The insecticide spinosad induces DNA damage and apoptosis in HEK293 and HepG2 cells

Accepted Manuscript Title: The insecticide spinosad induces DNA damage and apoptosis in HEK293 and HepG2 cells Author: Mingjun Yang Guanggang Xiang Diqiu Li Yang Zhang Wenping Xu Liming Tao PII: DOI: Reference:

S1383-5718(16)30036-5 http://dx.doi.org/doi:10.1016/j.mrgentox.2016.11.001 MUTGEN 402784

To appear in:

Mutation Research

Received date: Revised date: Accepted date:

1-2-2016 12-10-2016 2-11-2016

Please cite this article as: Mingjun Yang, Guanggang Xiang, Diqiu Li, Yang Zhang, Wenping Xu, Liming Tao, The insecticide spinosad induces DNA damage and apoptosis in HEK293 and HepG2 cells, Mutation Research/Genetic Toxicology and Environmental Mutagenesis http://dx.doi.org/10.1016/j.mrgentox.2016.11.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The insecticide spinosad induces DNA damage and apoptosis in HEK293 and HepG2 cells Mingjun Yang, Guanggang Xiang, Diqiu Li, Yang Zhang, Wenping Xu, Liming Tao  a

Shanghai Key Lab of Chemical Biology, School of Pharmacy, East China University of Science and Technology,

Shanghai 200237, China



Corresponding authors. Tel.: +86 2 164 253 756; fax: +86 2 159 883 730. E-mail addresses: [email protected] (L. Tao). 1

Highlights 1. This study evaluates the cytotoxicity of spinosad and its mode of action in HEK293 and HepG2 cells. 2. Spinosad induced increases on single- and double-strand DNA breaks in HEK293 and HepG2 cells. 3. DNA damage-induced p53 accumulation and modulation of Bcl-2/Bax ratio in HEK293 and HepG2 cells; 4. Spinosad induced disruption of the mitochondrial membrane potential in HEK293 and HepG2 cells. 5. Spinosad induced apoptosis in HEK293 and HepG2 cells through mitochondrial pathways.

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Abstract: Spinosad, a pesticide acting on the central nervous system of insects, is classified as a pesticide with reduced risk. However, spinosad-induced toxicological effects on non-target organisms must not be ignored. This study aimed to evaluate the cytotoxicity and potential genotoxicity of spinosad in HEK293 and HepG2 cell lines. The results showed that spinosad caused a concentration- and time-dependent decrease in cell viability of HEK293 and HepG2 cells. Spinosad-induced p53 accumulation thereby upregulates the expression of Bax and downregulates the expression of Bcl-2. Further studies confirmed that spinosad induced apoptosis in HEK293 and HepG2 cells, accompanied by a dissipation of the mitochondrial membrane potential and an increase in caspase-3 activity. The alkaline comet assay and γ-H2AX foci staining revealed that spinosad induced significant concentration-dependent increases of DNA strand breaks in HEK293 and HepG2 cells. Our results indicate that spinosad effectively induced DNA damage and apoptosis in HEK293 and HepG2 cells. Keywords: Spinosad; DNA damage; Comet assay; H2AX foci; Apoptosis; Cytotoxic

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1.

