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Assessment of the cytotoxicity of ionic liquids on Spodoptera frugiperda 9 (Sf-9) cell lines via in vitro assays
Journal of Hazardous Materials 348 (2018) 1–9
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Assessment of the cytotoxicity of ionic liquids on Spodoptera frugiperda 9 (Sf-9) cell lines via in vitro assays
T
Shuanggen Wub,1, Liangbin Zenga,1, Chaoyun Wanga, Yuanru Yanga, Wanlai Zhoua, Fenfang Lib, ⁎ Zhijian Tana, a b
Institute of Bast Fiber Crops and Center of Southern Economic Crops, Chinese Academy of Agricultural Sciences, Changsha, 410205, China College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, China
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Ionic liquids Cytotoxicity Sf-9 cells DNA agarose gel electrophoresis Flow cytometry
Cytotoxicity studies are important tools for the assessment of the toxicity of ionic liquids (ILs). In the present study, the cytotoxicity of eleven ILs against Spodoptera frugiperda 9 (Sf-9) cell lines were evaluated via 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The effect on cellular morphology, ultrastructural morphology, and nuclear morphology induced by 1-ethyl-3-methylimidazolium bromide ([C2mim] [Br]) was studied via inverted light microscopy observation, acridine orange staining, and transmission electron microscope (TEM) analysis, respectively. The effect on cell DNA fragmentation, cell apoptosis and cell cycle induced by [C2mim][Br] was also investigated via DNA agarose gel electrophoresis and flow cytometry analysis, respectively. The results showed that the cytotoxic effect of ILs on Sf-9 cells was related to the IL structures, concentrations, and length of exposure. The morphological features of apoptosis induced by [C2mim][Br] such as cell shrinkage and convolution, apoptotic bodies, pyknosis, and karyorrhesis were observed. All these phenomena confirmed that Sf-9 cells exposed to [C2mim][Br] died via apoptosis. This study complements the current knowledge about the cytotoxic properties of ILs on insect cells and highlights the mechanism by which ILs kill these cells. Furthermore, it provides a basis for further studies on the future applications of ILs as insecticides.
⁎
1
Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Tan). These authors contributed equally.
https://doi.org/10.1016/j.jhazmat.2018.01.028 Received 27 September 2017; Received in revised form 5 January 2018; Accepted 12 January 2018 0304-3894/ © 2018 Elsevier B.V. All rights reserved.
Journal of Hazardous Materials 348 (2018) 1–9
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1. Introduction
risk evaluation, however, the toxicity mechanism is not yet clear and still requires further investigation. Thus, it is of great importance to further realize the exact mechanisms underlying the toxic effect of ILs toward cell lines. Insects are the most diverse groups of animals on our planet, which include a million species, thus they were usually used as models assessing the toxicity of toxins [33]. In the present study, the cytotoxic effect of ILs toward Sf-9 insect cells was assessed. The Sf-9 cell lines were originally established from immature ovaries of the lepidopteran S. frugiperda pupae, otherwise known as the fall armyworm [34], which had previously been used as reliable tool for testing toxins and drug toxicity [33,35,36]. Larvae of S. frugiperda pupae live in the soil, and since a recent study showed that ILs were strongly absorbed and retained in soil for a period of time [37]. Thus, Sf-9 cells were chosen as a model to assess ILs toxicity on soil-dwelling invertebrate organisms in this work. Commonly used ILs containing assorted cations and anions were chosen, and their cytotoxicity toward Sf-9 cells was investigated via MTT assay. To our knowledge, the application of imidazolium-based ILs was the most for industry and academic research among the all known ILs. Moreover, This IL [C2mim][Br] is the least cytotoxic among the imidazolium ILs in this study. So, [C2mim][Br] was chosen for investigating its toxicity mechanism toward Sf-9 cell lines via various methods, such as acridine orange (AO) staining, DNA fragmentation assays, flow cytometry analysis, and transmission electron microscope (TEM) analysis. Information obtained from this study can complement the currently insufficient knowledge about IL cytotoxicity on insect cells by clarifying the mechanism of IL cytotoxicity. Moreover, it can provide some ideas and preliminary data for the future applications of ILs as insecticides.
Ionic liquids (ILs) are defined as molten salts with melting temperatures around or below 100 °C, which are usually constructed from organic cations and organic or inorganic anions [1]. Regarding the structures of ILs, cations are composed of large head groups, such as ammonium, imidazolium, pyridinium, piperidinium, pyrrolidinium, morpholinium or cholinium, and alkyl side chains that can vary in length, number or position; the anions type include halides, acetate, fluorine, and cyano derivatives [2]. Since the first synthesis of the airand water- stable ILs containing 1-ethyl-3-methylimidazolium cations [3], ILs have been praised by researchers and production personnel due to their unique advantages, including low volatility, non-flammability, high ionic conductivity, high thermal stability, excellent dissolving capacity, and wide electrochemical window [4–7]. Owing to these various properties, ILs have become versatile solvents and are used in different fields, such as extraction and separation [8,9], electrochemistry [10], analytical chemistry [11], synthesis and catalysis [12], and material science [13]. Most importantly, ILs are tunable, meaning that their cations and anions can be tailored to obtain various taskspecific ILs with different physical and chemical properties [14,15]. With the wide use of ILs in the chemical industry and in academic research, it is unavoidable that ILs would be released into the living environment. However, not enough attention has been paid to the effect of ILs on the environment. Therefore, extensive efforts are required to obtain data on the ecotoxicity of ILs. To our knowledge, ILs are less volatile than organic solvents, and are unlikely to enter the atmosphere or cause atmospheric toxicity. However, most ILs are water soluble and enter the water and soil easily [16]. At present, the toxicity of ILs toward microorganisms (bacteria, fungi, yeast) [17,18], algae [19,20], plants [21,22], vertebrates [23], invertebrates [24], and cells [25,26] had been studied. These toxicity studies generally focused on external data, such as minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), inhibitory concentration resulting in 50% of inhibition of the activity of biological or biochemical systems by tested compound (IC50), and effective concentration of tested compound causing 50% of reduction on processes, such as growth or reproductive activity (EC50), and median lethal dose (LD50). These data were mainly obtained via in vivo studies. Although in vivo studies provide useful data about the toxicity of ILs toward organisms, sometimes they are time consuming and laborious. In vitro assays have many advantages, including speed, quantitative results, reproducibility, and low cost. Therefore in vitro assays have been widely used to assess the toxic effect of ILs. Moreover, in vitro cytotoxicity studies can clarify the effect of ILs on internal cellular structures and microstructures, and the cellular mechanisms that underlie toxicity. In the past, increasing amounts of cytotoxic data were obtained through the study of ILs toxicity against rat [27,28], human [29–31], and fish [26,32] cell lines, in studies that they mainly used IC50 values to assess IL cytotoxicity. Studies regarding IL toxicity on insect cell lines and the effect of IL on internal cell structures have been rarely reported. In recent years, in order to clarify the mechanisms of the effect of ILs on organism, ILs toxicity toward selected cell lines has been studied via DNA fragmentation assays, flow cytometry analysis, and TEM analysis. These studies have indicated that ILs cause cell death via necrosis or apoptosis. For example, Li et al [28] investigated the cytotoxicity mechanism of imidazolium based ILs towards rat pheochromocytoma (PC12) cells via various methods, such as reactive oxygen species (ROS) levels detection, DNA fragmentation and lactate dehydrogenase release (LDH) analysis, and caspase-3 activity assay. The results showed that IL [C8mim][Br] may induce the apoptosis of PC12 cell lines. Radošević et al [26] assessed the cytotoxicity of imidazolium-based ILs towards Channel Catfish Ovary (CCO) cell lines via fluorescent microscopy observation and flow cytometry analysis, the results suggested that ILs with longer side chain length may cause the CCO cell lines necrosis. The work investigating toxic effect mechanism of ILs is necessary to their
2. Materials and methods 2.1. Materials and reagents The ILs tetramethylammonium bromide [N1,1,1,1][Br], tetrabutylphosphonium bromide [P4,4,4,4][Br], 1-methyl-ethylpiperidinium bromide [MEPip][Br], 1-methyl-1-ethylpyrrolidinium bromide [MEPyrro][Br], 1-ethyl-3-methylimidazolium bromide [C2mim][Br], 1butyl-3-methylimidazolium bromide [C4mim][Br], 1-hexyl-3-methylimidazolium bromide [C6mim][Br], 1-octyl-3-methylimidazolium bromide [C8mim][Br], 1-decyl-3-methylimidazolium bromide [C10mim] [Br], 1-butyl-3-methylimidazolium tetrafluoroborate [C4mim][BF4], 1butyl-3-methylimidazolium hexyfluoroborate [C4mim][BF6], and 1butyl-3-methylimidazolium chloride [C4mim][Cl] were purchased from Shanghai Cheng Jie Chemical Co., LTD. (Shanghai, China), each with purity > 99%. The chemical structures of these ILs are shown in Table 1. Sf-9 cells were obtained from the China Center for Type Culture Collection (CCTCC). 3-(4,5-Dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) was purchased from Amresco (USA). Acridine orange (AO) was purchased from Solarbio. Annexin V-EGFP Apoptosis and Cell Cycle Detection Kits were purchased from Jiangsu KGI Biotechnology Co., LTD. (Jiangsu, China). Fetal bovine serum (FBS) and gentamycin were purchased from Beijing North Carolina Souren Biotechnology Research Institute (Beijing, China). Insect DNA Kit (200) was obtained from OMEGA Bio-TEK (Doraville, GA, USA). Other reagents obtained from commercial sources were of analytical grade and were used without further treatment. 2.2. Cell culture Sf-9 cells were cultured in 25 cm2 T-flasks (Corning, USA) containing Trichoplusia ni medium-formulation Hink (TNM-FH) supplemented with 10% (v/v) fetal bovine serum (FBS) and 10 μg/mL gentamycin and maintained at 25 °C. To ensure the presence of sufficient nutrients, the medium was replaced every 2 – 3 days. 2
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2.5. Fluorescence microscopy analysis
Table 1 The full names and chemical structures of the ILs used in this study. Ionic liquids (full names)
Cations
The AO staining method was used to observe cellular morphologies. Sf-9 cellular suspension was treated as explained above (Section 2.4). After exposure to the IL (24 h or 48 h at 25 °C), the cells were washed twice with PBS; 50 μL AO (100 μg/mL in PBS) was added; and the plates were incubated at 25 °C for 5 min in the dark. The cells were analyzed using an Olympus fluorescent inverted microscope (model CKX53, Japan) at an excitation wavelength of 488 nm.
