- Email: [email protected]
Enantioselective cytotoxicity of isocarbophos is mediated by oxidative stress-induced JNK activation in human hepatocytes
Toxicology 276 (2010) 115–121
Contents lists available at ScienceDirect
Toxicology journal homepage: www.elsevier.com/locate/toxicol
Enantioselective cytotoxicity of isocarbophos is mediated by oxidative stress-induced JNK activation in human hepatocytes Huigang Liu a,1 , Jing Liu b,1 , Lihong Xu c , Shanshan Zhou a , Ling Li a , Weiping Liu b,a,∗ a
Research Center of Green Chirality, College of Biology and Environment, Zhejiang University of Technology, Hangzhou 310032, People’s Republic of China MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China c Department of Biochemistry and Genetics, School of Medicine, Zhejiang University, Hangzhou 310058, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 22 June 2010 Received in revised form 24 July 2010 Accepted 26 July 2010 Available online 3 August 2010 Keywords: Chiral pesticide Enantioselectivity Isocarbophos JNK pathway Cytotoxicity
a b s t r a c t Recent studies have shown the enantioselectivity of chiral pesticides in environmental fate, aquatic toxicity, endocrine disruption and cytotoxicity. Thus it is of significance to investigate the molecular mechanisms of chiral pesticides enantioselectivity in cytotoxicity. In the present study, we used Hep G2 cells as in vitro model to assay cytotoxicity of enantiomers of isocarbophos (ICP), a widely used chiral organophosphorus pesticide. The results of cell viability assay and cytoflow assay indicated an obvious enantioselective hepatocyte toxicity of ICP: (−)-ICP was about two times more toxic than (+)-ICP in Hep G2 cells. We found that (−)-ICP, but not (+)-ICP, up-regulated Bax protein expression and down-regulated Bcl-2 expression levels, which resulted in an increase in Bax/Bcl-2 ratio with the apoptosis co-ordination. Although (−)-ICP enantioselectively activated both ERK and JNK, only the specific inhibitor for JNK could completely reverse (−)-ICP-induced apoptosis of Hep G2 cells. It suggests that (−)-ICP-induced hepatocyte toxicity was more dominantly through the sustained activation of JNK pathway, but only partially via ERK cascade. Furthermore, (−)-ICP induced ROS production, while (+)-ICP had no effect on ROS generation. The antioxidant MnTBAP attenuated (–)-ICP-induced activation of JNK and ERK, indicating that the outcome from challenge with (−)-ICP enantiomer depends on the oxidative stress-induced activation of a series of signaling cascades that promote hepatocyte apoptosis. In conclusion, (−)-ICP enantioselectively causes the change of Bax/Bcl-2 ratio, triggers the generation of intracellular ROS and sequentially induces sustainable activation of JNK, which in turn, results in a decrease in cell viability and an increase in cell apoptosis. Our observations provide further insight into enantiomers toxicity pathway which is able to differentiate between enantiomer activities at molecular level. Crown Copyright © 2010 Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction A chiral molecule and its non-superimposable mirror image are special types of stereoisomers called enantiomers. Although pure enantiomers of chiral compounds have identical physical and chemical properties in achiral environments, their behavior in biochemical processes might be strikingly different and selective.
Abbreviations: OPs, organophosphorus pesticides; ICP, isocarbophos; HPLC, high-performance liquid chromatography; MAPK, mitogen-ctivated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PVDF, polyvinylidene difluoride. ∗ Corresponding author at: MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China. Tel.: +86 571 8832 0666; fax: +86 571 8832 0884. E-mail address: [email protected] (W. Liu). 1 These authors contributed equally to the article.
This enantioselectivity often results in differences in bioactivity and toxicity of the two enantiomers. Up to 30% of currently used pesticides are chiral molecules, as are some organophosphorus pesticides (OPs) and many other compounds. However, almost all chiral pesticides are introduced into the environment as mixtures of equal amounts of the two or more enantiomers (racemic mixtures). A number of studies have shown the enantioselectivity of chiral pesticides in environmental fate (Garrison, 2006), aquatic toxicity (Jin et al., 2010; Wang et al., 2009a; Zhao et al., 2009), endocrine disruption (Wang et al., 2009b; Zhao et al., 2008, 2010) and cytotoxicity (Lu et al., 2009; Hu et al., 2010; Zhao and Liu, 2009). Recently, we reported that bifenthrin, a typical chiral pesticide, enantioselectively induced apoptosis and DNA damage in human aminon epithelial cells (Liu et al., 2008). Importantly, our further finding indicated that the enantioselectivity of bifenthrin in cytotoxicity was mediated by the mitogen-activated protein kinase (MAPK) signaling pathway (Liu et al., 2009). Therefore, to make more accurate risk assessments for chiral pesticides, it is of significance to investigate the
0300-483X/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2010.07.018
116
H. Liu et al. / Toxicology 276 (2010) 115–121 2.2. Assessment of cell viability Hep G2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Scientific HyClone, San Jose, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 ◦ C in a 5% CO2 incubator. Hep G2 cells, at 5 × 106 cells mL−1 , were treated with ICP or vehicle for 24 h. At end of culture, cell viability was measured by MTT with a Bio-Rad Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The results were calculated as the ratio of each exposure group to the vehicle control (0.1% ethanol, onefold) and as the mean ± standard deviation (SD) of four independent measurements performed in three replicates. These data were then analyzed using the t-test with Origin (OriginLab, Northampton, MA, USA).
Fig. 1. Enantiomers of isocarbophos (the asterisk indicates the chiral center).
molecular mechanisms of their enantioselectivity in cytotoxicity. Organophosphorus pesticides (OPs) are the most widely used pesticides worldwide since the 1950s and their metabolites are widespread across different populations (Aprea et al., 2000; Barr et al., 2004, 2005; Curl et al., 2003; Lu et al., 2001). A large number of OPs are chiral compounds, and yet enantioselectivity in their environmental fate and toxic effects is rarely addressed. One of these chiral OPs, isocarbophos (ICP), is widely used to control a variety of leaf-eating and soil insects. ICP was introduced into China for agricultural use in 1981, and currently the annual production of pure isocarbophos in China is about 5000 t (Jia et al., 2006). This molecule consists of a pair of enantiomers with a chiral center at the phosphorus atom: (−)-isocarbophos and (+)-isocarbophos (Fig. 1). In our previous study, ICP showed enantiomer-specific acute toxicity toward Daphnia magna (Lin et al., 2008). However, the molecular mechanism of ICP-induced enantiomer-specific toxicity remains unclear. In particular, it is currently unknown what signaling pathways mediated cytotoxic response to individual isomers of ICP. Although the primary target organ for OPs is the nerve system, there is increasing evidence to show their possible toxic effects on non-target tissues such as hepatocytes. For instance, trichlorfon induced apoptosis in hepatocyte primary cultures of Carassius auratus gibelio (Xu et al., 2009). Hepatic ultrastructural alterations were observed in cockerels exposed to methylobromofenvinphos (Chishti and Rotkiewicz, 1992, 1993). Hreljac et al. (2008) reported that OPs caused DNA damage and affected expression of DNA damage responsive genes in human hepatoma Hep G2 cells. Given hepatocyte toxicity is one of important toxic effects of OPs, we used Hep G2 cells as in vitro model in this study to assay cytotoxicity of enantiomers of ICP. Furthermore, we evaluated the enantioselective activation of MAPK signaling in Hep G2 cells exposed to individual isomers of ICP.
