Article pubs.acs.org/JAFC
Imaging and Detection of Carboxylesterase in Living Cells and Zebrafish Pretreated with Pesticides by a New Near-Infrared Fluorescence Off−On Probe Dongyu Li,† Zhao Li,*,† Weihua Chen,‡ and Xingbin Yang*,† †
Shaanxi Engineering Laboratory for Food Green Processing and Safety Control, College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710062, China ‡ Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China S Supporting Information *
ABSTRACT: A new near-infrared fluorescence off−on probe was developed and applied to fluorescence imaging of carboxylesterase in living HepG-2 cells and zebrafish pretreated with pesticides (carbamate, organophosphorus, and pyrethroid). The probe was readily prepared by connecting (4-acetoxybenzyl)oxy as a quenching and recognizing moiety to a stable hemicyanine skeleton that can be formed via the decomposition of IR-780. The fluorescence off−on response of the probe to carboxylesterase is based on the enzyme-catalyzed spontaneous hydrolysis of the carboxylic ester bond, followed by a further fragmentation of the phenylmethyl unit and thereby the fluorophore release. Compared with the only existing near-infrared carboxylesterase probe, the proposed probe exhibits superior analytical performance, such as near-infrared fluorescence emission over 700 nm as well as high selectivity and sensitivity, with a detection limit of 4.5 × 10−3 U/mL. More importantly, the probe is cell membrane permeable, and its applicability has been successfully demonstrated for monitoring carboxylesterase activity in living HepG-2 cells and zebrafish pretreated with pesticides, revealing that pesticides can effectively inhibit the activity of carboxylesterase. The superior properties of the probe make it of great potential use in indicating pesticide exposure. KEYWORDS: fluorescent probe, carboxylesterase, pesticide, imaging analysis
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and pesticide residue assay. In particular, fluorescent probes with an NIR analytical wavelength for carboxylesterase are rather desirable for practical applications because of their good tissue penetration and low autofluorescence and biological damage.25−27 Yang et al. prepared a novel NIR fluorescent probe which has been used to detect endogenous carboxylesterases in living cells and mice.18 To the best of our knowledge, only one NIR fluorescent probe for carboxylesterase has been reported so far. Hence, NIR fluorescent probes are still necessary for carboxylesterase and pesticide residue assay. In this work, we report the probe (E)-2-(2-(6-((4acetoxybenzyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-propyl-3H-indolium (1; Scheme 1) as a new NIR fluorescence off−on probe for carboxylesterase and pesticide residue assay. The probe is constructed by introducing (4acetoxybenzyl)oxy as a quenching and reacting moiety to the hemicyanine skeleton (2), which can be generated via the decomposition of its unstable precursor of IR-780 but still possesses an NIR feature.28,29 As a result, probe 1 indeed exhibits a selective fluorescent response to carboxylesterase; both the color and fluorescence of the fluorophore (2) are recovered, which leads to the development of a highly sensitive and selective method for monitoring carboxylesterase activity as well as for imaging pesticide exposure in the living HepG-2 cells
INTRODUCTION Pesticides, such as organophosphates, pyrethroids, and carbamates, play an important role in agricultural fields by increasing the food production and decreasing the vector-borne diseases. However, the trace amounts of pesticides in the environment will result in a potential risk for human health.1−3 There have been a number of detection methods for pesticide residues.4−9 Among them, fluorescent probes based on the inhibition of acetylcholinesterase activity have shown satisfactory results for pesticide analysis. For example, Li et al. reported a fluorescent probe based on the aggregation-induced emission effect of tetraphenylethylene and the addition reaction capability of maleimide, which has been applied for acetylcholinesterase activity and organophosphorus pesticide detection in diluted human serum samples.9 Renard et al. developed two near-infrared (NIR) fluorescent probes to detect acetylcholinesterase and indirectly monitor organophosphates, which have been used