- Email: [email protected]
Enantioselective apoptosis induced by individual isomers of bifenthrin in Hep G2 cells
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 810–814
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Enantioselective apoptosis induced by individual isomers of bifenthrin in Hep G2 cells Huigang Liu ∗ , Juan Li Department of Public Health, School of Medicine, China Three Gorges University, 8# Daxue Avenue, Yichang 443002, China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Bifenthrin (BF) has been used in racemate for agricultural purposes against soil insects,
Received 5 December 2014
leading to increased inputs into soil environments. However, most of the studies about the
Received in revised form
toxicology research on BF were performed in its racemic form. The aim of the present study
9 February 2015
was to evaluate the enantiomer-specific cis-BF-induced apoptosis and intracellular reactive
Accepted 14 February 2015
oxygen species (ROS) generation on human hepatocarcinoma cells (Hep G2). The results
Available online 21 February 2015
of cell viability assay and cytoflow assay indicated an obvious enantioselective hepatocyte toxicity of 1S-cis-BF in Hep G2 cells. 1S-cis-BF also induced ROS production, up-regulated Bax
Keywords:
protein expression and down-regulated Bcl-2 expression levels. The present study suggested
Apoptosis
that enantioselective toxicity should be evaluated on currently used chiral pesticides, such
Enantioselective
as synthetic pyrethroids.
Pyrethroid pesticides
1.
Introduction
Synthetic pyrethroids (SPs) are widely used in most sectors of agricultural production to ensure high crop yields for decades (Tyler et al., 2000). Some pyrethroid pesticides have played a key role in providing reliable supplies of agricultural production at prices affordable to consumers and ensuring high profits to farmers. Recent works point out this ubiquity in the environment, even in vegetables (Velki and Hackenberger, 2013; Hall and Anderson, 2013; Phyu et al., 2013). Many SPs used as pesticides are chiral compounds (Carlsson et al., 2014). In recent years, people have become increasingly concerned about chiral pesticides use and particularly about their impact on human health and environmental quality (Liu et al., 2005). Bifenthrin is effective against a wide range of insect and has been used for plant protection (Solomon et al., 2001). Previous study indicated that 1S-cis-BF presented more toxic effects
∗
Corresponding author. Tel.: +86 07176396818. E-mail address: [email protected] (H. Liu).
http://dx.doi.org/10.1016/j.etap.2015.02.010 1382-6689/© 2015 Elsevier B.V. All rights reserved.
© 2015 Elsevier B.V. All rights reserved.
than 1R-cis-BF on human amnion epithelial (FL) cell lines. FL cells incubated with 1S-cis-BF exhibited a dose-dependent accumulation of ROS (Liu et al., 2008). Many studies reported that SPs have been associated with many human health hazards, ranging from short-term impacts such as allergies, and headaches to chronic impacts like reproductive disorders and neurological disorders (Wang et al., 2009; Guvenc et al., 2013). SPs are also known to be hepatotoxic and carcinogenic (Giray et al., 2001). Many of the toxic effects elicited by exposure to pesticides are mediated by the regulation of apoptosis (Park et al., 2013). It has been demonstrated that pyrethroid insecticides have an apoptotic effect on different organisms (Casco et al., 2006). Apoptosis can be induced by a wide range of chemical, physical and genetic factors. However, most of these studies were performed in their racemic form of these chiral pesticides. Hence, in the present study, we aim to evaluate the apoptosis and ROS generation induced by
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 810–814
individual isomers of cis-BF on human hepatocarcinoma cells (Hep G2).
2.
Materials and methods
2.1.
Chemicals
Racemic cis-BF (analytical standard, 99.5%, 2-methylbiphenyl3-ylmethyl-(Z)-(1RS)-cis-(2-chloro-3,3,3-trifluoroprop-1-enyl)2,2-dimethylcyclopropane carboxylate was from Sigma (St. Louis, MO, USA). Minimal essential medium (MEM) was purchased from Gibco (Paisley, UK), and fetal bovine serum (FBS) was from Shijiqing Reagent Company (Hangzhou, China). Thiazolyl blue (MTT) solution (5 mg/mL in phosphatebuffered saline (PBS)) was from Amresco (Solon, OH, USA). 2 7 -dichlorofluorescin diacetate (DCFH-DA) was from Sigma (St. Louis, MO, USA).
2.2.