Introduction

Spinosad is a mixture of secondary metabolites produced by the soil actinomycete Saccharopolyspora spinosa and is composed of two major active ingredients: spinosyn A and spinosyn D [1]. The naturally occurring spinosyn compounds are a novel kind of macrolides with a unique 21-carbon, 12-membered tetracyclic lactone to which the deoxy sugars for osamine and tri-O-methyl rhamnose are attached [2]. As a stomach and contact poison, spinosad triggers nicotinic acetylcholine receptors (nAChRs) and inhibits gamma-amino butyric acid (GABA) receptor in the central nervous system of insects [3, 4]. It has been reported that the insecticide has a potently effect against a series of species of Lepidoptera, Physopoda, Diptera and parasites, such as mites and fleas [5-8]. In addition, photodegradation and microbial degradation are the primary pathways of degradation of spinosad in the environment. Spinosad is rapidly degraded in soil, and the degradation rate might be accelerated in aqueous solutions [9, 10]. Due to its relatively low acute toxicity towards mammals (LD50 values: rats; between 2000 and 5000 mg/kg, mice; > 5000 mg/kg) and being environmentally friendly[11], spinosad has been widely used in integrated pest management (IPM) programmes for different insect pests of numerous crops[1]. However, absolute selectivity will be difficult to accomplish. Most pesticides, therefore, are more or less toxic towards non-target creatures, including humans. Some studies have suggested that high doses of spinosad exposure can cause toxic effects in mammals, which include cell necrosis, cytoplasmic vacuolisation, membrane permeability change, inflammatory response, sometimes accompanied by anaemia, disruption of hormone homeostasis and prevention of weight gain [12-16]. Recent in vitro or in vivo experiments also demonstrated that spinosad treatments can induce DNA fragmentation, structural chromosomal aberrations, oxidative stress, activation of caspase-3 and apoptosis [4, 15, 17-19]. Toxicological studies on the effects of spinosad on humans have been rarely conducted before, since an extensive search of the literature found only toxicological information obtained from laboratory animal studies [20]. Because spinosad exhibits high log Kow values in neutral pH, it can easily pass through the biological membrane. The kidney and liver are the organs where toxic metabolites accumulate in an organism; hence, marking these key locations where high toxicity occurs is important [26]. In this study, we investigated the cytotoxicity of spinosad towards normal (HEK293) and cancerous (HepG2) human cell lines. We also examined the apoptosis induced by spinosad in the target cells, which was initiated by p53, and then the alteration in the expression of Bcl-2-related proteins and the activity of caspase-3. Single- and double-strand DNA breaks in the target cells exposed to spinosad were measured using alkaline comet assay and γ-H2AX foci staining, respectively. 2.

Materials and methods 4

2.1 Chemicals and reagents Spinosad (98% purity); MTT, 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl-tetrazolium bromide; Tris, 2-Amino-2-(hydroxymethyl)-1,3-propanediol; TEMED, N,N,N’,N’-tetramethylethylenediamine; Rhodmine123; DMSO, dimethylsulfoxide and hydrogen peroxide (H2O2) were purchased from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA). Polyacrylamide (30%) was obtained from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). For the experiments, stock solution of spinosad was prepared in DMSO and diluted into the culture media to the desired concentrations. The final concentration of DMSO was < 0.1% in the cell culture media. DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; penicillin-streptomycin(10,000 U/mL); trypsin-EDTA solution (0.25%) were purchased from Thermo Fisher Scientific Inc (Grand Island, NY, USA). Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Yesen biological technology Co., LTD (Shanghai, China). Other reagents and chemicals used were of analytical grade and purchased locally. 2.2 Cell line and culture conditions The cell lines HEK293 and HepG2, were obtained from cellbank of chinese academy of sciences(Shanghai, China), and routinely maintained in DMEM high glucose culture medium supplemented with 10% FBS, 1% penicillin-streptomycin in 25 cm2 culture flasks (Corning, NY, USA). Subcultivation was accomplished by trypsination in a ratio of 1:3 every two or three days. Cells were maintained in an incubator at 37 °C in a humidified atmosphere of 5% CO2, and maintained in exponential and asynchronous phase of growth. 2.3 Cell viability test The MTT bioassay was performed to monitor cell viability [21]. Cell suspensions (3×104 cells/cm2) were pipetted in a 96-well plates. One-hundred microliters of spinosad was added to the final concentrations of 5, 10, 20, 40 and 80 μmol/L. The wells containing complete DMEM only served as blank, the wells containing complete DMEM and cell served as control. Four hours before the end of incubation, 10 μL of MTT solution (5 mg/mL in PBS) was added into each well. To dissolve the formazan crystals, supernatant was removed and 100 μL of DMSO was added to each well. The optical density (OD) was read on a microplate reader (BioTek, Winooski, VT, USA) at 570 nm with a reference wavelength at 630 nm. The percentage of relative inhibition rate was calculated as follows: Sample OD-Blank OD Relative cell viability= ×100% Control OD-Blank OD 2.4 Apoptosis assay Apoptosis was determined by flow cytometry using a Annexin V-FITC/PI Apoptosis Detection Kit according to the method of Wang, Zhao [22]. After exposure to 0, 10, 20 and 40 μmol/L spinosad for 24 h (HEK 293), and 0, 12.5, 25 and 50 5