to detect acetylcholinesterase in muscle.10 On the other hand, carboxylesterases are involved in biotransformation of various drugs, environmental toxicants, and carcinogens containing ester groups.11,12 These enzymes are of great importance for detoxication of pesticides and serve as indicators of pesticide exposure.13−15 Besides, these enzymes efficiently catalyze the cleavage of the ester bond in various carboxylic esters, releasing the corresponding carboxylic acid fragments.16−20 Moreover, fluorescence spectroscopy has attracted much attention because of its high spatiotemporal resolution.21−24 Thus, carboxylic ester compounds have been employed to develop fluorescent probes for carboxylesterases © 2017 American Chemical Society
Received: Revised: Accepted: Published: 4209
March 2, 2017 May 5, 2017 May 5, 2017 May 5, 2017 DOI: 10.1021/acs.jafc.7b00959 J. Agric. Food Chem. 2017, 65, 4209−4215
Article
Journal of Agricultural and Food Chemistry Scheme 1. Synthesis of Probe 1 and Its Reaction with Carboxylesterase
(100:1, v/v) as the eluent, affording (E)-2-(2-(6-hydroxy-2,3-dihydro1H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-propyl-3H-indol-1-ium (2) as a blue-green solid (67.3 mg, yield 52.6%). The 1H NMR and 13C NMR spectra of 2 are given in Figures S1 and S2 (Supporting Information), respectively. 1H NMR (400 MHz, 298 K, CDCl3): δ 8.03 (d, J = 14.2 Hz, 1H), 7.28−7.23 (m, 3H), 7.17(d, J = 9.2 Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.74 (d, J = 8.8 Hz, 1H), 6.51 (s, 1H), 5.61 (d, J = 14.2 Hz, 1H), 3.76 (t, J = 7.2 Hz, 2H), 2.67 (t, J = 6.0 Hz, 2H), 2.61 (t, J = 6.0 Hz, 2H), 1.91−1.78 (m, 4H), 1.66 (s, 6H), 1.06 (t, J = 7.6 Hz, 3H). 13C NMR (400 MHz, 298 K, CD3OD): δ 174.5, 173.6, 163.7, 158.4, 144.0, 142.2, 141.6, 140.4, 131.1, 130.0, 126.1, 123.5, 123.2, 121.9, 116.2, 115.9, 112.0, 103.6, 100.1, 50.4, 46.6, 30.0, 29.0, 25.3, 22.1, 21.8, 11.9. Second, to a solution of 2 (0.41 g, 1.0 mmol) in anhydrous DMF (5 mL) was added K2CO3 (0.21 g, 1.5 mmol), followed by stirring at 40 °C for 5 min under an Ar atmosphere. Then a solution of 4(chloromethyl)phenyl acetate (0.18g, 1.0 mmol) in DMF (5 mL) was added dropwise. The resulting mixture was stirred at 40 °C for 3 h and then diluted with dichloromethane (20 mL). The organic layer was separated, washed with water and brine, and then dried over dry Na2SO4. The solvent was removed by evaporation, and the residue was subjected to silica gel chromatography, eluted with petroleum ether (bp 60−90 °C)/ethyl acetate (1:1, v/v), affording (E)-2-(2-(6-((4acetoxybenzyl)oxy)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3,3-dimethyl1-propyl-3H-indolium (1) as a blue-green solid (0.29 g, 52%). The 1H NMR and 13C NMR spectra of 1 are given in Figures S3 and S4 (Supporting Information), respectively. 1H NMR (600 MHz, 298 K, CD3OD): δ 8.67 (d, J = 14.9 Hz, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.48− 7.40 (m, 4H), 7.40−7.33 (m, 2H), 7.29 (s, 1H), 7.07 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 2.1 Hz, 1H), 6.96 (dd, J = 8.6, 2.2 Hz, 1H), 6.42 (d, J = 14.9 Hz, 1H), 5.18 (s, 2H), 4.24 (t, J = 7.4 Hz, 2H), 2.72−2.66 (m, 2H), 2.62 (t, J = 6.0 Hz, 2H), 2.18 (s, 3H), 1.85 (dd, J = 14.6, 7.3 Hz, 4H), 1.73 (s, 6H), 0.98 (t, J = 7.4 Hz, 3H). 13C NMR (151 MHz, 298 K, CD3OD): δ 179.42, 171.13, 163.65, 163.10, 155.83, 152.22, 147.18, 143.56, 143.07, 135.44, 135.06, 130.23, 130.14, 129.84, 128.75, 128.43, 123.83, 123.07, 117.45, 115.71, 115.32, 113.99, 104.88, 102.75, 71.29, 52.08, 47.61, 30.10, 28.42, 25.12, 22.28, 21.67, 20.93, 11.61. General Procedure for Carboxylesterase Detection. Unless otherwise stated, the fluorescence of probe 1 (10 μM) reacting with carboxylesterase was determined in PBS (pH 7.4) as follows. The stock solution (1 mM) of probe 1 was made by dissolving probe 1 in DMSO. In a test tube, 4 mL of PBS and 50 μL of the stock solution of probe 1 were mixed, followed by addition of carboxylesterase and the pesticide (carbaryl, chlorpyrifos, and deltamethrin) sample solution. The final volume was adjusted to 5 mL with PBS. After incubation at 37 °C for 15 min in a shaker incubator, a 3 mL portion of the reaction solution was transferred to a 1 cm quartz cell to measure the absorbance or fluorescence with λex/em = 670/705 nm and excitation
and zebrafish pretreated with pesticides (carbamate, organophosphorus, and pyrethroid).