Anions
[C2mim][Br] (1-ethyl-3methylimidazolium bromide) [C4mim][Br] (1-butyl-3methylimidazolium bromide) [C6mim][Br] (1-hexyl-3methylimidazolium bromide) [C8mim][Br] (1-octyl -3methylimidazolium bromide) [C10mim][Br] (1-decyl -3methylimidazolium bromide) [C4mim][Cl] (1-butyl-3methylimidazolium chloride) [C4mim][BF4] (1-butyl-3methylimidazolium tetrafluoroborate) [N1,1,1,1][Br] (tetramethylammonium bromide) [P4,4,4,4][Br] (tetrabutylphosphonium bromide) [Pip][Br] (1-methylethylpiperidinium bromide)
Br−
[Pyr][Br] (1-methyl-1ethylpyrrolidinium bromide)
Br−
Br− Br− Br−
2.6. DNA agarose gel electrophoresis
Br−
Four milliliter Sf-9 cells suspension was seeded in T-flasks at initial cellular concentrations of 1 × 105 – 2 × 105 cells/mL and incubated for 24 h. The culture medium was removed and 3600 μL fresh medium supplemented with 400 μL [C2mim][Br] at different concentrations (initial concentration of 100, 25, 6.25, 1.5625, 0.3906 mg/mL, respectively) was added to the T-flasks. The T-flasks were incubated at 25 °C for 24 h, 48 h, or 72 h. The Sf-9 cells without [C2mim][Br] treatment were used as a negative control, and cells killed by incubating the T-flasks in hot water (100 °C) for 5 min were used as a positive control. After incubation, the cells were collected into a centrifuge tube and centrifuged at 800 rpm for 5 min, after which the cells were washed twice with PBS. DNA of Sf-9 cells was extracted using an insect DNA Kit according to the instructions of the manufacturer. DNA agarose gel electrophoresis was conducted according to a previous report [38]. The DNA fragments were visualized using a Tanon 2500 Gel imaging system (Hunan Kengene Instrument Co., LTD, Changsha, China).
Cl− BF4−
Br−
Br−
Br−
2.3. Cytotoxicity assay 2.7. TEM analysis In vitro cytotoxicity of ILs was assessed via MTT assay. Cell culture suspension (100 μL) at concentrations of 1.0 × 105 – 2.0 × 105 cells/ mL were transferred into 96-well microtiter plates and allowed to incubate for 24 h. The medium was then removed and replaced with 90 μL fresh medium supplemented with 10 μL IL solution at different concentration. After the time period (24 h, 48 h, and 72 h) to evaluate toxicity had elapsed, 10 μL (5.0 mg/mL) MTT reagent was added to each well and the plates were incubated at 25 °C for 4 h. After the culture medium being removed, the wells were washed with phosphate-buffered saline (PBS), then 100 μL dimethyl sulfoxide (DMSO) was added to dissolve the intracellular formazan crystals. The microtiter plate was then incubated at 25 °C for 10 min until the formazan crystals were almost completely dissolved. Absorption was measured at 570 nm via a microplate reader (Infinite 200 PRO, Tecan, Shanghai, China). Percentage cell viability was calculated according to the following formula:
Cell viability (%) = OD570 OD570 OD570
OD570 test −OD570 blank × 100 OD570 control −OD570 blank
Cell suspensions were prepared according to the procedure described above (Section 2.6), and then treated according to the method described previously [39]. The samples were observed via TEM (Tecnai G2 Spirit TWIN, USA). 2.8. Flow cytometry analysis The rate of Sf-9 apoptosis induced by different concentrations of [C2mim][Br] was assessed through flow cytometry. Briefly, 4 mL Sf-9 cells suspension was seeded in T-flasks at an initial concentration of 1 × 105 – 2 × 105 cells/mL. After 24 h, the culture medium was removed, and 3600 μL fresh medium supplemented with 400 μL [C2mim] [Br] (initial concentration of 100, 25, 6.25, 1.5625, 0.3906 mg/mL, respectively) was added into the T-flasks. The T-flasks were incubated at 25 °C for 24 h. The detailed procedures were as follows and performed according to the instructions of Annexin V-EGFP Apoptosis Detection Kit. The Sf-9 cells were collected into a centrifuge tube and centrifuged at 800 rpm for 5 min, after which the cells were rinsed twice with PBS, producing final cellular concentrations of 1 × 105 – 2 × 105 cells/mL. Binding buffer solution (500 μL), 5 μL Annexin VEGFP, and 5 μL propidium iodide (PI) were then added sequentially, and the cells were incubated for 10 min at room temperature in the dark. The treated cells were analyzed via flow cytometry (Bio-Rad Laboratories Ltd., Shanghai, China) at an excitation wavelength of 488 nm of laser and emission wavelength of 530 nm. In addition, the cell cycle of Sf-9 cells after treatment with [C2mim][Br] was analyzed using flow cytometry. The cells were prepared in the same manner as before, and the flow cytometry analysis was conducted according to the instructions of the Cell Cycle Detection Kit at an excitation wavelength of 488 nm.
(1)
test:
the absorbance of IL treated cells at 570 nm; the absorbance of DMSO at 570 nm; control: the absorbance of non-IL treated cells at 570 nm. blank:
2.4. Inverted light microscopy analysis Cell culture suspension (1000 μL) at concentrations of 1.0 × 105 – 2.0 × 105 cells/mL were plated in 12-well plates. After 24 h, the medium was removed and replaced with 900 μL fresh culture medium and 100 μL [C2mim][Br] (initial concentration of 100, 25, 6.25, 1.5625, 0.3906 mg/mL, respectively). The plates were incubated at 25 °C for 24 h or 48 h, and the cells were observed using an inverted light microscope (Shanghai Measuring Photoelectric technology Co., Ltd., Shanghai, China). Pictures were taken by using a digital camera (Shanghai Measuring Photoelectric Technology Co., Ltd.).