Preparation of BF enantiomers
The enantiomers of cis-BF were resolved on a Jasco LC2000 series HPLC system (Jasco, Tokyo, Japan) equipped with a PU-2089 intelligent quaternary gradient pump, an AS-1559 autosampler with a 100 L loop, a mobile-phase vacuum degasser, an UV-2075 ultraviolet-visible detector, a variable-wavelength CD-2095 circular dichroism (CD) detector, a CO-2060 column temperature-control compartment, and an LC-Net II/ADC data collector (Jasco, Tokyo, Japan). The mobile phase (-cyclodextrin) was made of n-hexane fortified with ethanol. The Chiralcel OJ [250 mm × 4.6 mm i.d., cellulose tris (4-methyl benzoate) column (Daicel Chemical Industries, Tokyo, Japan)] was used. The detection wavelength of the CD detector was 230 nm. The resolved enantiomers were individually collected at the HPLC outlet, evaporated to dryness, and used in the following experiment. The enantiomers concentrations were tested on an Agilent 6890N gas chromatograph (GC) equipped with an electron capture detector (ECD), assuming the same response factor for enantiomers as for the racemate of cis-BF.
2.3.
Cell culture and treatments
Hep G2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Scientific HyClone, San Jose, CA, USA) with 10% (v/v) fetal bovine serum (FBS) at 37 ◦ C in a 5% CO2 incubator. Cells were subcultured every 2–5 days and seeded in 96-well plates (Costar, Cambridge, MA, USA) at a density of 2 × 105 cells/mL and allowed to adhere for 24 h prior to assaying.
2.4.
Assessment of cell viability
Cell viability was measured by quantitative colorimetric with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5assay diphenyltetrazolium bromide). Hep G2 cells were incubated with five different concentrations of individual 1S-cis-BF, 1R-cis-BF, or cis-BF for 12 h. MTT at 500 g/mL (final concentration) was added to each well and the cells were incubated at 37 ◦ C for 4 h. After removing MTT, the cells were lysed with
811
dimethyl sulfoxide (DMSO) (150 L each well). The absorbance at 490 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Control cells were treated with the vehicle (0.1% ethanol, onefold).
2.5.
Assay of the cellular contents of ROS
Hep G2 cells were incubated with individual enantiomers or a racemate of cis-BF at concentrations of 0, 5, 10, 15, and 20 mg/L for 2 h. After this treatment, cells were washed three times with ice-cold PBS then incubated with 10 M DCFH-DA for 30 min at 37 ◦ C. A fluorescent spectrophotometer (Tecan Infinite M200, Switzerland) was used to assay the cellular ROS by measuring 2 7 -dichlorofluorescein fluorescence (excitation at 485 nm/emission at 535 nm). The resulting ROS levels were determined as relative fluorescence intensities.
2.6.
Cell apoptosis assay
Hep G2 cells at 5 × 106 cells mL−1 were treated with individual or racemate cis-BF at 10 or 20 mg/L for 6 h, respectively. Then cells were harvested and washed twice with PBS. The treated cells were suspended in binding buffer at 3 × 106 cells mL−1 , and supplemented with 5 L of FITC-Annexin-V and 10 L of propidium iodide (PI) (Annexin-V-FITC Apoptosis Detection Kit, Sigma). Cell apoptosis was assessed by flow cytometry (Beckman Coulter EPICSXL, USA) after incubated for 20 min at room temperature in the dark. All experiments were repeated at least three times.
2.7.
Western blot analysis
The lysate was cleared by centrifugation at 17,000 × g for 30 min at 4 ◦ C after cells were homogenized on ice in lysis buffer. 50 g protein from each sample were loaded on a 12% SDS-PAGE gel and transferred to PVDF membranes by electrophoretic transfer and blocked with 5% nonfat dry milk in TBST buffer. Then membranes were incubated with Bax and Bcl-2 (ProteinTech Group) overnight at 4 ◦ C. Enhanced chemiluminescence (Pierce) was used to detect HRP-labeled secondary anti-rabbit antibody (Cell Signal). Blots were exposed to X-ray film for radiographic detection of the bands after immersion in ECL chemiluminescence reagents.
2.8.
Statistical analysis
Statistical significance was determined by one-way analysis of variance (ANOVA). The statistical analysis of the data was performed with the statistical program package SPSS 11.0. A p-value <0.05 was considered significant.
3.
Results and discussion
3.1. cells
Enantioselective cytotoxicity in human Hep G2
The results of MTT assay show that treatment with 1S-cisBF reduced cell viability in a dose-dependent manner (Fig. 1).
812
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 810–814
Fig. 1 – Effects of individual enantiomers and racemate of cis-BF on the viability of Hep G2 cell lines (*p < 0.05; **p < 0.01, n = 4). Hep G2 cells were treated with different concentrations of individual 1R-cis-BF, 1S-cis-BF, or cis-BF for 12 h.