μmol/L spinosad for 24 h (HepG 2). Approximately 1 × 106 cells from each condition were collected by centrifugation (600 g, 5 min) and washed with PBS. The cell pellets were resuspended in 100 μL ice-cold binding buffer. 5 μL AnnexinV-FITC solution and 5 μL dissolved PI were added to cell suspensions, then mixed gently and incubated at room temperature for 10 min in the dark. After incubation, 400 μL of binging buffer was added and cells were kept on ice until analyzed on a flow cytometer (FACS Calibur, BD Biosciences) within 60 minutes. The fluorescence data was captured using an excitation wavelength of 488 nm and an emission wavelength of 525 nm for annexin V-FITC, an excitation wavelength of 535 nm and an emission wavelength of 615 nm for PI. Data analysis was performed using the Flowjo software program. 2.5 Measurement of caspase-3 activity Caspase-3 activity in cell lysates was determined using a colorimetric substrate Ac-DEVD-pNA (Santa Cruz, CA, USA). After incubation with Spinosad for 24 h, cells were collected and lysed using lysate buffer. The cell lysate was centrifuged (12,000 g, 4℃ for 5 min), and then 50 μl of the supernatant (cytosolic extracts, 100 mg) was mixed with 40 μl of reaction buffer (10 mmol/L Tris·HCl, 1mmol/L CaCl2, pH 8.0) and 10 μl of Ac-DEVD-pNA (2 μmol/L). After overnight incubation at 37 °C, the absorbance was measured with a plate reader at a wavelength of 405 nm (BioTeck, VT, USA). Caspase-3 activity was expressed as a percentage of control activity[23]. The experiment was repeated three times. 2.6 Alkaline comet assay Spinosad-induced DNA damage was measured with a comet assay that was conducted as previously described [24] with some minor modifications. In short, HEK293 cells were treated with 0, 10, 20, 40 and 80 μmol/L spinosad for 24 h, and HepG 2 cells were treated with 0, 12.5, 25, 50 and 100 μmol/L spinosad for 24 h. All cells treated by H2O2 (20 μmol/L) were used as positive controls. The cells were collected and washed with PBS, the pellets were resuspended in PBS. Cells suspension were mixed with prewarmed 1% low melting point agarose and layered on microscopic slides precoated with 0.8% normal melting point agarose. After incubation at 4 °C for solidification, the slides were immersed in lysis buffer (100 mmol/L Na2EDTA, 2.5 mol/L NaCl, 10mmol/L Tris-HCl, 1% Triton X-100 and 10% DMSO, pH10) for 2 h at 4 °C in the dark. Slides were washed in distilled water and submerged in ice cold electrophoresis buffer (0.3 mol/L NaOH, 1 mmol/L Na2EDTA, pH 13) for 30 minutes. An electric field was then applied at 20 V (1 V/cm) and 300 mA for 10 min. Slides were neutralised (0.4 mol/L Tris-HCl, pH 7.4) and washed with distilled water before staining with 30 μL of 20 μg/mL PI solution. Comets were photographed with a fluorescence microscope (Leica, Wetzlar and Mannheim, Gemany) with a filter of 515-560 nm. At least 100 cells were randomly selected from each group and analyzed using the Comet Assay Software Project (CASP). 2.7 H2AX foci staining Immunofluorescence microscopy was used to visualize histone H2AX 6

phosphorylation that as a marker of DNA double-strand breaks [25]. HEK293 and HepG2 cells were treated with various concentration spinosad and 20 μmol/L H2O2 for 24 h. Cells were fixed with 4% formaldehyde for 15 min, washed with PBST (PBS buffer and 0.1% Tween 20, pH 7.4), and permeabilized with 1% Triton X-100. After that, cells were blocked with 5% BSA for 1 h, samples were incubated with a rabbit monoclonal anti-γH2AX antibody (Abgent Inc, Suzhou, China, 1:1500) for 2 h, followed with Alexa Fluor 488-conjugated goat-anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories, West Grove, USA,1:360) for 1 h. To stain the nuclei, DAPI (1mg/mL) was added to the cells and incubated for another 30 min. Acquisition of images was carried out using a fluorescence microscope. Objectives were set at wavelengths of 594 nm for γH2AX and 350 nm for DAPI. To prevent bias in selection of cells that display foci, over 200 randomly selected cells were counted. Cells with four or more foci of any size were classified as positive. 2.8 Western blot analysis HEK293 cells were treated with 0, 10, 20 and 40 μmol/L spinosad for 48 h, and HepG 2 cells were treated with 0, 12.5, 25 and 50 μmol/L spinosad for 24 h. The cells were harvested by gentle centrifugation (600 g, 5 min) and washed with PBS. The cell pellets were lysed in lysis RIPA buffer (50 mmol/L Tris-HCl, pH 8.0, with 150 mmol/L sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) and protease inhibitor cocktail (Sigma, St. Louis, USA). Protein concentrations were determined by the BCA protein assay reagent kit (Thermo Scientific, Rockford, USA). The protein samples (40μg each) were resolved by 12% SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene-difluoride (PVDF) membrane. The membranes were blocked for 2 h at room temperature with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20. The primary antibodies against p53, Bax and Bcl-2 (Cell signaling technology, Beverly, USA) were added and detected with secondary antibody conjugated with horseradish peroxidase and analyzed by enhanced chemiluminescence (Tanon, Shanghai, China). 2.9 Mitochondrial membrane potential analysis The mitochondrial transmembrane potential (ΔΨm) was assessed using Rhodmine 123 dye[26]. Cells (1.5 × 106 cells per well) cultured in 6-well microtiter plates. The cells were incubated with different concentrations of spinosad for 24 h, Rhodmine 123 was added to cell suspension at a final concentration of 50 nmol/L at 37 °C for 30 minutes prior to harvest, and then cells were pelleted by centrifugation (600 g, 5 min) and washed twice with PBS. All the procedure was performed under dim light. The fluorescence intensity of the cells was determined by FACS Calibur flow cytometry (BD Biosciences) with excitation and emission settings of 490 and 535 nm, respectively. 2.10 Statistical analysis Data were shown as averages ± standard deviation of the three independent experiments. Data were analyzed by one-way analysis of variance (ANOVA) followed 7