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MATERIALS AND METHODS
Apparatus. 1H NMR and 13C NMR spectra were measured on a Bruker DMX-400 or Bruker DMX-600 spectrometer in CD3OD. Electrospray ionization mass spectrometry (ESI-MS) was performed in positive mode with a Shimadzu LC-MS 2010A instrument (Kyoto, Japan). pH measurements were made on a model HI-98128 pH meter (Hanna Instruments Inc., United States). Absorption spectra were measured with a TU-1900 spectrophotometer (Beijing, China) in 1 cm quartz cells. Fluorescence spectra were collected on a Hitachi F4600 spectrofluorimeter in 1 × 1 cm quartz cells with excitation and emission slit widths of 10 nm. The absorbance for 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis was recorded on a microplate reader (BIO-TEK Synergy HT, United States). Fluorescence imaging was performed on a TCS SP5 confocal laser scanning microscope (Leica, Germany) with excitation at 635 nm. Optical sections were acquired at 0.8 μm. Reagents. IR-780 iodide, resorcinol, 4-(chloromethyl)phenyl acetate, carboxylesterase, acetylcholinesterase, and butyrylcholinesterase were purchased from Sigma-Aldrich. A phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) solution was obtained from Invitrogen Co. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, penicillin (100 μg/mL), and streptomycin (100 μg/mL) were obtained from Invitrogen Corp. The lyophilized powder of carboxylesterase was dissolved in pure water, and the solution was divided into 20 parts as suitable amounts for daily experiments. All these enzyme solutions were frozen immediately at −20 °C for storage and allowed to thaw before use according to the known procedure,30 which results in no change of the enzyme activity. MTT was obtained from Serva Electrophoresis GmbH (Germany). The stock solution (1.0 mM) of the probe 1 was prepared by dissolving the requisite amount of 1 in deoxygenated dimethyl sulfoxide (DMSO), which should be used fresh. All other chemicals used were local products of analytical grade. Ultrapure water (over 18 MΩ·cm) from a Milli-Q reference system (Millipore) was used throughout. Synthesis of 1. First, the raw material 2 (Scheme 1) was prepared using the following procedure. Resorcinol (86.0 mg, 0.78 mmol) and K2CO3 (102 mg, 0.78 mmol) were mixed in a flask containing CH3CN (5 mL), and the mixture was stirred at room temperature under a nitrogen atmosphere for 5 min. Then a solution of IR-780 iodide (207 mg, 0.31 mmol) in CH3CN (1.0 mL) was added to the mixture via a syringe, and the reaction mixture was heated at 50 °C for 2 h. The solvent was then evaporated under reduced pressure, and the residue was purified by silica gel chromatography using CH2Cl2/MeOH 4210
DOI: 10.1021/acs.jafc.7b00959 J. Agric. Food Chem. 2017, 65, 4209−4215
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Journal of Agricultural and Food Chemistry
Figure 1. (A) Absorption spectra of probe 1 (10 μM) before (a) and after (b) reaction with carboxylesterase (1 U/mL). (B) Fluorescence spectra (λex = 670 nm) of probe 1 (10 μM) reacting with carboxylesterase at varied concentrations (0, 0.01, 0.025, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.6, and 1 U/ mL). The reactions were performed at 37 °C for 15 min in 10 mM PBS (pH 7.4). and emission slit widths of 10 nm. In the meantime, a blank solution containing no carboxylesterase (control) was prepared and measured under the same conditions for comparison. Cytotoxicity Assay. HepG-2 cells were seeded in 96-well Ubottom plates at a density of 7000 cells per well and incubated with 1 at varied concentrations (1−20 μM) at 37 °C for 24 h. Then the culture media were discarded, and 0.1 mL of the MTT solution (0.5 mg/mL in DMEM) was added to each well, followed by incubation at 37 °C for 4 h. The supernatant was abandoned, and 110 μL of DMSO was added to each well to dissolve the formed formazan. After the plates were shaken for 10 min, the absorbance values of the wells were read with a microplate reader at 490 nm. The cell viability rate (VR) was calculated according to the equation VR = A/A0 × 100, where A is the absorbance of the experimental group (i.e., the cells treated with probe 1) and A0 is the absorbance of the control group (i.e., the