While the reduction of cell viability by 1R-cis-BF has no significant difference. At the concentration of 20 mg/L, the order of viability of Hep G2 cells exposed to chemicals was 1Scis-BF (51%) < rac-cis-BF (63%) < 1R-cis-BF (83%) (p < 0.01); At the concentration of 15 mg/L, the order of viability of Hep G2 cells exposed to chemicals was 1S-cis-BF (68%) < rac-cis-BF (77%) < 1R-cis-BF (82%) (p < 0.05). There was no significant difference in other concentrations. Previous study indicated that 1S-cis-BF presented more toxic effects than 1R-cis-BF above the concentration of 7.5 mg/L on FL cell lines (Liu et al., 2008). In this study, the MTT assay showed that 1S-cis-BF and rac-cisBF induced cytotoxicity in concentration-dependent manners in Hep G2 cells. In contrast, 1R-cis-BF had no effect on MTT metabolism.
3.2. cis-BF
ROS production induced by individual isomer of
Hep G2 cells were incubated with individual enantiomers or a racemate of cis-BF for 2 h at concentrations of 0, 5, 10, 15, and 20 mg/L and the resulting ROS levels were determined as relative fluorescence intensities. Treatment with 1S-cis-BF or rac-cis-BF caused a dose-dependent accumulation of intracellular ROS in Hep G2 cells (Fig. 2). At the concentration of 20 and 15 mg/L, the order of ROS production of Hep G2 cells exposed to chemicals was 1S-cis-BF > rac-cis-BF > 1R-cisBF (p < 0.01). There was no significance compared to control and between the analyzed concentrations of chemicals in other concentrations. Oxidative stress has been implicated as the mechanism of cytotoxicity from numerous toxicants and many studies in humans or animals support that SPs can induce oxidative stress (Kale et al., 1999; Kumar et al., 2014; Narra, 2014). The generation of ROS plays an important role in apoptosis process induced by environmental pollutants (Xia et al., 2014). In the present study, 1S-cis-BF and rac-cis-BF
Fig. 2 – Effects of individual enantiomers and racemate of cis-BF on intracellular ROS production. Hep G2 cells exposed to different concentrations of individual enantiomers and racemate of cis-BF for 2 h, followed by ROS determination (*p < 0.05, **p < 0.01; n = 4).
induced ROS production, while 1R-cis-BF had no effect on ROS generation. It suggests that the ROS production induced by rac-cis-BF could be dominantly attributed to 1S-cis-BF but not 1R-cis-BF.
3.3.
Apoptosis induced by individual isomer of cis-BF
Hep G2 cells were treated with individual and racemate cis-BF at 10 and 20 mg/L, respectively, for 6 h. FITCAnnexin-V/propidium iodide staining assay was performed to determine the effects of cis-BF on Hep G2 cell apoptosis. Fig. 3 shows the results of the flow cytometric analysis of the cells. The bottom right quadrants: apoptotic cells. The bottom left quadrants: viable cells. The upper right quadrants: necrotic and late apoptotic cells. The percentage of cells in that category in ethanol (solvent) from cis-BF-treated samples were presented in each quadrant. The apoptosis rate in Hep G2 cells exposed to 1S-cis-BF, 1R-cis-BF and rac-cis-BF at concentration of 20 mg/L were 7.31%, 2.14% and 4.51%, respectively. To confirm the enantioselective apoptosis induced by individual isomers of cis-BF, the expression of the apoptotic proteins Bax and Bcl-2 were determined using Western blotting. 1S-cis-BF and rac-cis-BF treated Hep G2 cells showed an increase in pro-apoptotic Bax protein levels coupled with a decrease in Bcl-2 levels. However, 1R-cis-BF had no effect on Bax and Bcl-2 expression (Fig. 4). The Bcl-2 family of proteins has been implicated in either triggering or preventing apoptosis where a higher ratio of Bax to Bcl-2 has been shown to promote cell death. Bcl-2/Bax proteins are critical mediators of apoptosis (Manske et al., 2004). During apoptotic stimulation, these two proteins would affect mitochondrial Cyt c release, and the decrease in the Bcl-2/Bax ratio is considered a decisive correlation in apoptosis or cell death (Ouyang et al., 2012). SPs’ potential to induce apoptosis both in vitro and in vivo has been reported in previous studies (Hossain and Richardson, 2011).
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 810–814
Fig. 3 – Evaluation of apoptotic cells by the Annexin-V staining. (A) The percent of early apoptotic cells following treatment with individual enantiomers or racemate of BF was measured with flow cytometry (*p < 0.05, **p < 0.01; n = 4). (B) Apoptosis quantified by Annexin V (AV)-PI staining. Four subpopulations and their quantities are indicated. R1, non-apoptotic dead cells; R2, late apoptotic cells; R3, viable cells; R4, early apoptotic cells.