2.9. Statistical analysis Cell viability data were analyzed using the graphics program GraphPad Prism v5.0b (GraphPad Software Inc., San Diego, California, 3
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linear regression sigmoidal dose–response curve (GraphPad Prism v5.0b). The flow cytometric data were analyzed using Flowjo x 10.0.7r2 software. 3. Results and discussion 3.1. Viability of Sf-9 cells exposed to ILs with different cations and anions Many factors can influence IL cytotoxicity, including the IL substructures such as head groups, side chain length, and anion type. IC50 values after exposure of Sf-9 cells to a series of ILs with different structures for 24 h and 48 h were calculated from the dose–response plots. The dose–response curves of Sf-9 cells treated with a series of ILs (10−7 – 10 mg/mL) for 24 h were shown in Fig. 1(a). The corresponding IC50 and logIC50 values are shown in Table 2. The IC50 of Sf-9 cells exposed to [C10mim][Br] was the lowest (IC50 = 0.0037 and 0.0001 mg/mL after 24 h and 48 h exposure, respectively) of the 11 tested ILs. In contrast, the IC50 of [N1,1,1,1][Br] was the highest (IC50 = 12.4400 and 15.6600 mg/mL after 24 h and 48 h exposure, respectively). The results confirmed that the structure of the ILs played a major role in their toxicity [40]. 3.1.1. Influence of head groups To study the influence of IL structures on cytotoxicity, cell viability after treatment with five ILs with cations containing different head groups but the same anion was evaluated. As shown in Fig. 1(b), a rough order of the cytotoxicity strengths was obtained for the tested ILs, as follows: [N1,1,1,1]+ < [Pip]+≈[P4,4,4,4]+ < [Pyr]+ < [C4mim]+. However, the actual order maybe different at specific concentrations of each IL. For example, the order of cytotoxicities was [N1,1,1,1]+ < [Pip]+ < [P4,4,4,4]+ < [Pyr]+ < [C4mim]+ at 1.0 mg/ mL ILs concentration, whereas at 0.0001 mg/mL ILs concentration the order of cytotoxicities was [N1,1,1,1]+ < [Pyr]+ < [P4,4,4,4]+ < [Pip]+ < [C4mim]+. The imidazolium-based ILs was most toxic, which was consistent with the present work [41]. The cytotoxic effect of [N1,1,1,1][Br] on Sf-9 cells was consistently the weakest of the five tested ILs, whereas the cytotoxic effect of [C4mim][Br] was the strongest except at the highest IL concentration (10 mg/mL). It was found that the cytotoxicity of [P4,4,4,4][Br] was stronger than that of [C4mim] [Br] at 10 mg/mL. The toxicities of ([N1,1,1,1][Br], [P4,4,4,4][Br], and [Pip][Br]) were roughly similar at low concentrations (0.0001 – 0.01 mg/mL), which were much higher than those of [C4mim][Br] and [Pyr][Br]. It can also be seen in Fig. 1(b), that overall cell viability decreased with increasing ILs concentration. For example, the cell viabilities after treatment with [P4,4,4,4][Br] were 103.66% and 18.64% at the lowest (0.0001 mg/mL) and highest concentrations (10 mg/mL), respectively. These results indicated that the type of head group present in the IL affects the cytotoxic effect on insect cells, a phenomenon that was also found in a previous study [42]. Moreover, this study showed how the concentration of the IL contributes to its cytotoxicity; the cytotoxic effect of ILs on Sf-9 cell lines increased with increasing concentration, this phenomenon may be that the destructive effect of ILs toward cell membrane was stronger under high concentration [43]. Fig. 1. (a) Dose–response curves of Sf-9 cells treated with eleven ionic liquids (10−7 – 10 mg/mL) for 24 h. The cell viability was assessed via MTT assay. (b) The cell viability (%) of Sf-9 cells exposed for 24 h to ionic liquids containing different head group cations and the same bromide anion. (c) IC50 values for Sf-9 cells exposed to imidazolium-based ionic liquids containing cations with different alkyl chain lengths (the carbon number n ranged from C2-C10) and a bromide anion for 24 h and 48 h, respectively. (d) The cell viability (%) of Sf-9 cells exposed to ionic liquids containing 1-butyl-3-methylimimdazolium cation and three different anions for 24 h.
3.1.2. Influence of side chain length The cytotoxic effect of imidazolium ILs with different alkyl chain lengths (carbon number (n) = 2, 4, 6, 8, or 10) on Sf-9 cells is shown in Fig. 1(c). The IC50 of the ILs generally decreased with increasing carbon atoms, except in the case of n = 4. This pattern occurred because the longer alkyl chain length led to a stronger lipid solubility, which allowed the IL to penetrate the cell membrane more easily [19]. In contrast, the IC50 of ILs decreased with increasing incubation time. As the number of carbon atoms (n) increased, the difference between the IC50 values for 24 h and 48 h decreased, except for n = 4. The results showed that ILs with long chain lengths were more toxic than ILs with short carbon chains. It was reported that the lipophilicity played an
USA). All data are represented as the mean of three independent experiments with error values expressed as the standard error of the mean (SEM). IC50 half maximum toxicity values were obtained from a non4
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Table 2 IC50 and logIC50 values of ILs exposed to sf-9 cells for 24 h and 48 h. ILs
24 h
[C2mim][Br] [C4mim][Br] [C6mim][Br] [C8mim][Br] [C10mim][Br] [C4mim][Cl] [C4mim][BF4] [N1,1,1,1][Br] [P4,4,4,4][Br] [Pip][Br] [Pyr][Br]
48 h
IC50 (mg /mL)
logIC50 (mg/mL)
IC50 (mg/mL)
logIC50 (mg/mL)
1.7070 ± 0.3781 0.1269 ± 0.0988 0.3261 ± 0.1701 0.0551 ± 0.0242 0.0037 ± 0.0013 0.1244 ± 0.0441 0.0244 ± 0.0025 ∼ 12.4400 0.9915 ± 0.1451 4.2580 ± 1.0895 0.6813 ± 0.3843
0.2323 −0.8967 −0.4867 −1.2590 −2.4290 −0.9052 −1.6130 ∼ 1.0950 −0.0037 0.6292 −0.1667
0.2792 ± 0.0322 0.0003 ± 0.0001 0.0631 ± 0.0048 0.0008 ± 0.0001 0.0001 ± 1.0255 × 10−5 0.0449 ± 0.0038 0.0131 ± 0.0010 15.6600 ± 0.8418 0.0547 ± 0.0012 3.1420 ± 0.2865 0.5910 ± 0.0564
−0.5541 −3.4850 −1.2000 −3.1000 −3.8990 −1.3470 −1.8840 1.1950 −1.2620 0.4971 −0.2284
membrane blebbing, and apoptotic bodies. Apoptotic characteristics became more severe when the IL concentration quadrupled to 0.6250 mg/mL with the same treatment time of 12 h. For example, the number of blebs and apoptotic bodies increased. When the treatment time was increased to 72 h, these necrotic phenomena, such as swelling of cell structures, rupture of the plasma membrane, organelle breakdown, and increased release of cytoplasmic contents, blurred cell membrane were observed (Fig. 2A4). The results above indicated that IL concentration and exposure time strongly affected its cytotoxicity. These phenomena suggested that the necrosis of Sf-9 cells caused by [C2mim][Br] under high IL concentration and long exposure time [51]. However, the observed apoptotic characteristics of cell shrinkage, membrane blebbing, and apoptotic bodies suggested that cell death induced by ILs occurs via apoptosis rather than necrosis under low tested concentration.
important in the cytotoxicity, the longer alkyl chain lengths tended to have stronger lipophilicity, which increased the odds of contact with the lipid bilayer and hydrophobic proteins of the membrane, thus disrupting the normal physiological function of the membrane, even leading to the cell death [43,44]. However, the toxicity did not increase after a certain number of carbons (for example n = 4), which is generally described as a “cut-off effect” [45,46]. 3.1.3. Influence of the anion Toxicity of ILs with the same cation and different anions was also assessed ([C4mim]Br, [C4mim]Cl, and [C4mim]BF4). As shown in Fig. 1(d), [C4mim][Br] was the least cytotoxic of the three tested ILs. Sf9 viabilities after treatment with [C4mim]Cl and [C4mim]BF4 were similar at low concentrations (0.0001 – 0.1 mg/mL), but differed at high concentrations (1.0–10 mg/mL). Overall, the cytotoxic strengths of the ILs followed this order: Br− < Cl− < BF4− at 0.1 mg/mL. For Br− and Cl−, the cytotoxic effect may be attributed to the molecular weight, which allows the molecule to enter the cells easily [47]. BF4−, in contrast, is hydrolyzed to HF at high concentration, which is more cytotoxic than either Br− or Cl− [48–50]. These results suggest that the species of anion affect the ILs cytotoxicity on Sf-9 cells [42].