2.3. FITC-Annexin-V/propidium iodide staining assay Cell apoptosis was assessed by flow cytometry in accordance with the manufacturer’s protocol (Annexin-V-FITC Apoptosis Detection Kit, Sigma). Briefly, Hep G2 cells, at 5 × 106 cells mL−1 , were treated with ICP or vehicle for 24 h at 37 ◦ C in a 5% CO2 incubator, and then 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 PI, and incubated for 20 min at room temperature in the dark. All experiments were repeated at least three times and flow cytometric analysis was performed using a FACScan flow cytometer (Beckman Coulter EPICS XL). 2.4. Western blot analysis Cell cultures were homogenized on ice in freshly prepared lysis buffer. The lysate was cleared by centrifugation at 17,000 × g for 30 min at 4 ◦ C. Equal amounts of protein (50 g) from each sample were fractionated on a 12% SDS-PAGE gel and electrophoretically transferred to PVDF membranes. Transfers were blocked with 5% nonfat dry milk in TBST buffer and incubated with phospho-ERK1/2 (Thr202/Tyr-204), phospho-JNK, phospho-p38 (Cell Signal), Bax and Bcl-2 (ProteinTech Group) overnight at 4 ◦ C. HRP-labeled secondary anti-rabbit antibody (Cell Signal) was detected by enhanced chemiluminescence (Pierce). Finally, following immersion in ECL chemiluminescence reagents and blots were exposed to X-ray film for radiographic detection of the bands. 2.5. Assay of the cellular contents of reactive oxygen species (ROS) Following treatment with ICP or vehicle for 6 h, the treated cells were washed three times with ice-cold PBS and then incubated with 10 M 2 ,7 dichlorofluorescin diacetate (DCFH-DA; 100 mM in dimethyl sulfoxide, St. Louis, MO, USA) for 30 min at 37 ◦ C. The cellular free radical content was assayed by measuring 2 ,7 -dichlorofluorescein fluorescence using a fluorescent spectrophotometer (excitation at 485 nm/emission at 535 nm, Tecan Infinite M200, Switzerland). 2.6. Statistical analysis Results were expressed as the mean ± SD from at least three independent experiments. Statistical analysis was performed by one-way analysis of variance. Asterisks denote statistically significant differences at p < 0.05.
3. Results 3.1. Enantioselective cytotoxicity in human Hep G2 cells
2. Materials and methods 2.1. Reagents Analytical standard racemic isocarbophos [(±)-ICP, >99.6%, (RS)-(O-2isopropoxycarbonylphenyl O-methyl phosphoramidothioate)] was obtained from Kefa New Technology Development (Shenyang, China). Individual pure enantiomers [(−)-enantiomer and (+)-enantiomer] of ICP were separated and prepared from (±)-ICP according to modified procedures as described previously (Lin et al., 2008). Briefly, enantiomeric preparation was carried out using a high-performance liquid chromatography (HPLC) machine equipped with a variable-wavelength CD-2095 circular dichroism (CD) detector. Separation was achieved on a Chiralcel® OD column (250 mm × 4.6 mm) at 25 ◦ C and the flow rate of mobile phase was 0.8 mL min−1 . The detection wavelength of CD was set at 230 nm. The rotation sign (“+” or “−”) was indicated by a positive or negative peak on the chromatogram form CD detector. The preparation of pure enantiomers was obtained by manually collecting the eluent corresponding to the resolved peaks at the HPLC outlet of the CD detector while observing the UV absorbance. The collected individual enantiomer solution was evaporated to dryness, redissolved in ethanol, and used as the stock solution for bioassays (the final amount of ethanol in assay solution was <0.04%).
The results of MTT assay showed that treatment with (−)-ICP at concentrations of 2.5–40 mg L−1 reduced cell viability in a dosedependent manner and racemate of ICP significantly decreased viability of Hep G2 cells only at the highest concentration. But the reduction of cell viability by (+)-ICP is not significant. At the concentration of 40 mg L−1 , the order of viability of Hep G2 cells exposed to chemicals was (−)-ICP (55%) < rac-ICP (60%) < (+)-ICP (82%) (p < 0.05) (Fig. 2). 3.2. Enantioselective apoptosis in human Hep G2 cells FITC-Annexin-V/propidium iodide staining assay was performed to determine the effects of ICP on Hep G2 cell apoptosis. Fig. 3A shows the typical results of the flow cytometric analysis of the cells. The bottom left quadrants represent viable cells, the bottom right quadrants represent apoptotic cells and the upper right
H. Liu et al. / Toxicology 276 (2010) 115–121
117
Fig. 2. The effect of individual enantiomers and racemates of isocarbophos on the viability of human Hep G2 cell lines. Hep G2 cells were incubated with different concentrations of individual enantiomers and racemates of isocarbophos for 24 h, followed by analysis using the MTT assay. Different letters above adjacent bars indicate significant differences (p < 0.05, n = 3) between individual enantiomers and racemate, while the same letter indicates no significant difference.
quadrants represent necrotic and late apoptotic cells. The numbers in each quadrant are the percentage of cells in that category in ethanol (solvent) from ICP-treated samples. The apoptosis rate in Hep G2 cells exposed to rac-ICP, (+)-ICP and (−)-ICP at concentration of 20 mg L−1 were 3.32%, 2.28% and 7.95%, respectively (Fig. 3B). In addition, the expression of the apoptotic proteins Bax and Bcl-2 were determined using Western blotting. (−)-ICP and racICP increased the expression levels of Bax protein and reduced Bcl-2 expression. However, (+)-ICP had no effect on Bax and Bcl-2 expression (Fig. 4). 3.3. Expression and activation of MAP kinases by isocarbophos To determine whether specific MAPK signal transduction pathways are involved in cellular responses to rac-ICP, the expression of phosphorylation of ERK1/2, JNK and p38 were measured by Western blotting. Both ERK1/2 and JNK exhibited sustained activation in response to 20 mg L−1 rac-ICP exposure (Fig. 5). No significant change in the active levels of p38 kinase was observed throughout the 12 h treatment. The total levels of each MAPK did not change during the 12 h incubation period.