Long-term exposure to deltamethrin induced Cyt c release and altered expression of Bcl-2/Bax, leading to apoptotic cell death (Kote et al., 2006). In the present study, we found that 1S-cis-BF significantly up-regulated Bax protein expression and downregulated Bcl-2 expression levels. However, 1R-cis-BF had no significant effect on Bax/Bcl-2 expression compared to control cells. In conclusion, the present study evaluates cytotoxicity and apoptosis induced by individual isomers of BF in Hep G2 cells. Our study indicated that the apoptotic effects induced by cisBF could be almost entirely attributed to 1S-cis-BF enantiomer. Thus, it is of great significance to evaluate the possible hazards and the influence of the single chiral monomers of pesticides on environment and human health.
813
Fig. 4 – Involvement of Bax and Bcl-2 protein expression in cis-BF induced apoptosis. In the western blot analysis of Bax and Bcl-2 protein expression, Hep G2 cells were treated with individual enantiomers or racemates of cis-BF at the concentration of 20 mg/L for 6 h, and cell lysates were subjected to western immunoblotting. Bax protein expression in Hep G2 cells treated with 1S-cis-BF showed a significant increase in its levels compared to cells treated with 1R-cis-BF or control protein GAPDH. Conversely, Hep G2 cells incubated with 1S-cis-BF showed a marked decrease in Bcl-2 protein levels. (A) A representative autoradiograph of Bax and Bcl-2 protein expression in Hep G2 cells. (B) Graphic representation of Bax and Bcl-2 protein expression in Hep G2 cells. Each value (mean ± SD, n = 4) represents the ratio of target protein level to its corresponding GADPH level (*p < 0.05, **p < 0.01; n = 4).
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgment This work was supported by the National Nature Science Foundation of China Projects, Grant Number 81141109.
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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 810–814
references
Carlsson, P., Warner, N.A., Hallanger, I.G., Herzke, D., Kallenborn, R., 2014. Spatial and temporal distribution of chiral pesticides in Calanus spp. from three Arctic fjords. Environ. Pollut. 192, 154–161. Casco, V.H., Izaguirre, M.F., Marin, L., Vergara, M.N., Lajmanovich, R.C., Peltzer, P., Soler, A.P., 2006. Apoptotic cell death in the central nervous system of Bufo arenarum tadpoles induced by cypermethrin. Cell Biol. Toxicol. 22, 199–211. Guvenc, D., Kabak, Y., Atmaca, E., Aksoy, A., Guvenc, T., 2013. Examination of caspase-dependent apoptotic and necrotic changes in rat kidney exposed to different doses of permethrin. Biotech. Histochem. 88, 76–85. Giray, B., Gürbay, A., Hincal, F., 2001. Cypermethrin-induced oxidative stress in rat brain and liver is prevented by vitamin E or allopurinol. Toxicol. Lett. 118, 139–146. Hall Jr., L.W., Anderson, R.D., 2013. Relationship between bifenthrin sediment toxic units and benthic community metrics in urban California streams. Arch. Environ. Contam. Toxicol. 65, 173–182. Hossain, M.M., Richardson, J.R., 2011. Mechanism of pyrethroid pesticide-induced apoptosis: role of calpain and the ER stress pathway. Toxicol. Sci. 122, 512–525. Kale, M., Rathore, N., John, S., Bhatnagar, D., 1999. Lipid peroxidative damage on pyrethroid exposure and alterations in antioxidant status in rat erythrocytes: a possible involvement of reactive oxygen species. Toxicol. Lett. 105, 197–205. Kote, P., Ravindra, V., Chauhan, R.S., Kote, P., Girish, V., 2006. Use of avian lymphocytes to detect toxicity: effects of a commonly utilized deltamethrin preparation. J. Immunotoxicol. 3, 101–109. Kumar, A., Sasmal, D., Sharma, N., 2014. Deltamethrin induced an apoptogenic signalling pathway in murine thymocytes: exploring the molecular mechanism. J. Appl. Toxicol. 34, 1303–1310. Liu, H.G., Zhao, M., Zhang, C., Ma, Y., Liu, W.P., 2008. Enantioselective cytotoxicity of the insecticide bifenthrin on a human amnion epithelial (FL) cell line. Toxicology 253, 89–96.