3.2.2. Ultrastructure changes in Sf-9 cell lines induced by [C2mim][Br] Apoptotic cells share a number of common features such as cell shrinkage, membrane blebbing, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies of condensed chromatin [52]. To further distinguish the cell death process induced by [C2mim] [Br] and to observe the toxic effect of ILs on cell ultrastructure, the treated cells were observed with a TEM. As can be seen in Fig. 3A, normal Sf-9 cells had continuous cell membrane integrity, uniform cytoplasm chromatin, proper organelle structures, and nuclei at the center of the cells. However, the cells treated with 0.6250 mg/mL [C2mim][Br] for 12 h had a large number of vacuoles and showed transformation of the cell membrane (Fig. 3B) [53]. Cells treated for 24 h showed some apoptotic characteristics, such as membrane blebbing, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies. Moreover, a shift of chromatin to one side of the cell and
3.2. Morphological changes in Sf-9 cells induced by [C2mim][Br] 3.2.1. Cellular morphology of Sf-9 cells Cellular morphological observations was reliable tool for distinguishing apoptotic and necrotic cell death. The changes in cell morphology induced by [C2mim][Br] at different concentrations were observed using inverted microscopy. As shown in Fig. 2, when the Sf-9 cells were treated with 0.1563 mg/mL [C2mim][Br] for 12 h, typical apoptotic characteristics could be observed, such as cell shrinkage,
Fig. 2. Morphological changes in Sf-9 cells induced by several concentrations and exposure times to [C2mim][Br], as seen via inverted microscopy. Magnification 400 × .
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Fig. 3. Transmission electron microscope images of Sf-9 cells. A shows normal cells; B, C, and E show cells treated with the 0.6250 mg/mL [C2mim][Br] for 12 h, 24 h, and 48 h, respectively. D shows Sf-9 cells treated with 2.5000 mg/mL [C2mim][Br] for 24 h.
nuclei of cells treated with 0.1563 mg/mL [C2mim][Br] were stained deeper green than those of cells treated with 0.0391 mg/mL [C2mim] [Br], suggesting that the DNA was highly condensed. Moreover, the nuclei of cells treated with high-concentration IL were stained orange, possibly due to the overlapping fluorescence between RNA in the cytoplasm and DNA in the nucleus. A similar phenomenon was observed in cells treated for longer time. These results indicate that the cell death induced by the IL occurred via apoptosis rather than necrosis.
nuclear membrane invagination were observed [51] (Fig. 3C) along with apoptotic bodies, organelle vacuolation, and chromatin content reduction (Fig. 3D). In Fig. 3E, a vanishing nuclear membrane, cell shrinkage, and chromatin reduction can be seen, which indicates the final stage of apoptosis [51,53] Taken together, we conclude that [C2mim][Br] induced apoptosis rather than necrosis in Sf-9 cells. 3.2.3. Nuclear morphology changes in Sf-9 cell lines induced by [C2mim] [Br] To study the effect of ILs on Sf-9 cell nuclei, AO stain was used. Following staining, intact cell nuclei were stained light green, where asnecrotic cell nuclei were stained deep green and orange due to a heat reaction with the stain. We observed that the amount of stained nuclei increased in the cell cytoplasm after treatment with [C2mim][Br], which may have been caused by the breakdown of the nuclear membrane (Fig. 4). The nuclei of cells treated with IL solution were stained deeper green than those of the controls, with a heterogeneous distribution. This phenomenon indicated that the cells were undergoing the typical apoptotic feature of chromatin condensation [51]. The
3.2.4. DNA fragmentation of Sf-9 cells DNA agarose gel electrophoresis is commonly used to differentiate between apoptosis and necrosis [51]. The results for the DNA agarose gel electrophoresis are shown in Fig. 5, where it can be easily seen that the DNA stripes in cells not treated with ILs or killed with heat were continuous (A1, B1, and C1). In contrast, a DNA ladder with a molecular spacing of approximately 180–200 base pairs, which is a hallmark of apoptosis, appeared in the cells treated with IL (A2-6, B2-6, and C26). These results also verified that the cell death induced by IL occurred via apoptosis rather than necrosis [51].
Fig. 4. Nuclear morphological changes in Sf-9 cells induced by different [C2mim][Br] concentrations over 24 h or 48 h using acridine orange staining. Sf-9 cells not treated with the IL were used as negative controls, and heat-killed cells with 100 °C boiling water for 5 min were used as positive controls. Magnification 400 × .
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Fig. 5. Agarose gel electrophoresis of DNA extracted from Sf-9 cells treated with different concentrations of [C2mim][Br] for 24 h, 48 h, and 72 h. The mark “m” indicates the DNA marker and “c” indicates the nontreated control group. Samples A, B, and C were treated for 24 h, 48 h, and 72 h, respectively. Sample number 1 was the negative control group treated by heating to 100 °C for 5 min. Sample numbers 2, 3, 4, 5, and 6 were treated with IL at final concentrations of 0.0391, 0.1563, 0.6250, 2.5000, and 10.0000 mg/mL, respectively.
Fig. 6. Flow cytometry analysis of apoptosis rates in Sf-9 cells after 24 h of treatment with various concentrations of [C2mim][Br].
2.5000 mg/mL IL concentration, and the minimum apoptosis rate was 29% (Q2 + Q3) at 0.0391 mg/mL IL concentration, from which it can be deduced that the effect of IL on Sf-9 cells was apoptotic rather than necrotic. The results was similar with the reported studies, which investigated the HepG2 cell apoptosis induced by 1-methyl-3-octylimidazolium bromide IL, the cell apoptosis rate increased from 7.52% (control) to 26.38% with increasing concentration of tested IL, indicating that the IL induce the cell apoptosis [30]. Moreover, early
3.3. Flow cytometry analysis of Sf-9 cell lines treated with [C2mim][Br] 3.3.1. Rate of apoptosis To further study the effect of ILs on Sf-9 cells, Annexin V-EGFP Apoptosis Detection Kit was used to measure the rate of apoptosis induced by 24 h of treatment with different concentrations of [C2mim] [Br]. It can be observed in Fig. 6 that the apoptosis rate of control was 9.1% (Q2 + Q3), the maximum apoptosis rate was 43% (Q2 + Q3) at 7
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Fig. 7. The cell cycle of Sf-9 cells exposed to different [C2mim][Br] concentrations over 24 h.
apoptosis increased from 9.1% (control) to the maximum value 43% (treated with 2.5000 mg/mL [C2mim][Br]). The cell cycle analysis revealed that [C2mim][Br] can disrupt the Sf-9 cell cycle. From all these results above, it can be concluded that [C2mim][Br] could cause the apoptosis of Sf-9 cells rather than necrosis. This study supplements the current insufficient knowledge of IL cytotoxicity in insect cells and clarifies the cytotoxicity mechanism. Moreover, it provides support for the potential future use of ILs as insecticides.
apoptosis was the main mechanism at lower IL concentrations, whereas late apoptosis was the main mechanism at higher IL concentrations. The results also show that the higher the IL concentration, the larger the destructive effect on the cells. 3.3.2. Effect of IL treatment on the Sf-9 cell cycle The cell cycle of Sf-9 cells exposed to various concentrations of IL for 24 h was examined using a Cell Cycle Kit. After exposure to [C2mim] [Br], the number of G0/G1 phase cells remained approximately constant, the number of S phase cells decreased, and the number of G2/M phase cells increased (Fig. 7). It can be concluded that the influence of [C2mim][Br] on the cell cycle was mainly in the S and G2/M phases, particularly in the G2/M phase. When the IL concentration increased to 10.0000 mg/mL, the cell cycle was similar to that of normal cells, which indicated that the excess concentration of IL led to cell necrosis.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21406262), Central Public-interest Scientific Institution Basal Research Fund (No. 1610242016006), and the Fundamental Research Funds for the Central Universities of Central South University (No. 2017zzts336).