Fig. 3. Evaluation of apoptotic cells by the Annexin-V staining assay. (A) Apoptosis quantified by Annexin-V-PI staining. Hep G2 cells were stained by Annexin-V and PI and then analyzed by flow cytometry. Four subpopulations and their quantities are indicated: nonapoptotic dead cells (R1), late apoptotic cells (R2), viable cells (R3), and early apoptotic cells (R4). Numbers in R4 are the percentage of early apoptotic cells contained. (B) The percent of early apoptotic cells following treatment with individual enantiomers or racemate of isocarbophos was measured with flow cytometer. At least three independent experiments were carried out for each condition, and a minimum of 300 cells were counted for each measurement.
3.4. Activation of ERK1/2 and JNK by single isomer of isocarbophos To further determine whether the activation of ERK1/2 and JNK by rac-ICP results from a single enantiomer of ICP, pure (−)-ICP and (+)-ICP were individually incubated with Hep G2 cells at a concentration of 20 mg L−1 for 6 h. The data showed that exposure to (−)-ICP resulted in increased levels of the phosphorylated forms of ERK1/2 and JNK, whereas exposure to (+)-ICP had no effect on MAPK phosphorylation. Additionally, both (+)-ICP and (−)-ICP did not activate p38 MAPKs (Fig. 6). 3.5. Inhibition of ERK1/2 and JNK attenuates (−)-ICP-induced cytotoxicity and apoptosis To test the hypothesis that the enantioselective activation of ERK1/2 and JNK by ICP contributes to rac-ICP-induced cytotoxicity and apoptosis, a series of kinase inhibitors were used to block
Fig. 4. Involvement of Bax and Bcl-2 protein expression in isocarbophos 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 isocarbophos at the concentration of 20 mg L−1 for 6 h, and cell lysates were subjected to western immunoblotting. Lysates for SDS-PAGE were prepared and western blotting was performed as described in Section 2. Bax protein expression in Hep G2 cells treated with (−)-isocarbophos showed a marked increase in its levels compared to cells treated with (+)-isocarbophos or control protein GAPDH. Conversely, Hep G2 cells incubated with (−)-isocarbophos showed a marked decrease in Bcl-2 protein levels.
118
H. Liu et al. / Toxicology 276 (2010) 115–121
Fig. 5. Time course of isocarbophos-induced accumulation of phosphorylated ERK (A), JNK (B), and p38 MAPK (C). Hep G2 cells were incubated with 20 mg L−1 of the racemate of isocarbophos for the indicated time. The cell lysates were analyzed by western blotting using respective antibodies. The data shown is from a typical experiment involving two representative experiments.
the activity of ERK1/2 and/or JNK. As shown in Fig. 7, treatment with SP600125 significantly attenuated (−)-ICP-induced cytotoxicity (Fig. 7A) and apoptosis (Fig. 7B). The inhibitor of ERK1/2 partially reversed the effects of (−)-ICP on cytotoxicity and apoptosis of Hep G2 cells. These data suggest that (−)-ICP-induced hepototoxicity is mainly mediated by JNK activation, but partially through ERK1/2 pathway. Fig. 7. Inhibition of ERK1/2 and JNK kinases attenuates isocarbophos-induced cytotoxicity and apoptosis. Hep G2 cells were pre-treated for 1 h with 50 M of the ERK1/2 inhibitor PD98059, the 10 M JNK inhibitor SP600125, or vehicle control, followed by 20 mg L−1 of the individual enantiomers and racemates of isocarbophos for 12 h treatment. Following incubation, cell viability was measured by MTT assay (A), and apoptosis was analyzed by using flow cytometry (B).
3.6. Involvement of reactive oxygen species in ERK and JNK signaling pathways
Fig. 6. The effect of individual enantiomers of isocarbophos on the activation of phosphorylated ERK 1/2 (A), JNK (B), and p38 MAPKs (C). Hep G2 cells were incubated with 20 mg L−1 of the individual enantiomers and racemates of isocarbophos for 6 h, and cell lysates were subjected to western blot analysis. Results shown are representative of two independent experiments.
Reactive oxygen species (ROS) have been implicated as potential modulators of apoptosis (Circu and Aw, 2010). In Hep G2 cells, treatment with (−)-ICP and rac-ICP caused a dosedependent accumulation of intracellular ROS. But ROS production in cells incubated with (+)-ICP only slightly increased, even at a concentration of 20 mg L−1 . The levels of ROS production in (−)-ICP-treated cells were two times higher than that of (+)-ICP at the highest concentration of 20 mg L−1 (p < 0.01) (Fig. 8A).To further determine the role of (−)-ICP-induced ROS generation in the activation of JNK and ERK1/2, Hep G2 cells were pretreated for 12 h with 200 M of MnTBAP, a specific inhibitor for ROS, and then incubated with 20 mg L−1 of rac-ICP, (+)-ICP and (−)-ICP for 6 h. As shown in Fig. 8B, MnTBAP markedly decreased the activity of ERK1/2 and JNK induced by (−)-ICP. However, MnTBAP had no effect on total ERK1/2 and JNK expression. These results suggest that (−)-ICP-induced ROS accumulation may contribute to the activation of ERK1/2 and JNK in hepatocytes.
H. Liu et al. / Toxicology 276 (2010) 115–121
Fig. 8. The effect of individual enantiomers and racemates of isocarbophos on intracellular ROS production (A). Hep G2 cells exposed to different concentrations of individual enantiomers and racemate of isocarbophos for 3 h, followed by ROS determination. Inhibition of ROS on ERK and JNK signaling pathways (B). Hep G2 cells were pre-treated for 12 h with 200 M of MnTBAP, and then stimulated for 3 h with 20 mg L−1 of the individual enantiomers and racemates. Phosphorylation of ERK1/2 and JNK were measured by western blot analysis.