Liu, W.P., Gan, J.Y., Schlenk, D., Jury, W.A., 2005. Enantioselectivity in environmental safety of current chiral insecticides. Proc. Natl. Acad. Sci. U. S. A. 102, 701–706. Manske, M.K., Beltz, L.A., Dhanwada, K.R., 2004. Low-level atrazine exposure decreases cell proliferation in human fibroblasts. Arch. Environ. Contam. Toxicol. 46, 438–444. Narra, M.R., 2014. Tissue-specific recovery of oxidative and antioxidant effects of chlorpyrifos in the freshwater crab, Barytelphusa guerini. Arch. Environ. Contam. Toxicol. 67, 158–166. Ouyang, L., Shi, Z., Zhao, S., Wang, F.T., Zhou, T.T., Liu, B., Bao, J.K., 2012. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 45, 487–498. Park, J.H., Lee, J.E., Shin, I.C., Koh, H.C., 2013. Autophagy regulates chlorpyrifos-induced apoptosis in SH-SY5Y cells. Toxicol. Appl. Pharmacol. 268, 55–67. Phyu, Y.L., Palmer, C.G., Warne, M.S., Dowse, R., Mueller, S., Chapman, J., Hose, G.C., Lim, R.P., 2013. Assessing the chronic toxicity of atrazine, permethrin, and chlorothalonil to the cladoceran Ceriodaphnia cf. dubia in laboratory and natural river water. Arch. Environ. Contam. Toxicol. 64, 419–426. Solomon, K.R., Giddings, J.M., Maund, S.J., 2001. Probabilistic risk assessment of cotton pyrethroids: I. Distributional analyses of laboratory aquatic toxicity data. Environ. Toxicol. Chem. 20, 652–659. Tyler, C.R., Beresford, N., Woning, M.V., Sumpter, J.P., Thorpe, K., 2000. Metabolism and environmental degradation of pyrethroid insecticides produce compounds with endocrine activities. Environ. Toxicol. Chem. 4, 801–809. Velki, M., Hackenberger, B.K., 2013. Different sensitivities of biomarker responses in two epigeic earthworm species after exposure to pyrethroid and organophosphate insecticides. Arch. Environ. Contam. Toxicol. 65, 498–509. Wang, X.Z., Liu, S.S., Sun, Y., Wu, J.Y., Zhou, Y.L., Zhang, J.H., 2009. Beta-cypermethrin impairs reproductive function in male mice by inducing oxidative stress. Theriogenology 72, 599–611. Xia, X., Su, C., Fu, J., Zhang, P., Jiang, X., Xu, D., Hu, L., Song, E., Song, Y., 2014. Role of ␣-lipoic acid in LPS/d-GalN induced fulminant hepatic failure in mice: studies on oxidative stress, inflammation and apoptosis. Int. Immunopharmacol. 22, 293–302.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Enantioselective apoptosis induced by individual isomers of bifenthrin in Hep G2 cells Huigang Liu ∗ , Juan Li Department of Public Health, School of Medicine, China Three Gorges University, 8# Daxue Avenue, Yichang 443002, China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Bifenthrin (BF) has been used in racemate for agricultural purposes against soil insects,
Received 5 December 2014
leading to increased inputs into soil environments. However, most of the studies about the
Received in revised form
toxicology research on BF were performed in its racemic form. The aim of the present study
9 February 2015
was to evaluate the enantiomer-specific cis-BF-induced apoptosis and intracellular reactive
Accepted 14 February 2015
oxygen species (ROS) generation on human hepatocarcinoma cells (Hep G2). The results
Available online 21 February 2015
of cell viability assay and cytoflow assay indicated an obvious enantioselective hepatocyte toxicity of 1S-cis-BF in Hep G2 cells. 1S-cis-BF also induced ROS production, up-regulated Bax
Keywords:
protein expression and down-regulated Bcl-2 expression levels. The present study suggested
Apoptosis
that enantioselective toxicity should be evaluated on currently used chiral pesticides, such
Enantioselective
as synthetic pyrethroids.
Pyrethroid pesticides
1.
Introduction
Synthetic pyrethroids (SPs) are widely used in most sectors of agricultural production to ensure high crop yields for decades (Tyler et al., 2000). Some pyrethroid pesticides have played a key role in providing reliable supplies of agricultural production at prices affordable to consumers and ensuring high profits to farmers. Recent works point out this ubiquity in the environment, even in vegetables (Velki and Hackenberger, 2013; Hall and Anderson, 2013; Phyu et al., 2013). Many SPs used as pesticides are chiral compounds (Carlsson et al., 2014). In recent years, people have become increasingly concerned about chiral pesticides use and particularly about their impact on human health and environmental quality (Liu et al., 2005). Bifenthrin is effective against a wide range of insect and has been used for plant protection (Solomon et al., 2001). Previous study indicated that 1S-cis-BF presented more toxic effects
∗
Corresponding author. Tel.: +86 07176396818. E-mail address: [email protected] (H. Liu).
http://dx.doi.org/10.1016/j.etap.2015.02.010 1382-6689/© 2015 Elsevier B.V. All rights reserved.