4. Conclusions References The cytotoxic effects of 11 different ILs on Sf-9 cells were evaluated via MTT assays, and the IC50 values were obtained. The results showed that the cation head groups, alkyl chain lengths, and type of anions played major roles in the degree of cytotoxicity caused by the ILs. Moreover, the tested ILs concentration and treated time also contributed to their cytotoxicity. In addition, the mechanism of Sf-9 cells death induced by [C2mim][Br] was determined using biochemical tools, including AO staining, DNA agarose gel electrophoresis, flow cytometry and observations of cell morphologies via inverted microscopy and TEM. The results of AO staining indicated the cells were undergoing the typical apoptotic feature of chromatin condensation. Moreover, a DNA ladder was found in the DNA agarose gel electrophoresis analysis. The morphological features of apoptosis such as cell shrinkage and convolution, apoptotic bodies, pyknosis, and karyorrhesis were observed by inverted microscopy and TEM. The rate of cell
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Assessment of the cytotoxicity of ionic liquids on Spodoptera frugiperda 9 (Sf-9) cell lines via in vitro assays
T
Shuanggen Wub,1, Liangbin Zenga,1, Chaoyun Wanga, Yuanru Yanga, Wanlai Zhoua, Fenfang Lib, ⁎ Zhijian Tana, a b
Institute of Bast Fiber Crops and Center of Southern Economic Crops, Chinese Academy of Agricultural Sciences, Changsha, 410205, China College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, China
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Ionic liquids Cytotoxicity Sf-9 cells DNA agarose gel electrophoresis Flow cytometry
Cytotoxicity studies are important tools for the assessment of the toxicity of ionic liquids (ILs). In the present study, the cytotoxicity of eleven ILs against Spodoptera frugiperda 9 (Sf-9) cell lines were evaluated via 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The effect on cellular morphology, ultrastructural morphology, and nuclear morphology induced by 1-ethyl-3-methylimidazolium bromide ([C2mim] [Br]) was studied via inverted light microscopy observation, acridine orange staining, and transmission electron microscope (TEM) analysis, respectively. The effect on cell DNA fragmentation, cell apoptosis and cell cycle induced by [C2mim][Br] was also investigated via DNA agarose gel electrophoresis and flow cytometry analysis, respectively. The results showed that the cytotoxic effect of ILs on Sf-9 cells was related to the IL structures, concentrations, and length of exposure. The morphological features of apoptosis induced by [C2mim][Br] such as cell shrinkage and convolution, apoptotic bodies, pyknosis, and karyorrhesis were observed. All these phenomena confirmed that Sf-9 cells exposed to [C2mim][Br] died via apoptosis. This study complements the current knowledge about the cytotoxic properties of ILs on insect cells and highlights the mechanism by which ILs kill these cells. Furthermore, it provides a basis for further studies on the future applications of ILs as insecticides.
⁎
1
Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Tan). These authors contributed equally.
https://doi.org/10.1016/j.jhazmat.2018.01.028 Received 27 September 2017; Received in revised form 5 January 2018; Accepted 12 January 2018 0304-3894/ © 2018 Elsevier B.V. All rights reserved.
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1. Introduction
risk evaluation, however, the toxicity mechanism is not yet clear and still requires further investigation. Thus, it is of great importance to further realize the exact mechanisms underlying the toxic effect of ILs toward cell lines. Insects are the most diverse groups of animals on our planet, which include a million species, thus they were usually used as models assessing the toxicity of toxins [33]. In the present study, the cytotoxic effect of ILs toward Sf-9 insect cells was assessed. The Sf-9 cell lines were originally established from immature ovaries of the lepidopteran S. frugiperda pupae, otherwise known as the fall armyworm [34], which had previously been used as reliable tool for testing toxins and drug toxicity [33,35,36]. Larvae of S. frugiperda pupae live in the soil, and since a recent study showed that ILs were strongly absorbed and retained in soil for a period of time [37]. Thus, Sf-9 cells were chosen as a model to assess ILs toxicity on soil-dwelling invertebrate organisms in this work. Commonly used ILs containing assorted cations and anions were chosen, and their cytotoxicity toward Sf-9 cells was investigated via MTT assay. To our knowledge, the application of imidazolium-based ILs was the most for industry and academic research among the all known ILs. Moreover, This IL [C2mim][Br] is the least cytotoxic among the imidazolium ILs in this study. So, [C2mim][Br] was chosen for investigating its toxicity mechanism toward Sf-9 cell lines via various methods, such as acridine orange (AO) staining, DNA fragmentation assays, flow cytometry analysis, and transmission electron microscope (TEM) analysis. Information obtained from this study can complement the currently insufficient knowledge about IL cytotoxicity on insect cells by clarifying the mechanism of IL cytotoxicity. Moreover, it can provide some ideas and preliminary data for the future applications of ILs as insecticides.
Ionic liquids (ILs) are defined as molten salts with melting temperatures around or below 100 °C, which are usually constructed from organic cations and organic or inorganic anions [1]. Regarding the structures of ILs, cations are composed of large head groups, such as ammonium, imidazolium, pyridinium, piperidinium, pyrrolidinium, morpholinium or cholinium, and alkyl side chains that can vary in length, number or position; the anions type include halides, acetate, fluorine, and cyano derivatives [2]. Since the first synthesis of the airand water- stable ILs containing 1-ethyl-3-methylimidazolium cations [3], ILs have been praised by researchers and production personnel due to their unique advantages, including low volatility, non-flammability, high ionic conductivity, high thermal stability, excellent dissolving capacity, and wide electrochemical window [4–7]. Owing to these various properties, ILs have become versatile solvents and are used in different fields, such as extraction and separation [8,9], electrochemistry [10], analytical chemistry [11], synthesis and catalysis [12], and material science [13]. Most importantly, ILs are tunable, meaning that their cations and anions can be tailored to obtain various taskspecific ILs with different physical and chemical properties [14,15]. With the wide use of ILs in the chemical industry and in academic research, it is unavoidable that ILs would be released into the living environment. However, not enough attention has been paid to the effect of ILs on the environment. Therefore, extensive efforts are required to obtain data on the ecotoxicity of ILs. To our knowledge, ILs are less volatile than organic solvents, and are unlikely to enter the atmosphere or cause atmospheric toxicity. However, most ILs are water soluble and enter the water and soil easily [16]. At present, the toxicity of ILs toward microorganisms (bacteria, fungi, yeast) [17,18], algae [19,20], plants [21,22], vertebrates [23], invertebrates [24], and cells [25,26] had been studied. These toxicity studies generally focused on external data, such as minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), inhibitory concentration resulting in 50% of inhibition of the activity of biological or biochemical systems by tested compound (IC50), and effective concentration of tested compound causing 50% of reduction on processes, such as growth or reproductive activity (EC50), and median lethal dose (LD50). These data were mainly obtained via in vivo studies. Although in vivo studies provide useful data about the toxicity of ILs toward organisms, sometimes they are time consuming and laborious. In vitro assays have many advantages, including speed, quantitative results, reproducibility, and low cost. Therefore in vitro assays have been widely used to assess the toxic effect of ILs. Moreover, in vitro cytotoxicity studies can clarify the effect of ILs on internal cellular structures and microstructures, and the cellular mechanisms that underlie toxicity. In the past, increasing amounts of cytotoxic data were obtained through the study of ILs toxicity against rat [27,28], human [29–31], and fish [26,32] cell lines, in studies that they mainly used IC50 values to assess IL cytotoxicity. Studies regarding IL toxicity on insect cell lines and the effect of IL on internal cell structures have been rarely reported. In recent years, in order to clarify the mechanisms of the effect of ILs on organism, ILs toxicity toward selected cell lines has been studied via DNA fragmentation assays, flow cytometry analysis, and TEM analysis. These studies have indicated that ILs cause cell death via necrosis or apoptosis. For example, Li et al [28] investigated the cytotoxicity mechanism of imidazolium based ILs towards rat pheochromocytoma (PC12) cells via various methods, such as reactive oxygen species (ROS) levels detection, DNA fragmentation and lactate dehydrogenase release (LDH) analysis, and caspase-3 activity assay. The results showed that IL [C8mim][Br] may induce the apoptosis of PC12 cell lines. Radošević et al [26] assessed the cytotoxicity of imidazolium-based ILs towards Channel Catfish Ovary (CCO) cell lines via fluorescent microscopy observation and flow cytometry analysis, the results suggested that ILs with longer side chain length may cause the CCO cell lines necrosis. The work investigating toxic effect mechanism of ILs is necessary to their
2. Materials and methods 2.1. Materials and reagents The ILs tetramethylammonium bromide [N1,1,1,1][Br], tetrabutylphosphonium bromide [P4,4,4,4][Br], 1-methyl-ethylpiperidinium bromide [MEPip][Br], 1-methyl-1-ethylpyrrolidinium bromide [MEPyrro][Br], 1-ethyl-3-methylimidazolium bromide [C2mim][Br], 1butyl-3-methylimidazolium bromide [C4mim][Br], 1-hexyl-3-methylimidazolium bromide [C6mim][Br], 1-octyl-3-methylimidazolium bromide [C8mim][Br], 1-decyl-3-methylimidazolium bromide [C10mim] [Br], 1-butyl-3-methylimidazolium tetrafluoroborate [C4mim][BF4], 1butyl-3-methylimidazolium hexyfluoroborate [C4mim][BF6], and 1butyl-3-methylimidazolium chloride [C4mim][Cl] were purchased from Shanghai Cheng Jie Chemical Co., LTD. (Shanghai, China), each with purity > 99%. The chemical structures of these ILs are shown in Table 1. Sf-9 cells were obtained from the China Center for Type Culture Collection (CCTCC). 3-(4,5-Dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) was purchased from Amresco (USA). Acridine orange (AO) was purchased from Solarbio. Annexin V-EGFP Apoptosis and Cell Cycle Detection Kits were purchased from Jiangsu KGI Biotechnology Co., LTD. (Jiangsu, China). Fetal bovine serum (FBS) and gentamycin were purchased from Beijing North Carolina Souren Biotechnology Research Institute (Beijing, China). Insect DNA Kit (200) was obtained from OMEGA Bio-TEK (Doraville, GA, USA). Other reagents obtained from commercial sources were of analytical grade and were used without further treatment. 2.2. Cell culture Sf-9 cells were cultured in 25 cm2 T-flasks (Corning, USA) containing Trichoplusia ni medium-formulation Hink (TNM-FH) supplemented with 10% (v/v) fetal bovine serum (FBS) and 10 μg/mL gentamycin and maintained at 25 °C. To ensure the presence of sufficient nutrients, the medium was replaced every 2 – 3 days. 2
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2.5. Fluorescence microscopy analysis
Table 1 The full names and chemical structures of the ILs used in this study. Ionic liquids (full names)
Cations
The AO staining method was used to observe cellular morphologies. Sf-9 cellular suspension was treated as explained above (Section 2.4). After exposure to the IL (24 h or 48 h at 25 °C), the cells were washed twice with PBS; 50 μL AO (100 μg/mL in PBS) was added; and the plates were incubated at 25 °C for 5 min in the dark. The cells were analyzed using an Olympus fluorescent inverted microscope (model CKX53, Japan) at an excitation wavelength of 488 nm.
