each), a good cell suspension was established. Utilizing these cultures, the metabolic behaviors of six insecticides, including two organophosphates (dimethoate, omethoate) and four neonicotinoids (thiamethoxam, imidacloprid, acetamiprid, and imidaclothiz) were compared. The results showed that thiamethoxam, dimethoate, and omethoate were easily metabolized by tea cells, with degradation ratios after 75 days of 55.3%, 90.4%, and 100%, respectively. Seven metabolites of thiamethoxan and two metabolites of dimethoate were found in treated cell cultures using mass-spectrometry, compared to only two metabolites for thiamethoxam and one for dimethoate in treated intact plants. KEYWORDS: tea cell suspension, insecticide, plant metabolism, mass spectrometry

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INTRODUCTION Pesticides used in food production can remain as residues (the parent compound or its degradation products) in plant parts from either direct application, soil uptake, or atmospheric drift.1−3 Studies on the metabolism of pesticides by the crop plant are important for predicting the degradation behavior of the parent pesticide, determining the nature and extent of its metabolites, and assessing any potential human, animal, and environmental hazards.4,5 The manner and rate of metabolism of a pesticide chemical vary with the plant species and characteristics of the target organism.6,7 Tea is one of the most highly consumed drinks throughout the world, second only to water.8,9 Tea is made from leaves of the tea tree (Camellia sinensis L.), which is an evergreen, perennial, woody plant.10 Neonicotinoid and organophosphorus insecticides are often used as systemic insecticides11 to protect tea plants from pests such as leaf hoppers, aphids, whiteflies, and some lepidopteran species.1,12,13 These systemic insecticides may encounter metabolic enzymes during their distribution within the plant, by which they can be transformed through oxidation, hydrolysis, or reduction reactions. These metabolic reactions of Phase II generally yield products that are more polar and less toxic products than the parent compound. However, the bioactivation of compounds is common for those that have only undergone the Phase I © XXXX American Chemical Society

reactions, for example, some organophosphates, such as dimethoate is metabolized into the more toxic omethoate,14,15 and acephate into methamidophos.16,17 Plant metabolic studies are thus important in determining the lifetime and fate of a pesticide.18 Plant cell suspension cultures can be used to investigate the plant metabolism of xenobiotics. Assays can be performed in the absence of interfering microbial activity and abiotic transformations since cell suspensions are largely homogeneous concerning morphological and physiological development. Cell suspensions can provide consistent materials for metabolic studies, independent of seasonal and geographical variations. Suspended cells absorb and metabolize xenobiotics more rapidly than intact plants. Additionally, the metabolites can be isolated easily with less matrix interference from cell suspension compared to from intact plants. It has been shown that the metabolism of xenobiotics in plant tissue cultures is qualitatively similar to that in vivo.19 Cell suspension cultures can also be easily used to investigate related compounds within a series, as well as new classes of molecules.20 Plant tissue Received: May 12, 2018 Revised: July 22, 2018 Accepted: July 25, 2018

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DOI: 10.1021/acs.jafc.8b02417 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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directly in the laboratory with a Milli-Q water purification system (Millipore, Bedford, MA). Optimization of Culture Conditions for Induction of Tea Callus. Concentration of Plant Hormones for Tea Callus Induction. The sterile leaf explants36 were inoculated on MS basal medium containing 2,4-D (1.0 mg L−1) and different concentrations of KT (0.05, 0.1, or 0.5 mg L−1). Each media was inoculated with 60 explants (12 explants per flask with five replications). The growth rate, color, and texture of the callus were recorded within 28 days to optimize the appropriate concentration of KT. After optimizing the appropriate KT concentration, 60 explants (12 explants per flask with five replications) were similarly inoculated onto media containing different concentrations of 2,4-D (0.02, 0.5, 1, 2, or 3 mg L−1). The appropriate concentration of 2,4-D was finally selected according to the growth rate, color, and texture of the callus within 28 days. The growth rate of callus was calculated by the following equation:37