4. Discussion In the previous study, we demonstrated that ICP could enantioselectively cause toxicity toward Daphnia magna (Lin et al., 2008). But the enantioselective toxic effects of ICP on mammalian cells remain unknown. In this study, we investigate the role of individual isomers of ICP in inducing apoptosis of Hep G2 cells and further clarify the underlying molecular mechanisms. To the best of our knowledge, this study is the first report in which the role of MAPK signaling pathways and ROS generation involved in ICP-induced hepatocytic apoptosis. In our present study, the results of cell viability assay indicated an obvious enantioselective hepatocyte toxicity of ICP: (−)-ICP was about twofold more toxic than (+)-ICP in Hep G2 cells. In agreement with this data, the cytoflow assay showed that the amount of apoptotic cell treated with (−)-ICP was twofold higher than that of (+)-ICP at the concentration of 20 mg L−1 . It is well-known that the Bcl-2 protein is a suppressor of apoptosis that homodimerizes with itself and forms heterodimers with a homologous protein Bax, a promoter of apoptosis (Chen et al., 2005; Fletcher and Huang, 2008). These two proteins are critical mediators of apoptosis and their expression ratio are changed by apoptosis inducer. In this study, we found that (−)-ICP and rac-ICP up-regulated Bax protein expression and down-regulated Bcl-2 expression levels, which resulted in an increase in Bax/Bcl-2 ratio with the apoptosis co-ordination. However, (+)-ICP had no significant effect on
119
Bax/Bcl-2 expression compared to control cells. These data suggest that the isomers of ICP enantioselectively caused hepatic apoptosis resulting, at least in part, from their different actions on Bax/Bcl-2 ratio. Our previous observation indicated that MAPK signaling pathway was involved in the enantioselectivity of chiral pesticides in cytotoxicity (Liu et al., 2009). It is of importance to investigate the roles of MAPK signaling molecules in ICP-induced enantioselective hepatocyte toxicity. MAPK family includes the extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 kinase, which are involved in cell survival, proliferation and apoptosis in response to various growth or stress stimuli. The activation of ERK has been implicated in cell proliferation and cell cycle progression (Mebratu and Tesfaigzi, 2009), while JNK and p38 are more commonly activated in response to stress and toxicants which induce cell apoptosis (Xia et al., 1995). Importantly, a number of studies supported the concept that a sustained activation of JNK leads to apoptosis (Roos and Kaina, 2006; Wullaert et al., 2006). This idea was also proved by the present study, as evidenced by the fact that treatment with racemates of ICP caused a sustained JNK activation and the specific inhibitor of JNK significantly blocked (−)-ICP-induced apoptosis of Hep G2 cells. Notably, the activation of JNK was enantioselectively stimulated by (−)-ICP but not (+)-ICP. Although ERK1/2 was also activated by ICP, the inhibition of ERK activity only partially reversed (−)-ICP-induced hepatocyte toxicity. These results demonstrated that ICP enantioselectively induced hepatocyte toxicity dominantly through the sustained activation of JNK pathway, but only partially via ERK cascade. Interestingly, this observation is also consistent with our previous report that JNK mediated the enantioselectivity of bifenthrin in cytotoxicity of Hep G2 cells (Liu et al., 2009). It is conceivable that JNK is a common signaling mediator of enantioselective toxicity of chiral chemicals in mammalian cells or tissues. Oxidative stress has been implicated as the mechanism of hepatocytic toxicity from numerous toxicants (Czaja, 2007; Singh and Czaja, 2007). In the present study, (−)-ICP and rac-ICP induced ROS production, while (+)-ICP had no effect on ROS generation. It suggests that the ROS production induced by rac-ICP could be dominantly attributed to (−)-ICP but not (+)-ICP. This enantioselectivity of ICP in oxidative stress further confirmed the enantioselective toxicity of (−)-ICP enantiomer. Recent studies have demonstrated the mechanistic involvement of alterations in signal transduction cascades in response to ROS generation (Czaja, 2007; Singh and Czaja, 2007). We also found in this study that antioxidant MnTBAP attenuated (−)-ICP induced activation of JNK and ERK, indicating that the outcome from challenge with (−)-ICP enantiomer dependents on the oxidative stress-induced activation of a series of signaling cascades that promote hepatocyte apoptosis. As summarized in Fig. 9, we hypothesize the intracellular response in Hep G2 cells following exposure to individual enantiomers of ICP: (−)-ICP enantioselectively causes the change of Bax/Bcl-2 ratio, triggers the generation of intracellular ROS and sequentially induces sustainable activation of JNK, which in turn, results in a decrease in cell viability and an increase in cell apoptosis. This is the first report that estimates possible risks of the enantioselectivity of ICP for mammalian cells at the cytotoxic and molecular levels. Our observations provide further insight into enantiomers toxicity pathways which is able to differentiate between enantiomer activities at molecular level. Our enantiomer-specific researches may develop a predictive capability for enantioselectivity, so that these evidences may guide manufacturers toward the production of more single- or enrichedenantiomer pesticides, which are environmentally friendly and simultaneously efficient.
120
H. Liu et al. / Toxicology 276 (2010) 115–121
Fig. 9. A hypothetical model of the intracellular response in Hep G2 cells following exposure to individual enantiomers of ICP.
Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was supported by the National Natural Science Foundations of China (Nos. 20837002, 40973077), the National Basic Research Program of China (Nos. 2009CB421603, 2010CB126101). References Aprea, C., Strambi, M., Novelli, M.T., Lunghini, L., Bozzi, N., 2000. Biologic monitoring of exposure to organophosphorus pesticides in 195 Italian children. Environ. Health Perspect. 108, 521–525. Barr, D.B., Bravo, R., Weerasekera, G., Caltabiano, L.M., Whitehead Jr., R.D., Olsson, A.O., Caudill, S.P., Schober, S.E., Pirkle, J.L., Sampson, E.J., Jackson, R.J., Needham, L.L., 2004. Concentrations of dialkyl phosphate metabolites of organophosphorus pesticides in the U.S. population. Environ. Health Perspect. 112, 186–200. Barr, D.B., Allen, R., Olsson, A.O., Bravo, R., Caltabiano, L.M., Montesano, A., Nguyen, J., Udunka, S., Walden, D., Walker, R.D., Weerasekera, G., Whitehead Jr., R.D., Schober, S.E., Needham, L.L., 2005. Concentrations of selective metabolites of organophosphorus pesticides in the United States population. Environ. Res. 99, 314–326. Chen, L., Willis, S.N., Wei, A., Smith, B.J., Fletcher, J.I., Hinds, M.G., Colman, P.M., Day, C.L., Adams, J.M., Huang, D.C., 2005. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol. Cell. 17, 393–403. Chishti, M.A., Rotkiewicz, T., 1992. Ultrastructural alterations produced in cockerels after mercuric chloride toxicity and subsequent interaction with an organophosphate insecticide. Arch. Environ. Contam. Toxicol. 22, 445–451. Chishti, M.A., Rotkiewicz, T., 1993. Hepatic and renal ultrastructural changes in cockerels exposed to cadmium chloride and subsequent interaction with organophosphate insecticide. J. Environ. Pathol. Toxicol. Oncol. 12, 35–45. Circu, M.L., Aw, T.Y., 2010. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic. Biol. Med. 48, 749–762. Curl, C.L., Fenske, R.A., Elgethun, K., 2003. Organophosphorus pesticide exposure of urban and suburban preschool children with organic and conventional diets. Environ. Health Perspect. 111, 377–382. Czaja, M.J., 2007. Cell signaling in oxidative stress-induced liver injury. Semin. Liver Dis. 27, 378–389. Fletcher, J.I., Huang, D.C., 2008. Controlling the cell death mediators Bax and Bak: puzzles and conundrums. Cell Cycle 7, 39–44.