Anions
[C2mim][Br] (1-ethyl-3methylimidazolium bromide) [C4mim][Br] (1-butyl-3methylimidazolium bromide) [C6mim][Br] (1-hexyl-3methylimidazolium bromide) [C8mim][Br] (1-octyl -3methylimidazolium bromide) [C10mim][Br] (1-decyl -3methylimidazolium bromide) [C4mim][Cl] (1-butyl-3methylimidazolium chloride) [C4mim][BF4] (1-butyl-3methylimidazolium tetrafluoroborate) [N1,1,1,1][Br] (tetramethylammonium bromide) [P4,4,4,4][Br] (tetrabutylphosphonium bromide) [Pip][Br] (1-methylethylpiperidinium bromide)
Br−
[Pyr][Br] (1-methyl-1ethylpyrrolidinium bromide)
Br−
Br− Br− Br−
2.6. DNA agarose gel electrophoresis
Br−
Four milliliter Sf-9 cells suspension was seeded in T-flasks at initial cellular concentrations of 1 × 105 – 2 × 105 cells/mL and incubated for 24 h. The culture medium was removed and 3600 μL fresh medium supplemented with 400 μL [C2mim][Br] at different concentrations (initial concentration of 100, 25, 6.25, 1.5625, 0.3906 mg/mL, respectively) was added to the T-flasks. The T-flasks were incubated at 25 °C for 24 h, 48 h, or 72 h. The Sf-9 cells without [C2mim][Br] treatment were used as a negative control, and cells killed by incubating the T-flasks in hot water (100 °C) for 5 min were used as a positive control. After incubation, the cells were collected into a centrifuge tube and centrifuged at 800 rpm for 5 min, after which the cells were washed twice with PBS. DNA of Sf-9 cells was extracted using an insect DNA Kit according to the instructions of the manufacturer. DNA agarose gel electrophoresis was conducted according to a previous report [38]. The DNA fragments were visualized using a Tanon 2500 Gel imaging system (Hunan Kengene Instrument Co., LTD, Changsha, China).
Cl− BF4−
Br−
Br−
Br−
2.3. Cytotoxicity assay 2.7. TEM analysis In vitro cytotoxicity of ILs was assessed via MTT assay. Cell culture suspension (100 μL) at concentrations of 1.0 × 105 – 2.0 × 105 cells/ mL were transferred into 96-well microtiter plates and allowed to incubate for 24 h. The medium was then removed and replaced with 90 μL fresh medium supplemented with 10 μL IL solution at different concentration. After the time period (24 h, 48 h, and 72 h) to evaluate toxicity had elapsed, 10 μL (5.0 mg/mL) MTT reagent was added to each well and the plates were incubated at 25 °C for 4 h. After the culture medium being removed, the wells were washed with phosphate-buffered saline (PBS), then 100 μL dimethyl sulfoxide (DMSO) was added to dissolve the intracellular formazan crystals. The microtiter plate was then incubated at 25 °C for 10 min until the formazan crystals were almost completely dissolved. Absorption was measured at 570 nm via a microplate reader (Infinite 200 PRO, Tecan, Shanghai, China). Percentage cell viability was calculated according to the following formula:
Cell viability (%) = OD570 OD570 OD570
OD570 test −OD570 blank × 100 OD570 control −OD570 blank
Cell suspensions were prepared according to the procedure described above (Section 2.6), and then treated according to the method described previously [39]. The samples were observed via TEM (Tecnai G2 Spirit TWIN, USA). 2.8. Flow cytometry analysis The rate of Sf-9 apoptosis induced by different concentrations of [C2mim][Br] was assessed through flow cytometry. Briefly, 4 mL Sf-9 cells suspension was seeded in T-flasks at an initial concentration of 1 × 105 – 2 × 105 cells/mL. After 24 h, the culture medium was removed, and 3600 μL fresh medium supplemented with 400 μL [C2mim] [Br] (initial concentration of 100, 25, 6.25, 1.5625, 0.3906 mg/mL, respectively) was added into the T-flasks. The T-flasks were incubated at 25 °C for 24 h. The detailed procedures were as follows and performed according to the instructions of Annexin V-EGFP Apoptosis Detection Kit. The Sf-9 cells were collected into a centrifuge tube and centrifuged at 800 rpm for 5 min, after which the cells were rinsed twice with PBS, producing final cellular concentrations of 1 × 105 – 2 × 105 cells/mL. Binding buffer solution (500 μL), 5 μL Annexin VEGFP, and 5 μL propidium iodide (PI) were then added sequentially, and the cells were incubated for 10 min at room temperature in the dark. The treated cells were analyzed via flow cytometry (Bio-Rad Laboratories Ltd., Shanghai, China) at an excitation wavelength of 488 nm of laser and emission wavelength of 530 nm. In addition, the cell cycle of Sf-9 cells after treatment with [C2mim][Br] was analyzed using flow cytometry. The cells were prepared in the same manner as before, and the flow cytometry analysis was conducted according to the instructions of the Cell Cycle Detection Kit at an excitation wavelength of 488 nm.
(1)
test:
the absorbance of IL treated cells at 570 nm; the absorbance of DMSO at 570 nm; control: the absorbance of non-IL treated cells at 570 nm. blank:
2.4. Inverted light microscopy analysis Cell culture suspension (1000 μL) at concentrations of 1.0 × 105 – 2.0 × 105 cells/mL were plated in 12-well plates. After 24 h, the medium was removed and replaced with 900 μL fresh culture medium and 100 μL [C2mim][Br] (initial concentration of 100, 25, 6.25, 1.5625, 0.3906 mg/mL, respectively). The plates were incubated at 25 °C for 24 h or 48 h, and the cells were observed using an inverted light microscope (Shanghai Measuring Photoelectric technology Co., Ltd., Shanghai, China). Pictures were taken by using a digital camera (Shanghai Measuring Photoelectric Technology Co., Ltd.).