cultures have been used to investigate pesticide metabolism, with the identified metabolites matching those metabolized and extracted from intact plants.21−25 Ultraperformance liquid chromatography tandem mass spectrometry is now commonly used for identification and/ or quantification of trace levels of pesticide residues due to the precision, robustness, and high sensitivity of the technology.26,27 Despite these advantages, the matrix effect is a severe phenomenon in liquid chromatography−mass spectrometry (LC−MS) analysis,28,29 especially for dirty or difficult matrices such as tea extract, which contains large quantities of polar polyphenols, phenolic acids, pigments, and flavanols. Although there have been many studies conducted on the metabolism of pesticides in a variety of plants, there are currently few reports identifying the trace levels of pesticides and their polar breakdown products in tea plants using in vivo or in vitro methods.30−33 There is no reported systemic comparison of the metabolic behavior of different classes of pesticides in tea leaves. Therefore, there are many knowledge gaps regarding both the interaction between pesticides and the tea plant and the metabolism of different pesticides in the tea plant. This study describes the establishment of cell suspension cultures from a friable callus, which was obtained from tea leaf (Camellia sinensis L.). The optimized culture conditions were then used in tissue culture experiments to compare the dissipation behaviors of two organophosphates (dimethoate and omethoate) and four neonicotinoid (thiamethoxam, imidacloprid, acetamiprid, and imidaclothiz) pesticides, as well as the formation and identification of their metabolites. This is the first comparative study of the metabolism of different pesticides in tea using a cell suspension culture technique. Several pesticide metabolites were identified, and some of them were further investigated in intact tea plants. This new method could serve as a model for pesticide metabolic studies in tea or other plants.

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growth rate (%) = [(fresh weight of callus after inoculation − fresh weight of callus before inoculation) /fresh weight of callus before inoculation] × 100

Tea Callus Subculture Cycle Number and Length. Twelve pieces of calluses were inoculated on the MS basal medium containing 2,4-D (1.0 mg L−1) and KT (0.1 mg L−1). Four different subculture cycles of 21, 28, 35, and 42 days were tested for effect on the callus color, texture, and growth of leaf-derived callus. Once the subculture cycle of 28 days was chosen, the callus color and the rate of callus growth and browning were observed after four different subculture cycles (2, 3, 4, or 5 subcultures). The optimal number of subculture cycles and the optimal number of days of each subculture cycle were selected through experiments containing ten replications. Establishment of the Tea Cell Suspension System. Vigorous, friable, and loose calluses from the solid medium were cut into small pieces using a sterile surgical blade under sterile conditions. Small pieces of callus were weighed (about 3 g) and inoculated into B5 liquid media containing 2,4-D (1.0 mg L−1) and KT (0.1 mg L−1) to be in a liquid suspension culture at a constant temperature (25 ± 1 °C) and shaking incubator speed (120 r min−1) in the dark. After 7 to 10 days of culturing, the precipitated, large calluses were removed. The suspension liquid was taken as seed material for subculture in fresh medium. The subculture ratio was 15 g of mother liquid: 40 mL of total volume of culture liquid (v/v) in a 150 mL flask. After 3−4 subculture cycles of 28 d each, the final well-grown cell suspension culture was obtained. All the above procedures were conducted under sterile condition. Metabolic Behavior of Organophosphorus and Neonicotinoid Insecticides in Tea Cell Suspension Cultures and Intact Tea Plant. An aliquot of 400 μL of stock solution (500 μg mL−1) of four neonicotinoid insecticides (thiamethoxam, imidacloprid, acetamiprid, and imidaclothiz) or two organophosphorus insecticides (dimethoate and omethoate) was added into the vigorous cell suspension cultures derived from the same mother batch, respectively. The cell suspensions with insecticides were cultured at constant temperature (25 ± 1 °C) and shaking incubator speed (120 r/min). Samples were taken on different days between 0 to 75 d. Samples from cultures containing a neonicotinoid insecticide were taken as a 1 mL aliquot of the homogeneous cell culture and placed into a 1.5 mL plastic centrifuge tubes, which were centrifuged at 4000 rpm for 2 min. The supernatants were passed through a 0.22 μm poresize filter membrane before analysis by HPLC-UV (high-performance liquid chromatography paired with ultraviolet detector) and UPLCQTOF (ultrahigh-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer). Samples from cultures containing an organophosphorus insecticide were taken as a 500 μL aliquot of the cell suspension culture and placed into a 35 mL centrifuge tube or a 1.5 mL plastic centrifuge tube (the latter of which was prepared as above). For the 500 μL samples placed into the 35 mL centrifuge tube, 0.1 g of sodium