cells not treated with probe 1). The cell survival rate from the control group was considered as 100%. Fluorescence Imaging of Carboxylesterase in HepG-2 Cells. The HepG-2 cells were grown on glass-bottom culture dishes (MatTek Co.) in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin (100 μg/mL), and streptomycin (100 μg/mL) at 37 °C in a 5% CO2 incubator. Before use, the adherent cells were washed with fetal bovine serum (FBS)-free DMEM. For fluorescence imaging, the cells were pretreated with 5 μM carbaryl, chlorpyrifos, and deltamethrin for 10 min. After that, the cells were further incubated with 10 μM probe 1 in FBS-free DMEM at 37 °C for 20 min and then washed three times with the PBS buffer (pH 7.4) to remove the free probe. Fluorescence Imaging of Carboxylesterase in Zebrafish. Zebrafish were maintained in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 0.7 mM NaHCO3, 5−10% methylene blue, pH 7.5). In fluorescence imaging experiments, zebrafish grown for 5 days were pretreated with 5 μM carbaryl, chlorpyrifos, and deltamethrin for 10 min. After that, the zebrafish were incubated with 10 μM probe 1 in E3 embryo media for 20 min at 28 °C and then washed with PBS (pH 7.4) to remove the remaining probe 1. Fluorescence imaging experiments were performed on a TCS SP5 confocal laser scanning microscope (Leica, Germany) for excitation at 635 nm. The fluorescence was collected in the ranges of 650−750 nm.
NIR region due to the hydroxyl alkylation of compound 2, which is favorable to achieving a low background signal.26,28 Upon reaction with carboxylesterase, the carboxylic ester bond is cleaved by the enzyme as a consequence of the specific substrate recognition, leading to a further fragmentation of the phenylmethyl unit and thereby the release of the hemicyanine skeleton.14−18 Moreover, addition of carboxylesterase produces a large fluorescence enhancement at 705 nm, which resembles the characteristic emission of fluorophore 2. This sharp fluorescence off−on response is rather desirable for sensitive detection (Figure 1B). These observations suggest that the reaction of probe 1 with carboxylesterase causes the release of fluorophore 2, and the ESI-MS analysis further proves the generation of 2 (m/z 412.3 [M]+; Figure S5, Supporting Information). The effects of pH and temperature on the fluorescence of 1 were examined (Figure S6, Supporting Information), revealing that the changes of both pH from 7 to 8 and temperature from 20 to 40 °C scarcely activate the fluorescence of 1 itself, but do turn on that of the reaction solution of 1 with carboxylesterase. This clearly indicates that the reaction of 1 with carboxylesterase can proceed efficiently under physiological conditions (pH 7.4 and 37 °C). Kinetic curves of probe 1 reacting with carboxylesterase at varied concentrations are given in Figure S7 (Supporting Informaiton), which indicates that higher concentrations of carboxylesterase result in faster reduction and a stronger fluorescence intensity. For carboxylesterase of ≤1 U/mL, this fluorescence increase could reach a plateau in about 15 min. In contrast, the fluorescence of probe 1 without carboxylesterase (control) hardly changes during the same period of time, also implying that the probe is stable in the detection system. Under the above determined condition, the fluorescence response of probe 1 exhibits a good linearity with carboxylesterase (Figure S8, Supporting Information) in the concentration range of 0.01−0.3 U/mL with an equation of ΔF = (2.7 × 103)(C (U/mL)) + 30.9 (R = 0.993), where ΔF is the fluorescence enhancement of probe 1 at 705 nm with and without carboxylesterase. The detection limit (S/N = 3) is determined to be 4.5 × 10−3 U/mL carboxylesterase. Next the reaction selectivity was investigated by examining various potential interfering substances in parallel under the same conditions, such as inorganic salts (KCl, CaCl2, MgCl2), glucose, reactive oxygen species (H2O2), biothiols (glutathione, cysteine, and homocysteine), human serum albumin (HSA),