2.9. Statistical analysis Cell viability data were analyzed using the graphics program GraphPad Prism v5.0b (GraphPad Software Inc., San Diego, California, 3
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linear regression sigmoidal dose–response curve (GraphPad Prism v5.0b). The flow cytometric data were analyzed using Flowjo x 10.0.7r2 software. 3. Results and discussion 3.1. Viability of Sf-9 cells exposed to ILs with different cations and anions Many factors can influence IL cytotoxicity, including the IL substructures such as head groups, side chain length, and anion type. IC50 values after exposure of Sf-9 cells to a series of ILs with different structures for 24 h and 48 h were calculated from the dose–response plots. The dose–response curves of Sf-9 cells treated with a series of ILs (10−7 – 10 mg/mL) for 24 h were shown in Fig. 1(a). The corresponding IC50 and logIC50 values are shown in Table 2. The IC50 of Sf-9 cells exposed to [C10mim][Br] was the lowest (IC50 = 0.0037 and 0.0001 mg/mL after 24 h and 48 h exposure, respectively) of the 11 tested ILs. In contrast, the IC50 of [N1,1,1,1][Br] was the highest (IC50 = 12.4400 and 15.6600 mg/mL after 24 h and 48 h exposure, respectively). The results confirmed that the structure of the ILs played a major role in their toxicity [40]. 3.1.1. Influence of head groups To study the influence of IL structures on cytotoxicity, cell viability after treatment with five ILs with cations containing different head groups but the same anion was evaluated. As shown in Fig. 1(b), a rough order of the cytotoxicity strengths was obtained for the tested ILs, as follows: [N1,1,1,1]+ < [Pip]+≈[P4,4,4,4]+ < [Pyr]+ < [C4mim]+. However, the actual order maybe different at specific concentrations of each IL. For example, the order of cytotoxicities was [N1,1,1,1]+ < [Pip]+ < [P4,4,4,4]+ < [Pyr]+ < [C4mim]+ at 1.0 mg/ mL ILs concentration, whereas at 0.0001 mg/mL ILs concentration the order of cytotoxicities was [N1,1,1,1]+ < [Pyr]+ < [P4,4,4,4]+ < [Pip]+ < [C4mim]+. The imidazolium-based ILs was most toxic, which was consistent with the present work [41]. The cytotoxic effect of [N1,1,1,1][Br] on Sf-9 cells was consistently the weakest of the five tested ILs, whereas the cytotoxic effect of [C4mim][Br] was the strongest except at the highest IL concentration (10 mg/mL). It was found that the cytotoxicity of [P4,4,4,4][Br] was stronger than that of [C4mim] [Br] at 10 mg/mL. The toxicities of ([N1,1,1,1][Br], [P4,4,4,4][Br], and [Pip][Br]) were roughly similar at low concentrations (0.0001 – 0.01 mg/mL), which were much higher than those of [C4mim][Br] and [Pyr][Br]. It can also be seen in Fig. 1(b), that overall cell viability decreased with increasing ILs concentration. For example, the cell viabilities after treatment with [P4,4,4,4][Br] were 103.66% and 18.64% at the lowest (0.0001 mg/mL) and highest concentrations (10 mg/mL), respectively. These results indicated that the type of head group present in the IL affects the cytotoxic effect on insect cells, a phenomenon that was also found in a previous study [42]. Moreover, this study showed how the concentration of the IL contributes to its cytotoxicity; the cytotoxic effect of ILs on Sf-9 cell lines increased with increasing concentration, this phenomenon may be that the destructive effect of ILs toward cell membrane was stronger under high concentration [43]. Fig. 1. (a) Dose–response curves of Sf-9 cells treated with eleven ionic liquids (10−7 – 10 mg/mL) for 24 h. The cell viability was assessed via MTT assay. (b) The cell viability (%) of Sf-9 cells exposed for 24 h to ionic liquids containing different head group cations and the same bromide anion. (c) IC50 values for Sf-9 cells exposed to imidazolium-based ionic liquids containing cations with different alkyl chain lengths (the carbon number n ranged from C2-C10) and a bromide anion for 24 h and 48 h, respectively. (d) The cell viability (%) of Sf-9 cells exposed to ionic liquids containing 1-butyl-3-methylimimdazolium cation and three different anions for 24 h.
3.1.2. Influence of side chain length The cytotoxic effect of imidazolium ILs with different alkyl chain lengths (carbon number (n) = 2, 4, 6, 8, or 10) on Sf-9 cells is shown in Fig. 1(c). The IC50 of the ILs generally decreased with increasing carbon atoms, except in the case of n = 4. This pattern occurred because the longer alkyl chain length led to a stronger lipid solubility, which allowed the IL to penetrate the cell membrane more easily [19]. In contrast, the IC50 of ILs decreased with increasing incubation time. As the number of carbon atoms (n) increased, the difference between the IC50 values for 24 h and 48 h decreased, except for n = 4. The results showed that ILs with long chain lengths were more toxic than ILs with short carbon chains. It was reported that the lipophilicity played an
USA). All data are represented as the mean of three independent experiments with error values expressed as the standard error of the mean (SEM). IC50 half maximum toxicity values were obtained from a non4
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Table 2 IC50 and logIC50 values of ILs exposed to sf-9 cells for 24 h and 48 h. ILs
24 h
[C2mim][Br] [C4mim][Br] [C6mim][Br] [C8mim][Br] [C10mim][Br] [C4mim][Cl] [C4mim][BF4] [N1,1,1,1][Br] [P4,4,4,4][Br] [Pip][Br] [Pyr][Br]
48 h
IC50 (mg /mL)
logIC50 (mg/mL)
IC50 (mg/mL)
logIC50 (mg/mL)
1.7070 ± 0.3781 0.1269 ± 0.0988 0.3261 ± 0.1701 0.0551 ± 0.0242 0.0037 ± 0.0013 0.1244 ± 0.0441 0.0244 ± 0.0025 ∼ 12.4400 0.9915 ± 0.1451 4.2580 ± 1.0895 0.6813 ± 0.3843
0.2323 −0.8967 −0.4867 −1.2590 −2.4290 −0.9052 −1.6130 ∼ 1.0950 −0.0037 0.6292 −0.1667
0.2792 ± 0.0322 0.0003 ± 0.0001 0.0631 ± 0.0048 0.0008 ± 0.0001 0.0001 ± 1.0255 × 10−5 0.0449 ± 0.0038 0.0131 ± 0.0010 15.6600 ± 0.8418 0.0547 ± 0.0012 3.1420 ± 0.2865 0.5910 ± 0.0564
−0.5541 −3.4850 −1.2000 −3.1000 −3.8990 −1.3470 −1.8840 1.1950 −1.2620 0.4971 −0.2284
membrane blebbing, and apoptotic bodies. Apoptotic characteristics became more severe when the IL concentration quadrupled to 0.6250 mg/mL with the same treatment time of 12 h. For example, the number of blebs and apoptotic bodies increased. When the treatment time was increased to 72 h, these necrotic phenomena, such as swelling of cell structures, rupture of the plasma membrane, organelle breakdown, and increased release of cytoplasmic contents, blurred cell membrane were observed (Fig. 2A4). The results above indicated that IL concentration and exposure time strongly affected its cytotoxicity. These phenomena suggested that the necrosis of Sf-9 cells caused by [C2mim][Br] under high IL concentration and long exposure time [51]. However, the observed apoptotic characteristics of cell shrinkage, membrane blebbing, and apoptotic bodies suggested that cell death induced by ILs occurs via apoptosis rather than necrosis under low tested concentration.
important in the cytotoxicity, the longer alkyl chain lengths tended to have stronger lipophilicity, which increased the odds of contact with the lipid bilayer and hydrophobic proteins of the membrane, thus disrupting the normal physiological function of the membrane, even leading to the cell death [43,44]. However, the toxicity did not increase after a certain number of carbons (for example n = 4), which is generally described as a “cut-off effect” [45,46]. 3.1.3. Influence of the anion Toxicity of ILs with the same cation and different anions was also assessed ([C4mim]Br, [C4mim]Cl, and [C4mim]BF4). As shown in Fig. 1(d), [C4mim][Br] was the least cytotoxic of the three tested ILs. Sf9 viabilities after treatment with [C4mim]Cl and [C4mim]BF4 were similar at low concentrations (0.0001 – 0.1 mg/mL), but differed at high concentrations (1.0–10 mg/mL). Overall, the cytotoxic strengths of the ILs followed this order: Br− < Cl− < BF4− at 0.1 mg/mL. For Br− and Cl−, the cytotoxic effect may be attributed to the molecular weight, which allows the molecule to enter the cells easily [47]. BF4−, in contrast, is hydrolyzed to HF at high concentration, which is more cytotoxic than either Br− or Cl− [48–50]. These results suggest that the species of anion affect the ILs cytotoxicity on Sf-9 cells [42].