MATERIALS AND METHODS

Chemicals and Reagents. Half-strength MS basal medium34 containing 0.7% (w/v) agar (Agar Strip; Mingfu; Fujian, China) and 30 g L−1 sucrose was used in callus induction experiments; B5 basal medium (Gamborg B5 medium)35 with 20 g L−1 sucrose was used in cell suspension culture experiments. 2,4-Dichlorophenoxyacetic acid (2,4-D, >98.0%, Biosharp, Guangzhou Saiguo Biotech Co., Ltd., China) and Kinetin (KT, 6-furfurylaminopurine, >98.0%, Solarbio Science and Technology Co., Ltd. Beijing, China) were prepared for a 2.0 mg mL−1 hormone stock solution by weighing 20 mg of each into a 10 mL of volumetric flask and first dissolving in ethanol, then filling to a volume of 10 mL with deionized water, respectively. The pH of the two kinds of media with added hormones was adjusted to 5.8 prior to autoclaving (121 °C, 20 min). Cell cultivation instruments, including tweezers, surgical blades, scissors, and glass dishes, also underwent autoclaving. Triphenyltetrazolium chloride (TTC, 98.0%) was from Beijing Solarbio Science and Technology Co., Ltd., China. Optical microscope (Y-TV55, Nikon, Japan) with photographic system (DS-Ri2, Nikon, Japan). Thiamethoxam (99.8%), clothianidin (99.8%), imidacloprid (99.8%), acetamiprid (99.8%), omethoate (98.5%), and dimethoate (98.5%) were supplied by Dr. Ehrenstorfer (Augsburg, Germany); Imidaclothiz (purity 99.5%) was obtained from the Toronto Research Chemical Inc. (Canada). Standard stock solutions (500 mg L−1) of each insecticide were prepared by weighing 5 mg of each analyte, which was dissolved into 10 mL of acetonitrile (ACN) before storage at −20 °C. Polyvinylpolypyrrolidone (PVPP) was purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). ACN, hypergrade (99.9%) for LC−MS, was purchased from Tedia Company, OH, USA. Water used for LC−MS/MS was produced B

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Figure 1. Growth rate (A) and growth status (C) of tea-leaf derived callus under different KT concentrations and 1 mg/L 2,4-D;. Growth rate (B) and growth status (D) of callus under different 2,4-D concentrations and 0.5 mg/L KT. UPLC-QTOF Analysis of Cell Culture. The UPLC system consisted of an Agilent mode 1290, column compartment, and autosampler equipped with a Waters HSS T3 C18 column (100 mm × 2.1 mm × 1.8 μm). The elution for neonicotinoid insecticides began with 85% mobile phase A (5 mM ammonium formate water) and 15% mobile phase B (acetonitrile). Over 10 min, the mobile phase increased to 38% B at a constant rate and returned to the starting condition over 1 min, which was then held for 9 min. The elution of organophosphorus insecticides began with 55% A (water/ 0.1% formic acid) and 45% B (acetonitrile). Over 5 min, the mobile phase increased to 70% B, then returned to the 45% B over 0.5 min, which was held for 2.5 min. The flow rate was 0.2 mL min−1. The injection volume was 10 μL. The UPLC system was connected to an Agilent model 6545 ultrahigh-definition QTOF equipped with a Dual AJS ESI Source, operating in positive ion mode. The QTOF operation parameters were drying gas (nitrogen), 10 L min−1; gas temperature, 325 °C; sheath gas flow, 11 L min−1; sheath gas temperature, 350 °C; capillary voltage, 4000 V; nozzle voltage, 1000 V; nebulizer pressure, 35 psi; fragmentor voltage, 110 V for organophosphorus insecticides or 100 V for neonicotinoid insecticides; skimmer voltage, 65 V; octupole RF, 750 V. The instrument was operated in full scan spectrum mode and target MS/MS. Accuracy and stability of the mass spectrometer were daily checked, and the instrument was recalibrated when the value of corrected residuals was above 2 -ppm error. The data was processed with accurate mass tools, including Database Searching (MassHunter Pesticide PCDL B. 07.01) and Mass Hunter software (B. 07. 00), from which the metabolites with no standard products were inferred from the MS/MS studies as well as the literature.18,24,41−43 Q-Exactive Analysis of Intact Plant Extract. The Ultimate 3000 UPLC system was connected to a Q-Exactive mass spectrometer equipped with a HESI-II Source (Thermo Scientific), operating in positive ion mode with a Waters HSS T3 C18 column (100 mm × 2.1 mm × 1.8 μm). The Q-Exactive operation parameters were gas temperature, 300 °C; sheath gas pressure, 35 arb; capillary temperature, 350 °C; nozzle voltage, 3.5 kV. The elution program was the same as the above UPLC-QTOF analysis of cell culture.