by a t-test, and p-values less than 0.05 were considered statistically significant. 3.

Results

3.1 The cytotoxic effects of spinosad HEK293 and HepG2 cells were treated with increasing concentrations of spinosad for 24, 48 and 72 h. Results showed that spinosad caused a concentrationand time-dependent decrease in cell viability of HEK293 and HepG2 cells (Fig. 1A, Fig. 1B). The data demonstrated that spinosad has cytotoxic effects on both normal and cancer cells (Fig. 1). The median inhibitory concentrations (IC50) of spinosad for HEK293 and HepG2 cells are given in Table S1. In general, the cytotoxic effects of spinosad were non-selective between HEK293 and HepG2 cells. 3.2 Spinosad exhibits cytotoxic effects through apoptotic cell death In apoptotic cells, the phosphatidyl serine (PS), which is located in the inner layer of the plasma membrane in normal cells, is translocated to the outer plasma membrane. Annexin V, a Ca2+-dependent protein with high affinity for PS, was used to identify apoptosis at an early stage [27]. Quantification of apoptotic cells was performed using FACS analysis of Annexin V-FITC/PI double staining after spinosad treatment for 24 h. The flow cytometric quantification of dual staining revealed that spinosad increased the apoptotic populations of HEK293 and HepG2 cells in a dose-dependent manner. Moreover, within HEK293 cells, there was a higher percentage of Annexin V-FITC single-positive cells compared to that in HepG2 cells (Fig. 1C). The ratios (%) of apoptosis in HEK293 and HepG2 cells after exposure to spinosad are shown in Fig. 1D. The data demonstrate that spinosad plays a strong role in inducing cell apoptosis in HEK293 and HepG2 cells. Caspase-3 has been identified as a major contributor to the initiation of apoptosis. Fig.1E shows that caspase-3 activity in HEK293 and HepG2 cells increased with increasing spinosad concentration. These results indicated that spinosad-induced apoptosis in HEK293 and HpeG2 cells was mediated by the caspase cascade. 3.3 Spinosad induces DNA single-strand breaks The alkaline comet assay was used to determine DNA single-strand breaks [28]. The amount of DNA that migrates away from the nuclei is used to assess the extent of DNA damage. The HEK293 and HepG2 cells exposed to spinosad for 24 h exhibited significant induction of DNA breakage, increasing the migration of DNA fragments, and a tail of comet-like shape appeared, which was similar to that in the positive control group. Fig. 2A shows the comet images for HEK293 and HepG2 cells; the comet tails were observed to be lengthened in a concentration-dependent manner. The negative control group cells showed a nucleoid core with zero or minimal DNA migration. Data on individual cell response using the tail length (TL), Olive tail moment (OTM) and Tail DNA% are presented in Table S2. The OTM values of alkaline comet assay in HEK293 and HepG2 cells showed significant increases (p < 0.05) at 20 and 25 mmol/L, compared to the control (Fig. 2B). 3.4 H2AX foci revealing DNA double-strand breaks induced by spinosad 8