3.2.2. Ultrastructure changes in Sf-9 cell lines induced by [C2mim][Br] Apoptotic cells share a number of common features such as cell shrinkage, membrane blebbing, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies of condensed chromatin [52]. To further distinguish the cell death process induced by [C2mim] [Br] and to observe the toxic effect of ILs on cell ultrastructure, the treated cells were observed with a TEM. As can be seen in Fig. 3A, normal Sf-9 cells had continuous cell membrane integrity, uniform cytoplasm chromatin, proper organelle structures, and nuclei at the center of the cells. However, the cells treated with 0.6250 mg/mL [C2mim][Br] for 12 h had a large number of vacuoles and showed transformation of the cell membrane (Fig. 3B) [53]. Cells treated for 24 h showed some apoptotic characteristics, such as membrane blebbing, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies. Moreover, a shift of chromatin to one side of the cell and
3.2. Morphological changes in Sf-9 cells induced by [C2mim][Br] 3.2.1. Cellular morphology of Sf-9 cells Cellular morphological observations was reliable tool for distinguishing apoptotic and necrotic cell death. The changes in cell morphology induced by [C2mim][Br] at different concentrations were observed using inverted microscopy. As shown in Fig. 2, when the Sf-9 cells were treated with 0.1563 mg/mL [C2mim][Br] for 12 h, typical apoptotic characteristics could be observed, such as cell shrinkage,
Fig. 2. Morphological changes in Sf-9 cells induced by several concentrations and exposure times to [C2mim][Br], as seen via inverted microscopy. Magnification 400 × .
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Fig. 3. Transmission electron microscope images of Sf-9 cells. A shows normal cells; B, C, and E show cells treated with the 0.6250 mg/mL [C2mim][Br] for 12 h, 24 h, and 48 h, respectively. D shows Sf-9 cells treated with 2.5000 mg/mL [C2mim][Br] for 24 h.
nuclei of cells treated with 0.1563 mg/mL [C2mim][Br] were stained deeper green than those of cells treated with 0.0391 mg/mL [C2mim] [Br], suggesting that the DNA was highly condensed. Moreover, the nuclei of cells treated with high-concentration IL were stained orange, possibly due to the overlapping fluorescence between RNA in the cytoplasm and DNA in the nucleus. A similar phenomenon was observed in cells treated for longer time. These results indicate that the cell death induced by the IL occurred via apoptosis rather than necrosis.
nuclear membrane invagination were observed [51] (Fig. 3C) along with apoptotic bodies, organelle vacuolation, and chromatin content reduction (Fig. 3D). In Fig. 3E, a vanishing nuclear membrane, cell shrinkage, and chromatin reduction can be seen, which indicates the final stage of apoptosis [51,53] Taken together, we conclude that [C2mim][Br] induced apoptosis rather than necrosis in Sf-9 cells. 3.2.3. Nuclear morphology changes in Sf-9 cell lines induced by [C2mim] [Br] To study the effect of ILs on Sf-9 cell nuclei, AO stain was used. Following staining, intact cell nuclei were stained light green, where asnecrotic cell nuclei were stained deep green and orange due to a heat reaction with the stain. We observed that the amount of stained nuclei increased in the cell cytoplasm after treatment with [C2mim][Br], which may have been caused by the breakdown of the nuclear membrane (Fig. 4). The nuclei of cells treated with IL solution were stained deeper green than those of the controls, with a heterogeneous distribution. This phenomenon indicated that the cells were undergoing the typical apoptotic feature of chromatin condensation [51]. The
3.2.4. DNA fragmentation of Sf-9 cells DNA agarose gel electrophoresis is commonly used to differentiate between apoptosis and necrosis [51]. The results for the DNA agarose gel electrophoresis are shown in Fig. 5, where it can be easily seen that the DNA stripes in cells not treated with ILs or killed with heat were continuous (A1, B1, and C1). In contrast, a DNA ladder with a molecular spacing of approximately 180–200 base pairs, which is a hallmark of apoptosis, appeared in the cells treated with IL (A2-6, B2-6, and C26). These results also verified that the cell death induced by IL occurred via apoptosis rather than necrosis [51].
Fig. 4. Nuclear morphological changes in Sf-9 cells induced by different [C2mim][Br] concentrations over 24 h or 48 h using acridine orange staining. Sf-9 cells not treated with the IL were used as negative controls, and heat-killed cells with 100 °C boiling water for 5 min were used as positive controls. Magnification 400 × .
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Fig. 5. Agarose gel electrophoresis of DNA extracted from Sf-9 cells treated with different concentrations of [C2mim][Br] for 24 h, 48 h, and 72 h. The mark “m” indicates the DNA marker and “c” indicates the nontreated control group. Samples A, B, and C were treated for 24 h, 48 h, and 72 h, respectively. Sample number 1 was the negative control group treated by heating to 100 °C for 5 min. Sample numbers 2, 3, 4, 5, and 6 were treated with IL at final concentrations of 0.0391, 0.1563, 0.6250, 2.5000, and 10.0000 mg/mL, respectively.
Fig. 6. Flow cytometry analysis of apoptosis rates in Sf-9 cells after 24 h of treatment with various concentrations of [C2mim][Br].
2.5000 mg/mL IL concentration, and the minimum apoptosis rate was 29% (Q2 + Q3) at 0.0391 mg/mL IL concentration, from which it can be deduced that the effect of IL on Sf-9 cells was apoptotic rather than necrotic. The results was similar with the reported studies, which investigated the HepG2 cell apoptosis induced by 1-methyl-3-octylimidazolium bromide IL, the cell apoptosis rate increased from 7.52% (control) to 26.38% with increasing concentration of tested IL, indicating that the IL induce the cell apoptosis [30]. Moreover, early
3.3. Flow cytometry analysis of Sf-9 cell lines treated with [C2mim][Br] 3.3.1. Rate of apoptosis To further study the effect of ILs on Sf-9 cells, Annexin V-EGFP Apoptosis Detection Kit was used to measure the rate of apoptosis induced by 24 h of treatment with different concentrations of [C2mim] [Br]. It can be observed in Fig. 6 that the apoptosis rate of control was 9.1% (Q2 + Q3), the maximum apoptosis rate was 43% (Q2 + Q3) at 7
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Fig. 7. The cell cycle of Sf-9 cells exposed to different [C2mim][Br] concentrations over 24 h.
apoptosis increased from 9.1% (control) to the maximum value 43% (treated with 2.5000 mg/mL [C2mim][Br]). The cell cycle analysis revealed that [C2mim][Br] can disrupt the Sf-9 cell cycle. From all these results above, it can be concluded that [C2mim][Br] could cause the apoptosis of Sf-9 cells rather than necrosis. This study supplements the current insufficient knowledge of IL cytotoxicity in insect cells and clarifies the cytotoxicity mechanism. Moreover, it provides support for the potential future use of ILs as insecticides.
apoptosis was the main mechanism at lower IL concentrations, whereas late apoptosis was the main mechanism at higher IL concentrations. The results also show that the higher the IL concentration, the larger the destructive effect on the cells. 3.3.2. Effect of IL treatment on the Sf-9 cell cycle The cell cycle of Sf-9 cells exposed to various concentrations of IL for 24 h was examined using a Cell Cycle Kit. After exposure to [C2mim] [Br], the number of G0/G1 phase cells remained approximately constant, the number of S phase cells decreased, and the number of G2/M phase cells increased (Fig. 7). It can be concluded that the influence of [C2mim][Br] on the cell cycle was mainly in the S and G2/M phases, particularly in the G2/M phase. When the IL concentration increased to 10.0000 mg/mL, the cell cycle was similar to that of normal cells, which indicated that the excess concentration of IL led to cell necrosis.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 21406262), Central Public-interest Scientific Institution Basal Research Fund (No. 1610242016006), and the Fundamental Research Funds for the Central Universities of Central South University (No. 2017zzts336).
4. Conclusions References The cytotoxic effects of 11 different ILs on Sf-9 cells were evaluated via MTT assays, and the IC50 values were obtained. The results showed that the cation head groups, alkyl chain lengths, and type of anions played major roles in the degree of cytotoxicity caused by the ILs. Moreover, the tested ILs concentration and treated time also contributed to their cytotoxicity. In addition, the mechanism of Sf-9 cells death induced by [C2mim][Br] was determined using biochemical tools, including AO staining, DNA agarose gel electrophoresis, flow cytometry and observations of cell morphologies via inverted microscopy and TEM. The results of AO staining indicated the cells were undergoing the typical apoptotic feature of chromatin condensation. Moreover, a DNA ladder was found in the DNA agarose gel electrophoresis analysis. The morphological features of apoptosis such as cell shrinkage and convolution, apoptotic bodies, pyknosis, and karyorrhesis were observed by inverted microscopy and TEM. The rate of cell
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