chloride and 5 mL of acetone/ethyl acetate (3:7, v/v) were added. The mixture was vortexed for 1 min, and then allowed to rest for 10 min. A portion of supernatant (2.5 mL) was taken into a 10 mL glass tube and evaporated to near-dryness using a nitrogen evaporator at 40 °C. The residue was dissolved with 1 mL of acetone, passed through a 0.22 μm filter membrane, and analyzed by GC-FPD (gas chromatography paired with flame photometric detector). The intact plant trial was conducted using tea seedlings grown in a hydroponic system using 50 mL of nutrient solution38 in an experimental greenhouse under light-dark cycle conditions (12 h of light/12 h of darkness) at 20 °C at Anhui Agricultural University. Six plants per treatment were grown in plastic pots for 15 days in nutrient solution supplemented with 0 ppm (control) or 100 ppm of thiamethoxam or dimethoate, respectively. After the roots were washed to remove the surface compounds using tap water for 2 min, the intact plants were frozen in liquid nitrogen, ground with a mortar and pestle, extracted according to our previous method except for presoaking,40 and then analyzed by Q-Exactive for an accurate mass spectrum (described below). HPLC Analysis of Neonicotinoids. An Agilent 1260 HPLC system equipped with an Agilent UV detector at 254 nm (for thiamethoxam, acetamiprid) and 270 nm (for imidacloprid, imidaclothiz) was used. The chromatography column and the elution program were the same as our previous study.40 GC Analysis of Organophosphates. Dimethoate and omethoate were detected by a Shimadu GC-2010 Plus equipped with an FPD detector. The chromatography column was an Agilent CYCLOSIL-B (30 m × 0.25 mm × 0.25 μm). The carrier gas was nitrogen, and the flow rate was 1.0 mL min−1. The initial temperature was 120 °C, which was held for 5 min. The temperature rose over three increments: to 150 °C at 30 °C min−1 and then held for 3 min, to 170 °C at 10 °C min−1 and then held for 7 min, and to 210 °C at 30 °C min−1 and then held for 5 min. The injection temperature was 200 °C in splitless mode; the detector temperature was 250 °C; the hydrogen flow rate was 62.5 mL min−1; the air flow rate was 90.0 mL min−1. The injection volume was 1 μL. C

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Figure 2. Callus status after different numbers of subculture cycles (A) and different lengths of subculture cycles (B) on MS media with 2,4-D (1.0 mg L−1) and KT (0.1 mg L−1).