© 2015 Elsevier B.V. All rights reserved.
than 1R-cis-BF on human amnion epithelial (FL) cell lines. FL cells incubated with 1S-cis-BF exhibited a dose-dependent accumulation of ROS (Liu et al., 2008). Many studies reported that SPs have been associated with many human health hazards, ranging from short-term impacts such as allergies, and headaches to chronic impacts like reproductive disorders and neurological disorders (Wang et al., 2009; Guvenc et al., 2013). SPs are also known to be hepatotoxic and carcinogenic (Giray et al., 2001). Many of the toxic effects elicited by exposure to pesticides are mediated by the regulation of apoptosis (Park et al., 2013). It has been demonstrated that pyrethroid insecticides have an apoptotic effect on different organisms (Casco et al., 2006). Apoptosis can be induced by a wide range of chemical, physical and genetic factors. However, most of these studies were performed in their racemic form of these chiral pesticides. Hence, in the present study, we aim to evaluate the apoptosis and ROS generation induced by
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 810–814
individual isomers of cis-BF on human hepatocarcinoma cells (Hep G2).
2.
Materials and methods
2.1.
Chemicals
Racemic cis-BF (analytical standard, 99.5%, 2-methylbiphenyl3-ylmethyl-(Z)-(1RS)-cis-(2-chloro-3,3,3-trifluoroprop-1-enyl)2,2-dimethylcyclopropane carboxylate was from Sigma (St. Louis, MO, USA). Minimal essential medium (MEM) was purchased from Gibco (Paisley, UK), and fetal bovine serum (FBS) was from Shijiqing Reagent Company (Hangzhou, China). Thiazolyl blue (MTT) solution (5 mg/mL in phosphatebuffered saline (PBS)) was from Amresco (Solon, OH, USA). 2 7 -dichlorofluorescin diacetate (DCFH-DA) was from Sigma (St. Louis, MO, USA).
2.2.
Preparation of BF enantiomers
The enantiomers of cis-BF were resolved on a Jasco LC2000 series HPLC system (Jasco, Tokyo, Japan) equipped with a PU-2089 intelligent quaternary gradient pump, an AS-1559 autosampler with a 100 L loop, a mobile-phase vacuum degasser, an UV-2075 ultraviolet-visible detector, a variable-wavelength CD-2095 circular dichroism (CD) detector, a CO-2060 column temperature-control compartment, and an LC-Net II/ADC data collector (Jasco, Tokyo, Japan). The mobile phase (-cyclodextrin) was made of n-hexane fortified with ethanol. The Chiralcel OJ [250 mm × 4.6 mm i.d., cellulose tris (4-methyl benzoate) column (Daicel Chemical Industries, Tokyo, Japan)] was used. The detection wavelength of the CD detector was 230 nm. The resolved enantiomers were individually collected at the HPLC outlet, evaporated to dryness, and used in the following experiment. The enantiomers concentrations were tested on an Agilent 6890N gas chromatograph (GC) equipped with an electron capture detector (ECD), assuming the same response factor for enantiomers as for the racemate of cis-BF.
2.3.
Cell culture and treatments
Hep G2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Scientific HyClone, San Jose, CA, USA) with 10% (v/v) fetal bovine serum (FBS) at 37 ◦ C in a 5% CO2 incubator. Cells were subcultured every 2–5 days and seeded in 96-well plates (Costar, Cambridge, MA, USA) at a density of 2 × 105 cells/mL and allowed to adhere for 24 h prior to assaying.
2.4.
Assessment of cell viability
Cell viability was measured by quantitative colorimetric with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5assay diphenyltetrazolium bromide). Hep G2 cells were incubated with five different concentrations of individual 1S-cis-BF, 1R-cis-BF, or cis-BF for 12 h. MTT at 500 g/mL (final concentration) was added to each well and the cells were incubated at 37 ◦ C for 4 h. After removing MTT, the cells were lysed with
811
dimethyl sulfoxide (DMSO) (150 L each well). The absorbance at 490 nm was measured using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Control cells were treated with the vehicle (0.1% ethanol, onefold).
2.5.