Garrison, A.W., 2006. Probing the enantioselectivity of chiral pesticides. Environ. Sci. Technol. 40, 16–23. Hreljac, I., Zajc, I., Lah, T., Filipic, M., 2008. Effects of model organophosphorous pesticides on DNA damage and proliferation of HepG2 cells. Environ. Mol. Mutagen. 49, 360–367. Hu, F., Li, L., Wang, C., Zhang, Q., Zhang, X.F., Zhao, M.R., 2010. Enantioselective induction of oxidative stress by permethrin in rat adrenal pheochromocytoma (PC12) cells. Environ. Toxicol. Chem. 29, 683–690. Jia, Z.Q., Li, Y., Lu, S., Peng, H.Z., Ge, J.Y., Chen, S.D., 2006. Treatment of organophosphate-contaminated wastewater by acidic hydrolysis and precipitation. J. Hazard. Mater. 129, 234–238. Jin, M.Q., Zhang, Y., Ye, J., Huang, C.J., Zhao, M.R., Liu, W.P., 2010. Dual enantioselective effect of the insecticide bifenthrin on locomotor behavior and development in embryo-larval zebrafish. Environ. Toxicol. Chem. 29, 1561–1567. Lin, K., Liu, W., Li, L., Gan, J., 2008. Single and joint acute toxicity of isocarbophos enantiomers to Daphnia magna. J. Agric. Food Chem. 56, 4273–4277. Liu, H., Zhao, M., Zhang, C., Ma, Y., Liu, W., 2008. Enantioselective cytotoxicity of the insecticide bifenthrin on a human amnion epithelial (FL) cell line. Toxicology 253, 89–96. Liu, H., Xu, L., Zhao, M., Liu, W., Zhang, C., Zhou, S., 2009. Enantiomer-specific, bifenthrin-induced apoptosis mediated by MAPK signalling pathway in Hep G2 cells. Toxicology 261, 119–125. Lu, C., Knutson, D.E., Fisker-Andersen, J., Fenske, R.A., 2001. Biological monitoring survey of organophosphorus pesticide exposure among pre-school children in the Seattle metropolitan area. Environ. Health Perspect. 109, 299–303. Lu, X., Hu, F., Ma, Y., Wang, C., Zhang, Y., Zhao, M., 2009. The role of oxidative stress in enantiomer-specific, bifenthrin-induced cytotoxicity in PC12 cells. Environ. Toxicol. (Epub ahead of print). Mebratu, Y., Tesfaigzi, Y., 2009. How ERK1/2 activation controls cell proliferation and cell death: Is subcellular localization the answer? Cell Cycle 8, 1168– 1175. Roos, W.P., Kaina, B., 2006. DNA damage-induced cell death by apoptosis. Trends Mol. Med. 12, 440–450. Singh, R., Czaja, M.J., 2007. Regulation of hepatocyte apoptosis by oxidative stress. J. Gastroenterol. Hepatol. 22 (Suppl. 1), S45–S48. Wang, L., Ye, W., Zhou, S., Lin, K., Zhao, M., Liu, W., 2009a. Acute and chronic toxicity of organophosphate monocrotophos to Daphnia magna. J. Environ. Sci. Health B 44, 38–43. Wang, L., Zhou, S., Lin, K., Zhao, M., Liu, W., Gan, J., 2009b. Enantioselective estrogenicity of o,p -dichlorodiphenyltrichloroethane in the MCF-7 human breast carcinoma cell line. Environ. Toxicol. Chem. 28, 1–8. Wullaert, A., Heyninck, K., Beyaert, R., 2006. Mechanisms of crosstalk between TNFinduced NF-kappaB and JNK activation in hepatocytes. Biochem. Pharmacol. 72, 1090–1101. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J., Greenberg, M.E., 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326–1331.
H. Liu et al. / Toxicology 276 (2010) 115–121 Xu, W.N., Liu, W.B., Liu, Z.P., 2009. Trichlorfon-induced apoptosis in hepatocyte primary cultures of Carassius auratus gibelio. Chemosphere 77, 895–901. Zhao, M., Liu, W., 2009. Enantioselectivity in the immunotoxicity of the insecticide acetofenate in an in vitro model. Environ. Toxicol. Chem. 28, 578–585. Zhao, M., Zhang, Y., Liu, W., Xu, C., Wang, L., Gan, J., 2008. Estrogenic activity of lambda-cyhalothrin in the MCF-7 human breast carcinoma cell line. Environ. Toxicol. Chem. 27, 1194–1200.
121
Zhao, M., Wang, C., Liu, K.K., Sun, L., Li, L., Liu, W., 2009. Enantioselectivity in chronic toxicology and accumulation of the synthetic pyrethroid insecticide bifenthrin in Daphnia magna. Environ. Toxicol. Chem. 28, 1475– 1479. Zhao, M., Chen, F., Wang, C., Zhang, Q., Gan, J., Liu, W., 2010. Integrative assessment of enantioselectivity in endocrine disruption and immunotoxicity of synthetic pyrethroids. Environ. Pollut. 158, 1968–1973.