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RESULTS AND DISCUSSION Initiation of Tea Leaf Callus. Different parts of a plant, such as buds, leaves, stems, flowers, and seeds, can be used as explants to initiate callus.39 In this study, tea leaves were chosen because they are the edible part of the crop and the main target of pesticide application. Tea leaves picked from plants grown in the field and from sterile plantlets were used as explants placed on solid media. The rates of contamination, browning, and callus initiation were compared after 28 days of cultivation, using the experimental design shown in Supporting Information (SI) Figure S1. The result revealed that leaves from sterile plantlets had much lower rates of contamination and browning and a higher rate of callus initiation compared to the leaves picked from field-grown tea plants (Figure S1A). The growth condition of the tea callus was recorded at 20, 37, 62, and 90 days of culture (Figure S1B). The growth of the callus derived from the sterile plantlet was more vigorous than that from the field-grown plants during the whole 90 days of cultivation. The callus derived from sterile leaves was always bright yellowish, but the callus derived from picked leaves was white with brown spots. Therefore, sterile plantlet leaves were more suitable for induction of tea callus. Optimization of the Conditions for Initiation of Tea Callus. The crucial parameters for culturing loose and friable callus are primarily the levels of plant growth regulators and the length and number of subculture cycles.37,39,44 At a concentration of 1.0 mg L−1 2,4-D, the concentration of KT was varied (Figure 1A,C). When the KT concentration was 0.05 mg L−1, the callus color was white, the growth rate was slow, and the texture was a little compact; when KT concentration was increased to 0.1 mg L−1, the callus was yellowish in color and loose in texture, and the growth rate of callus was the highest, up to the 61.5%; when the KT was further increased to 0.5 mg L−1, the callus was irregular, the texture was compact, and the callus center was brown. At 0.5 mg L−1 KT concentration, the concentration of 2,4-D was varied (Figure 1B,D). When the concentration of 2,4-D was 1 mg L−1, the callus was loose and friable, and the growth rate of callus was the highest, reaching 46.9%. Therefore, the tea callus could grow well on a combination of 1 mg L−1 2,4-D and 0.1 mg L−1 KT. Optimization of the Subculture Conditions of Tea Callus. Subculture cycle length and number of subcultures are also important for callus culture.39 When the callus was

subcultured 2 to 4 times after 28 days for each cycle, the callus was loose, yellowish, and showed no browning (Figure 2A). The callus completely covered the leaves by the fourth subculture. However, during the fifth subculture, the callus texture began to become compact, accompanied by some white and brown spots appearing from the bottom of the callus. Therefore, the growth of callus was most prolific but not yet brown after the fourth subculture. When the subculture cycle was 21 days long, the callus was vigorous, but did not yet reach the greatest amount of growth, indicating that such frequent subculture would lead to lower experimental efficiency (Figure 2B). When the subculture cycle was 28 days, the callus grew vigorously with yellowish color and loose texture, but no browning. The callus began to brown from the center after 35 days. At 42 days, the callus was seriously brown and had almost stopped growing. The average growth rate of callus after the fourth subculture of 28 days was the highest, up to 66.02%. The optimized protocol for initiation of loose and friable callus includes placing tea leaf explants from sterile plantlets on media containing 1 mg L−1 2,4-D and 0.1 mg L−1 KT and subculturing the callus after 28 days for a total of four subculture cycles. Tea Cell Suspension Culture. For plant tissue culture, the most common media are Gamborg’s B5 and Murashige and Skoog (MS).39 Generally, for a certain plant, the optimal solid medium for callus growth is also suitable for its cell suspension in liquid culture without agar.37 However, tea produces large quantities of polyphenols in response to the high concentration of inorganic salts in MS basal medium, leading to browning of the callus.45,46 Therefore, in this study, two kinds of media were tested to compare the growth status of callus and the color of the cell suspension. The average growth rate of cells was 16.66% in B5 basal media and 15.77% in MS basal media (Figure S2); however, the cell suspension culture liquid and part of the callus were brown in MS basal media. Therefore, B5 basal media was selected for tea cell suspension culture. Plant cells in liquid medium must maintain a certain cell density to meet the physiological environment required for cell growth. Cells cannot grow normally when the cell density is lower or higher than the effective density.47 The OD value of the homogeneous suspension culture was used to represent the amount of cell growth. The cell yielded was highest when 15 g of mother liquid was made to a total volume of 40 mL of culture liquid (v/v) in 75 days of cultivation (Figure S3A). D

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Figure 3. Process of establishing a tea cell suspension culture at constant temperature (25 ± 1 °C) in a dark incubator: Sterile culture of tea plantlets as source of leaf explant (a). Tea leaf inoculated onto MS medium with 2,4-D (1.0 mg L−1) and KT (0.1 mg L−1) (b). Initial cultured callus after 28 days (c). Callus suitable for cell suspension after four subculture cycles of 28 days (d). Remaining steps were at the same temperature but at a constant speed of (120 r/min) in a shaking incubator: Callus inoculated into B5 medium for 7 to 10 days (e). Seeded cell suspension after removing the precipitated and large callus (f). Subculture of cell suspension after 1 cycle of 28 days (g). Mature cell suspension after 3−4 subculture cycles of 28 days each (h).