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RESULTS AND DISCUSSION Spectroscopic Response of 1 to Carboxylesterase. Absorption and fluorescence spectra of 1 before and after reaction with carboxylesterase in 10 mM PBS (pH 7.4) are shown in Figure 1. As is seen from Figure 1A, probe 1 displays a weak absorption in the visible region, but its reaction solution with carboxylesterase shows a strong one at about 670 nm. Furthermore, probe 1 itself has almost no fluorescence in the 4211
DOI: 10.1021/acs.jafc.7b00959 J. Agric. Food Chem. 2017, 65, 4209−4215
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Journal of Agricultural and Food Chemistry
be largely inhibited by pesticides, and thereby the fluorescence enhancement is attributed to the release of fluorophore 2 by the enzymatic cleavage reaction. To evaluate the potential toxicity of 1 to cells, a standard MTT assay was performed (Figure S10, Supporting Information). The results showed that the cell viability was not significantly changed upon treatment even with 10 μM 1 at 37 °C for 24 h, indicating the low cytotoxicity and good biocompatibility of the probe. Fluorescence Imaging of Carboxylesterase in HepG-2 Cells Pretreated with Pesticides. As mentioned above, the carboxylic ester bond of probe 1 is cleaved by the cellular carboxylesterase, leading to a further fragmentation of the phenylmethyl unit and thereby the release of the fluorophore. Thus, probe 1 is anticipated to be capable of imaging pesticide residues in HepG-2 cells via its reaction with the endogenous carboxylesterase. As shown in Figure 3, HepG-2 cells themselves show no background fluorescence (Figure 3A; control), which profits from the usage of an NIR excitation wavelength (670 nm). However, the 1-loaded HepG-2 cells (Figure 3B) display a strong fluorescence, suggesting that endogenous carboxylesterase can react with 1, producing a fluorescence response. In contrast, when HepG-2 cells were pretreated with 5 μM deltamethrin, carbaryl, and chlorpyrifos, a decreased fluorescence was observed (Figure 3C−E). This clearly indicates that the fluorescence change in HepG-2 cells arises from the cleavage reaction of probe 1 by carboxylesterase, releasing the free fluorophore 2. These studies indicate that the probe 1 is cell membrane permeable and suited for monitoring carboxylesterase in living cells. In addition, to compare quantitatively the inhibiting effects of three different kinds of pesticides (carbaryl, chlorpyrifos, and deltamethrin) on the activity of carboxylesterase, ImageJ software (version 1.37c, NIH) was used to analyze the pixel intensity of the cells (Figure 4). In doing so, the pixel intensities of 10 cells at least were averaged. All experiments were done in triplicate, and the error bars refer to the standard error of the mean of the triplicate experiments. As can be seen from Figure 4, the fluorescence intensity from the cells treated with carbaryl, chlorpyrifos, and deltamethrin is decreased to ca. 78%, 65%, and 48% with respect to that without pesticides (defined as 1.0), which corresponds to the inhibition of the carboxylester-
bovine serum albumin (BSA), acetylcholinesterase (AchE), and butyrylcholinesterase (BchE). The results (Figure S9, Supporting Information) show that probe 1 exhibits excellent selectivity for carboxylesterase and other substances do not significantly disturb the fluorescence intensity of the reaction solution, indicating the reliability of the detection system. Pyrethroid (deltamethrin), carbamate (carbaryl), and organophosphorus (chlorpyrifos) pesticides are used worldwide to control agricultural and household pests. The residues of these pesticides could pose a potential risk for human health due to their subacute and chronic toxicity.31 Besides, these three kinds of pesticides can inhibit carboxylesterase, which is useful as a biomarker of pesticide exposure.32−34 The influence of pesticides (deltamethrin, carbaryl, and chlorpyrifos) on the enzyme activity was also explored. As can be seen from Figure 2, the fluorescence intensity in the presence of 5 μM carbaryl,
Figure 2. Fluorescence emission spectra (λex = 670 nm) of different reaction systems: (a) probe 1 (10 μM) in PBS buffer of pH 7.4 (control); (b) the system in (a) + carboxylesterase (1 U/mL); (c) the system in (b) + carbaryl (5 μM); (d) the system in (b) + chlorpyrifos (5 μM); (e) the system in (b) + deltamethrin (5 μM). All the reactions were performed at 37 °C for 15 min.