Contents lists available at ScienceDirect
Toxicology journal homepage: www.elsevier.com/locate/toxicol
Enantioselective cytotoxicity of isocarbophos is mediated by oxidative stress-induced JNK activation in human hepatocytes Huigang Liu a,1 , Jing Liu b,1 , Lihong Xu c , Shanshan Zhou a , Ling Li a , Weiping Liu b,a,∗ a
Research Center of Green Chirality, College of Biology and Environment, Zhejiang University of Technology, Hangzhou 310032, People’s Republic of China MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China c Department of Biochemistry and Genetics, School of Medicine, Zhejiang University, Hangzhou 310058, People’s Republic of China b
a r t i c l e
i n f o
Article history: Received 22 June 2010 Received in revised form 24 July 2010 Accepted 26 July 2010 Available online 3 August 2010 Keywords: Chiral pesticide Enantioselectivity Isocarbophos JNK pathway Cytotoxicity
a b s t r a c t Recent studies have shown the enantioselectivity of chiral pesticides in environmental fate, aquatic toxicity, endocrine disruption and cytotoxicity. Thus it is of significance to investigate the molecular mechanisms of chiral pesticides enantioselectivity in cytotoxicity. In the present study, we used Hep G2 cells as in vitro model to assay cytotoxicity of enantiomers of isocarbophos (ICP), a widely used chiral organophosphorus pesticide. The results of cell viability assay and cytoflow assay indicated an obvious enantioselective hepatocyte toxicity of ICP: (−)-ICP was about two times more toxic than (+)-ICP in Hep G2 cells. We found that (−)-ICP, but not (+)-ICP, up-regulated Bax protein expression and down-regulated Bcl-2 expression levels, which resulted in an increase in Bax/Bcl-2 ratio with the apoptosis co-ordination. Although (−)-ICP enantioselectively activated both ERK and JNK, only the specific inhibitor for JNK could completely reverse (−)-ICP-induced apoptosis of Hep G2 cells. It suggests that (−)-ICP-induced hepatocyte toxicity was more dominantly through the sustained activation of JNK pathway, but only partially via ERK cascade. Furthermore, (−)-ICP induced ROS production, while (+)-ICP had no effect on ROS generation. The antioxidant MnTBAP attenuated (–)-ICP-induced activation of JNK and ERK, indicating that the outcome from challenge with (−)-ICP enantiomer depends on the oxidative stress-induced activation of a series of signaling cascades that promote hepatocyte apoptosis. In conclusion, (−)-ICP enantioselectively causes the change of Bax/Bcl-2 ratio, triggers the generation of intracellular ROS and sequentially induces sustainable activation of JNK, which in turn, results in a decrease in cell viability and an increase in cell apoptosis. Our observations provide further insight into enantiomers toxicity pathway which is able to differentiate between enantiomer activities at molecular level. Crown Copyright © 2010 Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction A chiral molecule and its non-superimposable mirror image are special types of stereoisomers called enantiomers. Although pure enantiomers of chiral compounds have identical physical and chemical properties in achiral environments, their behavior in biochemical processes might be strikingly different and selective.
Abbreviations: OPs, organophosphorus pesticides; ICP, isocarbophos; HPLC, high-performance liquid chromatography; MAPK, mitogen-ctivated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PVDF, polyvinylidene difluoride. ∗ Corresponding author at: MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China. Tel.: +86 571 8832 0666; fax: +86 571 8832 0884. E-mail address: [email protected] (W. Liu). 1 These authors contributed equally to the article.
This enantioselectivity often results in differences in bioactivity and toxicity of the two enantiomers. Up to 30% of currently used pesticides are chiral molecules, as are some organophosphorus pesticides (OPs) and many other compounds. However, almost all chiral pesticides are introduced into the environment as mixtures of equal amounts of the two or more enantiomers (racemic mixtures). A number of studies have shown the enantioselectivity of chiral pesticides in environmental fate (Garrison, 2006), aquatic toxicity (Jin et al., 2010; Wang et al., 2009a; Zhao et al., 2009), endocrine disruption (Wang et al., 2009b; Zhao et al., 2008, 2010) and cytotoxicity (Lu et al., 2009; Hu et al., 2010; Zhao and Liu, 2009). Recently, we reported that bifenthrin, a typical chiral pesticide, enantioselectively induced apoptosis and DNA damage in human aminon epithelial cells (Liu et al., 2008). Importantly, our further finding indicated that the enantioselectivity of bifenthrin in cytotoxicity was mediated by the mitogen-activated protein kinase (MAPK) signaling pathway (Liu et al., 2009). Therefore, to make more accurate risk assessments for chiral pesticides, it is of significance to investigate the
0300-483X/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2010.07.018
116
H. Liu et al. / Toxicology 276 (2010) 115–121 2.2. Assessment of cell viability Hep G2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Scientific HyClone, San Jose, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 ◦ C in a 5% CO2 incubator. Hep G2 cells, at 5 × 106 cells mL−1 , were treated with ICP or vehicle for 24 h. At end of culture, cell viability was measured by MTT with a Bio-Rad Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The results were calculated as the ratio of each exposure group to the vehicle control (0.1% ethanol, onefold) and as the mean ± standard deviation (SD) of four independent measurements performed in three replicates. These data were then analyzed using the t-test with Origin (OriginLab, Northampton, MA, USA).
Fig. 1. Enantiomers of isocarbophos (the asterisk indicates the chiral center).
molecular mechanisms of their enantioselectivity in cytotoxicity. Organophosphorus pesticides (OPs) are the most widely used pesticides worldwide since the 1950s and their metabolites are widespread across different populations (Aprea et al., 2000; Barr et al., 2004, 2005; Curl et al., 2003; Lu et al., 2001). A large number of OPs are chiral compounds, and yet enantioselectivity in their environmental fate and toxic effects is rarely addressed. One of these chiral OPs, isocarbophos (ICP), is widely used to control a variety of leaf-eating and soil insects. ICP was introduced into China for agricultural use in 1981, and currently the annual production of pure isocarbophos in China is about 5000 t (Jia et al., 2006). This molecule consists of a pair of enantiomers with a chiral center at the phosphorus atom: (−)-isocarbophos and (+)-isocarbophos (Fig. 1). In our previous study, ICP showed enantiomer-specific acute toxicity toward Daphnia magna (Lin et al., 2008). However, the molecular mechanism of ICP-induced enantiomer-specific toxicity remains unclear. In particular, it is currently unknown what signaling pathways mediated cytotoxic response to individual isomers of ICP. Although the primary target organ for OPs is the nerve system, there is increasing evidence to show their possible toxic effects on non-target tissues such as hepatocytes. For instance, trichlorfon induced apoptosis in hepatocyte primary cultures of Carassius auratus gibelio (Xu et al., 2009). Hepatic ultrastructural alterations were observed in cockerels exposed to methylobromofenvinphos (Chishti and Rotkiewicz, 1992, 1993). Hreljac et al. (2008) reported that OPs caused DNA damage and affected expression of DNA damage responsive genes in human hepatoma Hep G2 cells. Given hepatocyte toxicity is one of important toxic effects of OPs, we used Hep G2 cells as in vitro model in this study to assay cytotoxicity of enantiomers of ICP. Furthermore, we evaluated the enantioselective activation of MAPK signaling in Hep G2 cells exposed to individual isomers of ICP.