Figure 4. Metabolism of 5 μg/mL of six insecticides in tea cell suspension culture and in media (CK) incubated at constant temperature (25 ± 1 °C) and shaking incubator speed (120 r/min) over 75 days. (A): thiamethoxan (a), dimethoate (b), omethoate (c), acetamiprid (d), imidaclothiz (e), imidacloprid (f); b1, production over time of the metabolite of dimethoate (omethoate) produced in dimethoate-treated cell culture (Cell MD) and media (CK MD). (B) The metabolite ratios of (a) thiamethoxam and (b) omethoate and their respective metabolites over 75 days.

Single tea cell in suspension culture was round with a 2−3 μm diameter under an optical microscope (Figure S3B). Cell

viability within the tea cell suspension culture was tested by TTC staining, and the specific assay was shown in SI Figure E

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Figure 5. Total ion chromatograms (TICs) of the extracts from untreated control cell culture, thiamethoxam-treated cell culture, thiamethoxamtreated media (cell-free) after 75 d. Peaks 1−5, 7, and 8 were metabolites of thiamethoxam and Peak 6 was thiamethoxam (A). TICs of the extracts from dimethoate-treated cell culture, dimethoate-treated media (cell-free), and untreated control cells after 60 d. Peaks 1 and 2 were metabolites of dimethoate, and Peak 3 was dimethoate (B). TICs of the extracts from thiamethoxam-treated (upper) and untreated (lower) intact plants (C). TICs of the extracts from dimethoate-treated (upper) and untreated (lower) intact plants (D). The metabolite of dimethoate at tR 1.86 min in intact plants (D1); no compounds detected at tR 1.86 min in untreated plants (D2).

S3. Tea cells maintained a good growth status during the whole culture process using this proposed method. A good tea cell suspension system was successfully established from sterile callus, derived from tea leaves excised from sterile plantlets, after 3−4 subcultures in B5 liquid media (Figure 3). Metabolism of Insecticides in Tea Cell Suspension. Tea cell suspension cultures reduced matrix interference during sample analysis compared to the extract from fresh leaves (the extract method was shown in Figure S4). The extract from fresh tea leaves showed more matrix interference peaks than that of the cell suspension culture (Figure S4A), which would cause problems for detection of insecticides and their metabolites. The neonicotinoid insecticides thiamethoxam, imidacloprid, acetamiprid, and imidaclothiz and the organophosphorus insecticides dimethoate and omethoate were added into the tea cell suspension cultures for the metabolic studies, respectively. The results showed that dimethoate, omethoate, and thiamethoxam were easily metabolized compared to the other three neonicotinoids. (Figure 4A). Thiamethoxam was not metabolized in the cell suspension culture for the first 10 days, which might be due to an adaptive period after absorption of the xenobiotic, after which thiamethoxam was metabolized by tea cell (Figure 4A, a). The main metabolite of thiamethoxam began to appear on day 3 and increased slowly. By day 75, 55.3% of the thiamethoxam was degraded, and 33.5% of it was converted to its major metabolite (Figure 4B, a). The cell suspension showed high metabolic capacity for two other insecticides, dimethoate and omethoate (Figure 4A, b, c). In the cell suspension, the omethoate was completely

degraded in 30 days. The dimethoate was degraded more slowly, starting from day 10 and was 90.4% degraded by day 75. The main metabolite of dimethoate in cell suspension, omethoate, was the highest on the day 10, but was then also decreased by the cells (Figure 4A, b1). With the degradation of dimethoate, the content of omethoate increased again on days 30 and 60, reaching 2.02% on day 60 (Figure 4B, b and Figure S4B2), and gradually decreasing to zero on day 65. Omethoate (the main metabolite of dimethoate) was also metabolized by the cells (Figure 4A, c); therefore, the content of omethoate in the cell culture presented an increase−decrease−increase phenomenon. Interestingly, the metabolism rates of the neonicotinoids in tea suspension cells were much slower than previously observed in tomato cell suspension,24 which might be caused by the differences in plant cell species or experiment conditions. The organophosphorus insecticides were more rapidly metabolized than the neonicotinoids. It is also interesting that insecticides in the same structure classes showed diverse metabolic behaviors. This result highlights the diversity of biological metabolic pathways and smart regulation of metabolism within the tea cell, and the specific mechanism still needs to be further investigated. Metabolic Analysis of Thiamethoxam and Dimethoate in Tea Leaf-Derived Cell Suspension Culture and Tea Plant. Comparison of the total ion chromatograms (TICs) generated by LC-QTOF of the untreated control cells, thiamethoxam-treated cell culture, and thiamethoxam-treated media (cell-free) indicated that the thiamethoxam-treated cell culture had seven novel peaks (Figure 5A). However, the extract from thiamethoxam-treated intact plant showed only two peaks of identified metabolites (Figure 5C); Dimethoate F