chlorpyrifos, and deltamethrin is decreased to ca. 88%, 69%, and 51% with respect to that without pesticides (defined as 1.0). The results showed that the fluorescence intensity in the presence of pesticides is much lower than that in the absence of the inhibitor, illustrating that the carboxylesterase activity can
Figure 3. Confocal fluorescence images of HepG-2 cells: (A) HepG-2 cells only; (B) HepG-2 cells incubated with 10 μM 1 for 20 min; (C) HepG-2 cells pretreated with 5 μM carbaryl for 10 min and then incubated with 10 μM 1 for 20 min; (D) HepG-2 cells pretreated with 5 μM chlorpyrifos for 10 min and then incubated with 10 μM 1 for 20 min; (E) HepG-2 cells pretreated with 5 μM deltamethrin for 10 min and then incubated with 10 μM 1 for 20 min. The differential interference contrast (DIC) images of the corresponding samples are shown in panels F−J. Scale bar = 20 μm. 4212
DOI: 10.1021/acs.jafc.7b00959 J. Agric. Food Chem. 2017, 65, 4209−4215
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Journal of Agricultural and Food Chemistry
carboxylesterase in zebrafish, the carboxylic ester bond of probe 1 is cleaved by the enzyme-catalyzed spontaneous hydrolysis, leading to a further fragmentation of the phenylmethyl unit and thereby the release of the fluorophore. Interestingly, it is noted that the fluorescence intensity is not uniform in zebrafish, and especially the zebrafish yolk sac shows the strongest fluorescence. The probe 1 may be uniformly distributed in zebrafish,28 whereas carboxylesterase may not, though current evidence does not allow us to completely exclude the nonuniform distribution of the probe in zebrafish. Furthermore, we investigated the inhibitor effect of the three different kinds of pesticides (5 μM deltamethrin, carbaryl, and chlorpyrifos) on carboxylesterase activity. As shown in Figure 5C−E, the fluorescence from zebrafish pretreated with the three different kinds of pesticides is much weaker than that from the untreated zebrafish, clearly indicating that the three different kinds of pesticides can effectively inhibit the activity of carboxylesterase in zebrafish. Meanwhile, to compare quantitatively the inhibiting effects of three different kinds of pesticides (carbaryl, chlorpyrifos, and deltamethrin) on the activity of carboxylesterase in vivo, the pixel intensity of the zebrafish was analyzed by using ImageJ software. As can be seen from Figure 6, the fluorescence intensity from the zebrafish treated with carbaryl, chlorpyrifos, and deltamethrin is decreased to ca. 79%, 57%, and 39% with respect to that without pesticides (defined as 1.0), which corresponds to the inhibition of the carboxylesterase activity of ca. 21%, 43%, and 61%, respectively. These results are also the same as those from in vitro experiments (Figure 2). As can be seen from fluorescence images in living HepG-2 cells and zebrafish, the fluorescence intensity in the presence of pesticides is lower than that without the inhibitor, illustrating that the carboxylesterase activity can be largely inhibited by pesticides, and thereby the fluorescence enhancement is attributed to the release of fluorophore 2 by the enzymatic cleavage reaction. Moreover, the fluorescence intensity from cells and zebrafish pretreated with deltamethrin is the weakest, while that from cells and zebrafish pretreated with carbaryl is the strongest. The reason for the above results may be that carboxylesterase can be largely inhibited during the enzymecatalyzed hydrolysis procedure by deltamethrin, which is considered the most powerful of the synthetic pyrethroids.