2.3. FITC-Annexin-V/propidium iodide staining assay Cell apoptosis was assessed by flow cytometry in accordance with the manufacturer’s protocol (Annexin-V-FITC Apoptosis Detection Kit, Sigma). Briefly, Hep G2 cells, at 5 × 106 cells mL−1 , were treated with ICP or vehicle for 24 h at 37 ◦ C in a 5% CO2 incubator, and then 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 PI, and incubated for 20 min at room temperature in the dark. All experiments were repeated at least three times and flow cytometric analysis was performed using a FACScan flow cytometer (Beckman Coulter EPICS XL). 2.4. Western blot analysis Cell cultures were homogenized on ice in freshly prepared lysis buffer. The lysate was cleared by centrifugation at 17,000 × g for 30 min at 4 ◦ C. Equal amounts of protein (50 g) from each sample were fractionated on a 12% SDS-PAGE gel and electrophoretically transferred to PVDF membranes. Transfers were blocked with 5% nonfat dry milk in TBST buffer and incubated with phospho-ERK1/2 (Thr202/Tyr-204), phospho-JNK, phospho-p38 (Cell Signal), Bax and Bcl-2 (ProteinTech Group) overnight at 4 ◦ C. HRP-labeled secondary anti-rabbit antibody (Cell Signal) was detected by enhanced chemiluminescence (Pierce). Finally, following immersion in ECL chemiluminescence reagents and blots were exposed to X-ray film for radiographic detection of the bands. 2.5. Assay of the cellular contents of reactive oxygen species (ROS) Following treatment with ICP or vehicle for 6 h, the treated cells were washed three times with ice-cold PBS and then incubated with 10 M 2 ,7 dichlorofluorescin diacetate (DCFH-DA; 100 mM in dimethyl sulfoxide, St. Louis, MO, USA) for 30 min at 37 ◦ C. The cellular free radical content was assayed by measuring 2 ,7 -dichlorofluorescein fluorescence using a fluorescent spectrophotometer (excitation at 485 nm/emission at 535 nm, Tecan Infinite M200, Switzerland). 2.6. Statistical analysis Results were expressed as the mean ± SD from at least three independent experiments. Statistical analysis was performed by one-way analysis of variance. Asterisks denote statistically significant differences at p < 0.05.
3. Results 3.1. Enantioselective cytotoxicity in human Hep G2 cells
2. Materials and methods 2.1. Reagents Analytical standard racemic isocarbophos [(±)-ICP, >99.6%, (RS)-(O-2isopropoxycarbonylphenyl O-methyl phosphoramidothioate)] was obtained from Kefa New Technology Development (Shenyang, China). Individual pure enantiomers [(−)-enantiomer and (+)-enantiomer] of ICP were separated and prepared from (±)-ICP according to modified procedures as described previously (Lin et al., 2008). Briefly, enantiomeric preparation was carried out using a high-performance liquid chromatography (HPLC) machine equipped with a variable-wavelength CD-2095 circular dichroism (CD) detector. Separation was achieved on a Chiralcel® OD column (250 mm × 4.6 mm) at 25 ◦ C and the flow rate of mobile phase was 0.8 mL min−1 . The detection wavelength of CD was set at 230 nm. The rotation sign (“+” or “−”) was indicated by a positive or negative peak on the chromatogram form CD detector. The preparation of pure enantiomers was obtained by manually collecting the eluent corresponding to the resolved peaks at the HPLC outlet of the CD detector while observing the UV absorbance. The collected individual enantiomer solution was evaporated to dryness, redissolved in ethanol, and used as the stock solution for bioassays (the final amount of ethanol in assay solution was <0.04%).
The results of MTT assay showed that treatment with (−)-ICP at concentrations of 2.5–40 mg L−1 reduced cell viability in a dosedependent manner and racemate of ICP significantly decreased viability of Hep G2 cells only at the highest concentration. But the reduction of cell viability by (+)-ICP is not significant. At the concentration of 40 mg L−1 , the order of viability of Hep G2 cells exposed to chemicals was (−)-ICP (55%) < rac-ICP (60%) < (+)-ICP (82%) (p < 0.05) (Fig. 2). 3.2. Enantioselective apoptosis in human Hep G2 cells FITC-Annexin-V/propidium iodide staining assay was performed to determine the effects of ICP on Hep G2 cell apoptosis. Fig. 3A shows the typical results of the flow cytometric analysis of the cells. The bottom left quadrants represent viable cells, the bottom right quadrants represent apoptotic cells and the upper right
H. Liu et al. / Toxicology 276 (2010) 115–121
117
Fig. 2. The effect of individual enantiomers and racemates of isocarbophos on the viability of human Hep G2 cell lines. Hep G2 cells were incubated with different concentrations of individual enantiomers and racemates of isocarbophos for 24 h, followed by analysis using the MTT assay. Different letters above adjacent bars indicate significant differences (p < 0.05, n = 3) between individual enantiomers and racemate, while the same letter indicates no significant difference.
quadrants represent necrotic and late apoptotic cells. The numbers in each quadrant are the percentage of cells in that category in ethanol (solvent) from ICP-treated samples. The apoptosis rate in Hep G2 cells exposed to rac-ICP, (+)-ICP and (−)-ICP at concentration of 20 mg L−1 were 3.32%, 2.28% and 7.95%, respectively (Fig. 3B). In addition, the expression of the apoptotic proteins Bax and Bcl-2 were determined using Western blotting. (−)-ICP and racICP increased the expression levels of Bax protein and reduced Bcl-2 expression. However, (+)-ICP had no effect on Bax and Bcl-2 expression (Fig. 4). 3.3. Expression and activation of MAP kinases by isocarbophos To determine whether specific MAPK signal transduction pathways are involved in cellular responses to rac-ICP, the expression of phosphorylation of ERK1/2, JNK and p38 were measured by Western blotting. Both ERK1/2 and JNK exhibited sustained activation in response to 20 mg L−1 rac-ICP exposure (Fig. 5). No significant change in the active levels of p38 kinase was observed throughout the 12 h treatment. The total levels of each MAPK did not change during the 12 h incubation period.