Assay of the cellular contents of ROS
Hep G2 cells were incubated with individual enantiomers or a racemate of cis-BF at concentrations of 0, 5, 10, 15, and 20 mg/L for 2 h. After this treatment, cells were washed three times with ice-cold PBS then incubated with 10 M DCFH-DA for 30 min at 37 ◦ C. A fluorescent spectrophotometer (Tecan Infinite M200, Switzerland) was used to assay the cellular ROS by measuring 2 7 -dichlorofluorescein fluorescence (excitation at 485 nm/emission at 535 nm). The resulting ROS levels were determined as relative fluorescence intensities.
2.6.
Cell apoptosis assay
Hep G2 cells at 5 × 106 cells mL−1 were treated with individual or racemate cis-BF at 10 or 20 mg/L for 6 h, respectively. Then cells were harvested and washed twice with PBS. The treated cells were suspended in binding buffer at 3 × 106 cells mL−1 , and supplemented with 5 L of FITC-Annexin-V and 10 L of propidium iodide (PI) (Annexin-V-FITC Apoptosis Detection Kit, Sigma). Cell apoptosis was assessed by flow cytometry (Beckman Coulter EPICSXL, USA) after incubated for 20 min at room temperature in the dark. All experiments were repeated at least three times.
2.7.
Western blot analysis
The lysate was cleared by centrifugation at 17,000 × g for 30 min at 4 ◦ C after cells were homogenized on ice in lysis buffer. 50 g protein from each sample were loaded on a 12% SDS-PAGE gel and transferred to PVDF membranes by electrophoretic transfer and blocked with 5% nonfat dry milk in TBST buffer. Then membranes were incubated with Bax and Bcl-2 (ProteinTech Group) overnight at 4 ◦ C. Enhanced chemiluminescence (Pierce) was used to detect HRP-labeled secondary anti-rabbit antibody (Cell Signal). Blots were exposed to X-ray film for radiographic detection of the bands after immersion in ECL chemiluminescence reagents.
2.8.
Statistical analysis
Statistical significance was determined by one-way analysis of variance (ANOVA). The statistical analysis of the data was performed with the statistical program package SPSS 11.0. A p-value <0.05 was considered significant.
3.
Results and discussion
3.1. cells
Enantioselective cytotoxicity in human Hep G2
The results of MTT assay show that treatment with 1S-cisBF reduced cell viability in a dose-dependent manner (Fig. 1).
812
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Fig. 1 – Effects of individual enantiomers and racemate of cis-BF on the viability of Hep G2 cell lines (*p < 0.05; **p < 0.01, n = 4). Hep G2 cells were treated with different concentrations of individual 1R-cis-BF, 1S-cis-BF, or cis-BF for 12 h.
While the reduction of cell viability by 1R-cis-BF has no significant difference. At the concentration of 20 mg/L, the order of viability of Hep G2 cells exposed to chemicals was 1Scis-BF (51%) < rac-cis-BF (63%) < 1R-cis-BF (83%) (p < 0.01); At the concentration of 15 mg/L, the order of viability of Hep G2 cells exposed to chemicals was 1S-cis-BF (68%) < rac-cis-BF (77%) < 1R-cis-BF (82%) (p < 0.05). There was no significant difference in other concentrations. Previous study indicated that 1S-cis-BF presented more toxic effects than 1R-cis-BF above the concentration of 7.5 mg/L on FL cell lines (Liu et al., 2008). In this study, the MTT assay showed that 1S-cis-BF and rac-cisBF induced cytotoxicity in concentration-dependent manners in Hep G2 cells. In contrast, 1R-cis-BF had no effect on MTT metabolism.
3.2. cis-BF
ROS production induced by individual isomer of
Hep G2 cells were incubated with individual enantiomers or a racemate of cis-BF for 2 h at concentrations of 0, 5, 10, 15, and 20 mg/L and the resulting ROS levels were determined as relative fluorescence intensities. Treatment with 1S-cis-BF or rac-cis-BF caused a dose-dependent accumulation of intracellular ROS in Hep G2 cells (Fig. 2). At the concentration of 20 and 15 mg/L, the order of ROS production of Hep G2 cells exposed to chemicals was 1S-cis-BF > rac-cis-BF > 1R-cisBF (p < 0.01). There was no significance compared to control and between the analyzed concentrations of chemicals in other concentrations. Oxidative stress has been implicated as the mechanism of cytotoxicity from numerous toxicants and many studies in humans or animals support that SPs can induce oxidative stress (Kale et al., 1999; Kumar et al., 2014; Narra, 2014). The generation of ROS plays an important role in apoptosis process induced by environmental pollutants (Xia et al., 2014). In the present study, 1S-cis-BF and rac-cis-BF
Fig. 2 – Effects of individual enantiomers and racemate of cis-BF on intracellular ROS production. Hep G2 cells exposed to different concentrations of individual enantiomers and racemate of cis-BF for 2 h, followed by ROS determination (*p < 0.05, **p < 0.01; n = 4).
induced ROS production, while 1R-cis-BF had no effect on ROS generation. It suggests that the ROS production induced by rac-cis-BF could be dominantly attributed to 1S-cis-BF but not 1R-cis-BF.