Figure 4. Relative pixel intensity measurements obtained from the images of HepG-2 cells: (a) cells incubated with 10 μM probe 1 for 20 min; (b) cells pretreated with 5 μM carbaryl for 10 min and then incubated with 10 μM probe 1 for 20 min; (c) cells pretreated with 5 μM chlorpyrifos for 10 min and then incubated with 10 μM probe 1 for 20 min; (d) cells pretreated with 5 μM deltamethrin for 10 min and then incubated with 10 μM probe 1 for 20 min. The strongest fluorescence intensity from the image of HepG-2 cells incubated with 10 μM probe 1 for 20 min is defined as 1.0. The results are the mean ± standard deviation of three separate measurements.
ase activity of ca. 22%, 35%, and 52%, respectively. These results are close to those from in vitro experiments (Figure 2). Fluorescence Imaging of Carboxylesterase in Zebrafish Pretreated with Pesticides. Zebrafish, which is a favorable vertebrate model organism, has been widely used in the fields of developmental biology, aquaculture, and even human health research. Zebrafish have many unique features; especially a rapid development and transparency of their embryos facilitate the in vivo visualization of biologically relevant substances and processes by NIR fluorescent probes. However, no study has been reported to visualize carboxylesterase in zebrafish. In our experiments, zebrafish grew in E3 embryo media for 5 days and then were subjected to fluorescence imaging. As shown in Figure 5, zebrafish themselves show no fluorescence in the NIR region (Figure 5A), but the 1-loaded zebrafish display a strong fluorescence (Figure 5B). This implies that the probe is organism permeable, and surprisingly, these zebrafish contain endogenous carboxylesterase detectable by probe 1. Upon reaction with
Figure 5. Fluorescence images of carboxylesterase in living 5 day old zebrafish: (A) zebrafish only (control); (B) zebrafish treated with probe 1 (10 μM) for 20 min; (C) zebrafish preincubated with 5 μM carbaryl and treated with probe 1 (10 μM) for 20 min; (D) zebrafish preincubated with 5 μM chlorpyrifos and treated with probe 1 (10 μM) for 20 min; (E) zebrafish preincubated with 5 μM deltamethrin and treated with probe 1 (10 μM) for 20 min. The DIC images of the corresponding samples are shown in panels F−J. Scale bar = 200 μm. 4213
DOI: 10.1021/acs.jafc.7b00959 J. Agric. Food Chem. 2017, 65, 4209−4215
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Journal of Agricultural and Food Chemistry Notes
The authors declare no competing financial interest.
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Figure 6. Relative pixel intensity measurements obtained from the images of zebrafish: (a) zebrafish treated with probe 1 (10 μM) for 20 min; (b) zebrafish preincubated with 5 μM carbaryl and treated with probe 1 (10 μM) for 20 min; (c) zebrafish preincubated with 5 μM chlorpyrifos and treated with probe 1 (10 μM) for 20 min; (d) zebrafish preincubated with 5 μM deltamethrin and treated with probe 1 (10 μM) for 20 min. The strongest fluorescence intensity from the image of zebrafish incubated with 10 μM probe 1 for 20 min is defined as 1.0. The results are the mean ± standard deviation of three separate measurements.
Besides, carboxylesterase also can be phosphorylated or carbamylated by chlorpyrifos and carbaryl, while the stability of carbamylated esterase is less than that of phosphorylated esterase.35−37 Thus, compared with chlorpyrifos, carbaryl had a less marked effect on carboxylesterase activity.38 In summary, we have developed an NIR spectroscopic off− on probe for carboxylesterase assay, and its applicability has been demonstrated in fluorescence imaging of carboxylesterase in living HepG-2 cells and zebrafish pretreated with pesticides, revealing that pesticides can effectively inhibit the activity of carboxylesterase. The superior properties of the probe make it of great potential use in indicating pesticide exposure.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00959. Synthesis of probe 1 (Figures S1−S4), electrospray ionization mass spectrum of the reaction solution of 1 (Figure S5), effects of pH and temperature (Figure S6), fluorescence kinetic curves of 1 reacting with carboxylesterase (Figure S7), linear fitting curve (Figure S8), selectivity study results (Figure S9), and cytotoxicity assay (Figure S10) (PDF)
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*Phone: +86 10-85310517. Fax: +86 10-85310517. E-mail:
[email protected]. *Phone: +86 10-85310580. Fax: +86 10-399 85310580. E-mail:
[email protected]. ORCID
Xingbin Yang: 0000-0002-8039-0525 Funding
This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 31671823 and 21605099) and the Fundamental Research Funds for the Central Universities, China (Grant Nos. GK201603096 and 2016CSZ010). 4214
DOI: 10.1021/acs.jafc.7b00959 J. Agric. Food Chem. 2017, 65, 4209−4215
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