Phosphorylated H2AX is a marker of double-strand break formation and repair, and examination of γ-H2AX foci formation is a powerful tool to measure DNA double-strand break and cellular response to DNA damage [29]. Shown in Fig. 3A are representative images of HEK293 and HepG2 cells treated with positive control, various concentrations of spinosad for 24 h, which clearly demonstrate that spinosad can indeed induce γ-H2AX foci formation in a concentration-dependent manner. Detailed analyses (as shown in Fig. 3B) revealed that about 3.8% of HEK293 and 6.3% of HepG2 cells contained more than four foci. With the increase of the concentration of spinosad, the ratio of γ-H2AX-positive cells obviously increased. 3.5 Effects of spinosad on the expression of apoptotic proteins In response to DNA damage, p53 has been suggested to induce apoptosis through regulation of Bax and Bcl-2 expression, and the ratio of Bcl-2 to Bax expression determines the destiny of cells [30]. After exposure to different concentrations of spinosad, protein levels of p53, Bax and Bcl-2 in HEK293 and HepG2 cells were evaluated by Western blot analysis. Treatment of cells with spinosad resulted in a dose-dependent accumulation of p53 protein, which was positively related to Bax expression and negatively related to Bcl-2 expression (Fig.4). These results indicated that spinosad-induced apoptosis in HEK293 and HpeG2 cells was caused by apoptosis protein expression. 3.6 Depolarisation of the mitochondrial transmembrane potential Mitochondria have a pivotal role in the life and death of eukaryotic cells, and mitochondrial membrane potential changes as markers of mitochondrial function are often associated with apoptosis [31, 32]. HEK293 and HepG2 cells were treated with various concentrations of spinosad for 24 h. After staining with Rhodamine-123, the mitochondrial transmembrane potential was analysed by flow cytometry. The fluorescence intensity was decreased in a concentration-dependent manner, which demonstrated the occurrence of depolarisation of mitochondrial transmembrane potential (Fig. 5A). HEK293 and HepG2 cells exposed to spinosad for 24 h showed increased percentage of low-intensity fluorescence, suggesting depolarisation of the mitochondrial transmembrane potential following spinosad treatment (Fig. 5B). Obviously, spinosad treatment induces apoptosis of both HEK293 and HepG2 cells through disruption of the mitochondrial membrane potential. 4.

Discussion

Owing to its highly favorable environmental and toxicological profile, spinosad was classified as an insecticide with reduced risk. However, in recent years, a growing body of research has revealed that spinosad can cause variable toxic effects on nontarget organisms. Toxicological assessment of spinosad has been conducted using several species of laboratory animals with different concentrations (spinosad concentration ranging from 50 mg/kg to 400 mg/kg) [17, 33]. High dosages of spinosad may increase the likelihood of potentially significant toxic effects. In this study, the results showed that spinosad caused a decrease in cell viability, and 9

produced an induction of apoptotic cell death in HEK293 and HepG2 cells (Fig. 1). We also demonstrated that apoptosis may be associated with induction of DNA damage mediated via p53. Induction of apoptosis occurs in response to various stresses including DNA damage. The accumulation of p53 in the presence of DNA damage induced the expression of specific target genes, such as p21WAF, Bax, and Puma, to initiate cell cycle arrest, apoptosis, and DNA repair [34]. Our study demonstrated that spinosad treatment of HEK293 and HepG2 cells triggered apoptosis, which may be controlled by p53-mediated upregulation of Bax and downregulation of Bcl-2 (Fig. 4). In addition, HEK293 cells constitutively express adenovirus proteins E1A and E1B, which significantly inhibit the p53- and pRB-induced cellular senescence or apoptosis [35]. After treating with spinosad for 24 h, HEK293 cells had a nonsignificant upregulation of p53 and Bax, in contrast to HepG2 cells. When the exposure period was extended to 48 h, the expression of p53 protein was significantly upregulated in HEK293 cells (Fig. 4), which is consistent with the literature on olaquindox-induced DNA damage via p53 expression in HEK293 cells [36]. The Bax/Bcl-2 ratio determines the fate of cells in spinosad-induced apoptosis. Bax inserts into the mitochondrial outer membrane forming oligomers that lead to mitochondrial outer membrane permeabilization, release of mitochondrial apoptogenic proteins, and initiation of the caspase cascade [37]. The results of the present study indicated that spinosad treatment induced mitochondrial membrane potential collapse, which indicates increased permeability of the mitochondrial membrane (Fig. 5). Compared to the rate of apoptosis, caspase-3 activity exhibited a small increase, especially in HepG2 cells (Fig. 1, C–E). On this basis, we speculate that spinosad-induced apoptosis in HEK293 and HepG2 cells may be partly mediated through a caspase-independent and p53-dependent death pathway associated with mitochondrial damage [38]. There is extensive evidence regarding DNA damage induced by spinosad. Mansour et al. [15] demonstrated the genotoxic potential of spinosad in rats, showing a significant increase in chromosomal aberration and micronucleus frequency in rat bone marrow cells. However, another study has suggested that spinosad exposure causes DNA fragmentation in rat hepatocytes [17], which is in agreement with the findings of the present study. In response to DNA damage, cells trigger DNA damage response pathways to regulate DNA repair and cell death processes. In this study, we observed spinosad-induced γH2AX accumulation, indicating the formation of DNA double-strand breaks (DSBs) in HEK293 and HepG2 cells (Fig.3A). The formation of DSBs has previously been described for a number of DNA-interacting agents, including the DNA cross-linking agents, the monofunctional DNA alkylators, and the topoisomerase I inhibitor [39]. This shows that DSB formation may be a common cellular response to several different types of DNA damage. The comet assay can detect DNA damage, such as alkali-labile sites and strand breaks associated with the repair of these adducts [40]. Spinosad-induced DNA single-strand breaks in HEK293 10