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thiamethoxam for 75 days had the highest metabolite peak at 23.278 min, which was characterized as clothianidin according to the analytical standards. Clothianidin, another active neonicotinoid insecticide, was identified as the main metabolite of thiamethoxam in spinach.18 From the above results, it can be concluded that seven metabolites of thiamethoxam were detected in the tea cell suspension culture. These metabolites can be divided into two groups: one group with an intact oxadiazine ring (peaks 3, 5, and 7), oxidation of the oxadiazine ring and hydroxylation formed 6-hydroxy oxadiazine (peak 3), and thiamethoxam that underwent N-demethylation and no demethylation, proceeding to urea derivatives (peaks 5 and 7); the other group is ringopened metabolites, from which thiamethoxam was slowly converted to clothianidin (peak 8), which then either underwent N-demethylation and urea derivatives (peaks 1 and 4), or cleavage, yielding chlorothiazolylcarboxylic acid (peak 2).24,18 In comparison to the extract from cell suspensions, only two metabolite peaks, at tRs of 13.80 and 14.26 min, were detected in the intact plant extract, in addition to the thiamethoxam at tR of 12.65 min. Target MS/MS analysis of the peak at tR of 13.80 min gave a mass fragmentation pattern of 248.0256 (M + H), 174.9724 (M + H, −C3H7NO), 132.9744 (M + H, −C4H7N2O2) (Figure S5A2), which was the same as the metabolite at peak 7 in cell culture. Further analysis of the peak at the tR of 14.26 min showed that it was clothianidin (Figure S5A3), identical to the metabolite at peak 8 in the cell culture. The metabolites of dimethoate were compared in cell culture and intact plant (Figure 5B,D). In the extract from the cell culture, Peak 1 (tR of 2.893 min) gave mass fragmentation patterns of 202.1852 (M + H), 158.0269 (M + H, −CHO2), and 144.0479 (M + H, −C2H3O2), inferring it as (hydroxymethoxy-thiophosphorylsulfanyl)-acetic acid (Table 1). This peak was not detected in the dimethoate-treated intact plant. The metabolites at peak 2 in the cell suspension (Figure 5B, tR of 4.333 min) and at the only peak in the intact plant (Figure 5D1, tR of 1.86 min) showed mass fragmentations at 214.0336 (M + H), 124.9818 (M+, −C3H6NO2), and 182.9868 (M+, −CH4N), which matched that of the omethoate. Peak 3 in the cell suspension and at tR of 6.35 min in the intact plant was dimethoate according to the mass fragmentation pattern, which was 230.0103 (M + H), 124.9818 (M+, −C3H6NOS), and 198.9648 (M+, −CH4N), as well as the analytical standards (Table 1; Figure S6B2). Therefore, there was only one metabolite of dimethoate detected in intact plants. In conclusion, a tea cell suspension platform was established and provided a convenient in vitro research system for studying metabolism and metabolites of six insecticides. Two organophosphorus insecticides were metabolized faster than neonicotinoids by the tea cell suspension. Of the four neonicotinoid insecticides, only thiamethoxam was easily metabolized. In the extract from the cell culture, the metabolites of dimethoate and thiamethoxam could be well detected due to the reduction of external interference and matrix effects when they could not be observed in the extract from intact plant. It proved that a tea cell suspension culture could be a promising platform for the investigation of the metabolic behavior of xenobiotics in tea plant and for the study of the specific metabolic mechanisms that act on these insecticides.