3.3.
Apoptosis induced by individual isomer of cis-BF
Hep G2 cells were treated with individual and racemate cis-BF at 10 and 20 mg/L, respectively, for 6 h. FITCAnnexin-V/propidium iodide staining assay was performed to determine the effects of cis-BF on Hep G2 cell apoptosis. Fig. 3 shows the results of the flow cytometric analysis of the cells. The bottom right quadrants: apoptotic cells. The bottom left quadrants: viable cells. The upper right quadrants: necrotic and late apoptotic cells. The percentage of cells in that category in ethanol (solvent) from cis-BF-treated samples were presented in each quadrant. The apoptosis rate in Hep G2 cells exposed to 1S-cis-BF, 1R-cis-BF and rac-cis-BF at concentration of 20 mg/L were 7.31%, 2.14% and 4.51%, respectively. To confirm the enantioselective apoptosis induced by individual isomers of cis-BF, the expression of the apoptotic proteins Bax and Bcl-2 were determined using Western blotting. 1S-cis-BF and rac-cis-BF treated Hep G2 cells showed an increase in pro-apoptotic Bax protein levels coupled with a decrease in Bcl-2 levels. However, 1R-cis-BF had no effect on Bax and Bcl-2 expression (Fig. 4). The Bcl-2 family of proteins has been implicated in either triggering or preventing apoptosis where a higher ratio of Bax to Bcl-2 has been shown to promote cell death. Bcl-2/Bax proteins are critical mediators of apoptosis (Manske et al., 2004). During apoptotic stimulation, these two proteins would affect mitochondrial Cyt c release, and the decrease in the Bcl-2/Bax ratio is considered a decisive correlation in apoptosis or cell death (Ouyang et al., 2012). SPs’ potential to induce apoptosis both in vitro and in vivo has been reported in previous studies (Hossain and Richardson, 2011).
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Fig. 3 – Evaluation of apoptotic cells by the Annexin-V staining. (A) The percent of early apoptotic cells following treatment with individual enantiomers or racemate of BF was measured with flow cytometry (*p < 0.05, **p < 0.01; n = 4). (B) Apoptosis quantified by Annexin V (AV)-PI staining. Four subpopulations and their quantities are indicated. R1, non-apoptotic dead cells; R2, late apoptotic cells; R3, viable cells; R4, early apoptotic cells.
Long-term exposure to deltamethrin induced Cyt c release and altered expression of Bcl-2/Bax, leading to apoptotic cell death (Kote et al., 2006). In the present study, we found that 1S-cis-BF significantly up-regulated Bax protein expression and downregulated Bcl-2 expression levels. However, 1R-cis-BF had no significant effect on Bax/Bcl-2 expression compared to control cells. In conclusion, the present study evaluates cytotoxicity and apoptosis induced by individual isomers of BF in Hep G2 cells. Our study indicated that the apoptotic effects induced by cisBF could be almost entirely attributed to 1S-cis-BF enantiomer. Thus, it is of great significance to evaluate the possible hazards and the influence of the single chiral monomers of pesticides on environment and human health.
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Fig. 4 – Involvement of Bax and Bcl-2 protein expression in cis-BF induced apoptosis. In the western blot analysis of Bax and Bcl-2 protein expression, Hep G2 cells were treated with individual enantiomers or racemates of cis-BF at the concentration of 20 mg/L for 6 h, and cell lysates were subjected to western immunoblotting. Bax protein expression in Hep G2 cells treated with 1S-cis-BF showed a significant increase in its levels compared to cells treated with 1R-cis-BF or control protein GAPDH. Conversely, Hep G2 cells incubated with 1S-cis-BF showed a marked decrease in Bcl-2 protein levels. (A) A representative autoradiograph of Bax and Bcl-2 protein expression in Hep G2 cells. (B) Graphic representation of Bax and Bcl-2 protein expression in Hep G2 cells. Each value (mean ± SD, n = 4) represents the ratio of target protein level to its corresponding GADPH level (*p < 0.05, **p < 0.01; n = 4).
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgment This work was supported by the National Nature Science Foundation of China Projects, Grant Number 81141109.
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