and HepG 2 cells were assessed using the alkaline comet assay. Our results indicate that spinosad is able to induce DNA damage and apoptosis in human cells. In conclusion, the finding indicates the necessity for evaluating the toxic hazard of spinosad in humans.

Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was financially supported by the National Key Technology Research Development Program of China (NO.2011BAE06B04).

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Fig. 1. The cytotoxic effect of spinosad in HEK293 and HepG 2 cells. Cell viability of HEK293 (A) and HepG2 (B) cells after exposure to spinosad determined by MTT assay. (C) Representative dot plots of cells treated with the indicated concentrations of spinosad for 24. Fluorescence intensity of the untreated cells was adjusted such that the majority of the population was located in the lower left quadrant. Annexin V-FITC binding cells were presented on the horizontal axis and PI stained cells were distributed the vertical axis. Biparametric analysis revealed three distinct populations: untreated cells (FITC−/PI−); early apoptotic cells (FITC+/PI−); late apoptosis and/or necrotic cells (FITC+/PI+). (D) The percentage (%) of apoptosis cells. (E) The activity of caspase-3 was determined using a colorimetric substrate Ac-DEVD-pNA after 24h of exposure. The data are presented as mean ±SD of three independent experiments performed with triplicate measurements. *, ** p<0.05, 0.01 vs negative control of the same treatment group.

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Fig. 2. Spinosad induced strand breaks in cellular DNA of HEK293 and HepG2 cells by comet assay. HEK293 and HepG2 cells were treated by spinosad for 24h. (A) Comet images (200×); (B) the OTM values of alkaline comet assay. The data are presented as mean ± SD of three independent experiments performed with triplicate measurements. *, ** p<0.05, 0.01 vs control of the same treatment group.

Fig. 3. Spinosad induced DNA double strand breaks were illustrated by γH2AX foci formation in HEK293 and HepG2 cells. Cells were treated by spinosad for 24h. γH2AX-positive cells were classified as those with ≥4 foci/cell, 16

and the percentage of γH2AX positive cells was detected by visual scoring of at least 200 randomly selected cells after 24 h exposure to spinosad. (A) The immunofluorescent γH2AX foci images. Anti-γH2AX monoclonal antibody was used to detect DNA damage foci immunofluorescence and DAPI was for nuclei staining. (B) the percentage (%) of γH2AX positive cells. The data are presented as mean ± SD of three independent experiments performed with triplicate measurements. *, ** p<0.05, 0.01 vs control of the same treatment group.

Fig. 4. Effect of spinosad on the expression of apoptotic proteins in HEK293 and HepG2 cells. Cells were treated with the indicated concentrations of spinosad. Expression of p53, Bax and Bcl-2 in HEK293 and HepG2 cells after 48h and 24h-exposure period, respectively. β-actin was used for loading control.

Fig. 5. Depolarization of mitochondrial transmembrane potential in spinosad-treated HEK293 and HepG2 cells. Cells were treated with the indicated concentrations of spinosad for 24h. (A) Changes in mitochondrial membrane potential were detected by flow cytometric analysis of Rhodamine-123 stained cells. High intensity sub-populations mark as positive cells in the bimodal univariate histogram. (B) The ratio of cells in low-intensity fluorescence. The data are presented as mean ±SD of three independent experiments performed with triplicate measurements. *, ** p<0.05, 0.01 vs negative control of the same treatment group.

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