could be metabolized to omethoate both in cell culture and in intact plant; however, there was one other metabolite (peak 1) in the cell culture (Figure 5B,D). With the exception of the parent compounds (thiamethoxam and dimethoate) and their main metabolites, omethoate and clothianidin, other metabolites were presented in very small quantities and did not match any analytical standards. Their identities could only be inferred using high-sensitivity mass spectrometry (tR, [M+/M + H] and 35 Cl/37Cl ratio) on a preliminarily base but should be truly identified through a separation method in our future research. Compared with the published metabolites of thiamethoxam in other plant species,18,24 the mass spectra and structures of metabolites of thiamethoxam were shown in Figure S6A, and the mass fragmentation patterns of target MS/MS were shown in Table 1. Peak 7 at tR of 12.750 min had its mass Table 1. Chemical Names, Retention Time (tR), and Mass Fragmentation Patterns of Thiamethoxam and Dimethoate and Their Metabolites in Tea Cell Suspension Cultures as Identified by UPLC-QTOF chemical names of thiamethoxam and its metabolites (peak number)

retention time (min)

N-methyl-N-(2-chloro-1,3thiazolyl)-5-yl-methyl-urea (1)

5.302

2-chloro-thiazole-5-carboxylic acid (2) 3-(2-chloro-1,3-thiazol-5ylmethyl)-6-hydroxy-5-methyl1,3,5-oxadiazinan-4-ylidene (nitro) amine (3) N-(2-chloro-thiazol-5-ylmethyl)guanidine (4) 3-(2-chloro-thiazol-5-ylmethyl)[1,3,5] oxadiazinan-4-ketone (5)

7.768 7.901

8.796 9.919

thiamethoxam (6)

10.363

3-(2-chloro-1,3-thiazol-5ylmethyl)-5-methyl-1,3,5oxadiazin-4-one (7)

12.750

clothianidin (8)

13.075

Chemical names of dimethoate and its metabolites (peak number)

retention time (min)

(hydroxy-methoxythiophosphorylsulfanyl)acetic acid (1) omethoate (2)

2.893 4.333

Dimethoate (3)

5.839

mass fragmentation 205.0077 (M+), 147.0631 (M+, −C2H4NO), 171.0641 (M + H, −Cl) 164.0931 (M + H), 120.0432 (M + H, −CO2) 263.0372 (M+), 131.9670 (M+, −C4H7N2O3) 191.0044 (M + H), 131.9672 (M+, −CH4N3) 234.0104 (M + H), 131.9672 (M+, −C3H5N2O2), 174.9732 (M + H, −C2H5NO) 292.0347 (M + H), 211.0660 (M + H, −ClNO2), 181.0549 (M + H, −ClN2O3) 248.0256 (M + H), 174.9727 (M + H, −C3H7NO), 213.0148 (M + H, −Cl) 250.0229 (M + H), 176.9716 (M + H, −CHN2O2), 133.9640 (M + H, −C2H5N4O2) mass fragmentation

202.1852 (M + H), 158.0269 (M + H, −CHO2), 144.0479 (M + H, −C2H3O2) 214.0336 (M + H), 124.9818 (M+, −C3H6NO2), 182.9868 (M+, −CH4N) 230.0103 (M + H), 124.9818 (M+, −C3H6NOS), 198.9648 (M+, −CH4N)

fragmentation patterns of 248.0256 (M + H), 174.9727 (M + H, −C3H7NO) and 213.0148 (M + H, −Cl). Peak 8, at tR of 13.075 min, gave a mass fragmentation patterns as 250.0229 (M + H), 176.9716 (M + H, −CHN2O2), 133.9640 (M + H, −C2H5N4O2) and was identified as clothianidin, which was the main metabolite of thiamethoxam (Figure S4C1). HPLC analysis of the cell suspension extract after culturing with G

DOI: 10.1021/acs.jafc.8b02417 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b02417.

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Additional figures, chromatography, and spectrometry (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86-551-65786765. *E-mail: [email protected]. Tel: +86-551-65786765. ORCID

Lili He: 0000-0002-5046-9857 Rimao Hua: 0000-0002-5601-6568 Ruyan Hou: 0000-0003-4423-694X Author Contributions #

W.J. and Y.H. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Scientific Foundation of China (No. 31772076 and No. 31270728), National Key Research & Development Program (2016YFD0200900) of China, project of “Nutrition and Quality & Safety of Agricultural Products, Universities Leading Talent Team of Anhui Province”, and Natural Science Foundation for Distinguished Young Scholars of Anhui Province (1608085J08).

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