Fig. 3. Evaluation of apoptotic cells by the Annexin-V staining assay. (A) Apoptosis quantified by Annexin-V-PI staining. Hep G2 cells were stained by Annexin-V and PI and then analyzed by flow cytometry. Four subpopulations and their quantities are indicated: nonapoptotic dead cells (R1), late apoptotic cells (R2), viable cells (R3), and early apoptotic cells (R4). Numbers in R4 are the percentage of early apoptotic cells contained. (B) The percent of early apoptotic cells following treatment with individual enantiomers or racemate of isocarbophos was measured with flow cytometer. At least three independent experiments were carried out for each condition, and a minimum of 300 cells were counted for each measurement.
3.4. Activation of ERK1/2 and JNK by single isomer of isocarbophos To further determine whether the activation of ERK1/2 and JNK by rac-ICP results from a single enantiomer of ICP, pure (−)-ICP and (+)-ICP were individually incubated with Hep G2 cells at a concentration of 20 mg L−1 for 6 h. The data showed that exposure to (−)-ICP resulted in increased levels of the phosphorylated forms of ERK1/2 and JNK, whereas exposure to (+)-ICP had no effect on MAPK phosphorylation. Additionally, both (+)-ICP and (−)-ICP did not activate p38 MAPKs (Fig. 6). 3.5. Inhibition of ERK1/2 and JNK attenuates (−)-ICP-induced cytotoxicity and apoptosis To test the hypothesis that the enantioselective activation of ERK1/2 and JNK by ICP contributes to rac-ICP-induced cytotoxicity and apoptosis, a series of kinase inhibitors were used to block
Fig. 4. Involvement of Bax and Bcl-2 protein expression in isocarbophos 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 isocarbophos at the concentration of 20 mg L−1 for 6 h, and cell lysates were subjected to western immunoblotting. Lysates for SDS-PAGE were prepared and western blotting was performed as described in Section 2. Bax protein expression in Hep G2 cells treated with (−)-isocarbophos showed a marked increase in its levels compared to cells treated with (+)-isocarbophos or control protein GAPDH. Conversely, Hep G2 cells incubated with (−)-isocarbophos showed a marked decrease in Bcl-2 protein levels.
118
H. Liu et al. / Toxicology 276 (2010) 115–121
Fig. 5. Time course of isocarbophos-induced accumulation of phosphorylated ERK (A), JNK (B), and p38 MAPK (C). Hep G2 cells were incubated with 20 mg L−1 of the racemate of isocarbophos for the indicated time. The cell lysates were analyzed by western blotting using respective antibodies. The data shown is from a typical experiment involving two representative experiments.
the activity of ERK1/2 and/or JNK. As shown in Fig. 7, treatment with SP600125 significantly attenuated (−)-ICP-induced cytotoxicity (Fig. 7A) and apoptosis (Fig. 7B). The inhibitor of ERK1/2 partially reversed the effects of (−)-ICP on cytotoxicity and apoptosis of Hep G2 cells. These data suggest that (−)-ICP-induced hepototoxicity is mainly mediated by JNK activation, but partially through ERK1/2 pathway. Fig. 7. Inhibition of ERK1/2 and JNK kinases attenuates isocarbophos-induced cytotoxicity and apoptosis. Hep G2 cells were pre-treated for 1 h with 50 M of the ERK1/2 inhibitor PD98059, the 10 M JNK inhibitor SP600125, or vehicle control, followed by 20 mg L−1 of the individual enantiomers and racemates of isocarbophos for 12 h treatment. Following incubation, cell viability was measured by MTT assay (A), and apoptosis was analyzed by using flow cytometry (B).
3.6. Involvement of reactive oxygen species in ERK and JNK signaling pathways
Fig. 6. The effect of individual enantiomers of isocarbophos on the activation of phosphorylated ERK 1/2 (A), JNK (B), and p38 MAPKs (C). Hep G2 cells were incubated with 20 mg L−1 of the individual enantiomers and racemates of isocarbophos for 6 h, and cell lysates were subjected to western blot analysis. Results shown are representative of two independent experiments.
Reactive oxygen species (ROS) have been implicated as potential modulators of apoptosis (Circu and Aw, 2010). In Hep G2 cells, treatment with (−)-ICP and rac-ICP caused a dosedependent accumulation of intracellular ROS. But ROS production in cells incubated with (+)-ICP only slightly increased, even at a concentration of 20 mg L−1 . The levels of ROS production in (−)-ICP-treated cells were two times higher than that of (+)-ICP at the highest concentration of 20 mg L−1 (p < 0.01) (Fig. 8A).To further determine the role of (−)-ICP-induced ROS generation in the activation of JNK and ERK1/2, Hep G2 cells were pretreated for 12 h with 200 M of MnTBAP, a specific inhibitor for ROS, and then incubated with 20 mg L−1 of rac-ICP, (+)-ICP and (−)-ICP for 6 h. As shown in Fig. 8B, MnTBAP markedly decreased the activity of ERK1/2 and JNK induced by (−)-ICP. However, MnTBAP had no effect on total ERK1/2 and JNK expression. These results suggest that (−)-ICP-induced ROS accumulation may contribute to the activation of ERK1/2 and JNK in hepatocytes.
H. Liu et al. / Toxicology 276 (2010) 115–121
Fig. 8. The effect of individual enantiomers and racemates of isocarbophos on intracellular ROS production (A). Hep G2 cells exposed to different concentrations of individual enantiomers and racemate of isocarbophos for 3 h, followed by ROS determination. Inhibition of ROS on ERK and JNK signaling pathways (B). Hep G2 cells were pre-treated for 12 h with 200 M of MnTBAP, and then stimulated for 3 h with 20 mg L−1 of the individual enantiomers and racemates. Phosphorylation of ERK1/2 and JNK were measured by western blot analysis.
4. Discussion In the previous study, we demonstrated that ICP could enantioselectively cause toxicity toward Daphnia magna (Lin et al., 2008). But the enantioselective toxic effects of ICP on mammalian cells remain unknown. In this study, we investigate the role of individual isomers of ICP in inducing apoptosis of Hep G2 cells and further clarify the underlying molecular mechanisms. To the best of our knowledge, this study is the first report in which the role of MAPK signaling pathways and ROS generation involved in ICP-induced hepatocytic apoptosis. In our present study, the results of cell viability assay indicated an obvious enantioselective hepatocyte toxicity of ICP: (−)-ICP was about twofold more toxic than (+)-ICP in Hep G2 cells. In agreement with this data, the cytoflow assay showed that the amount of apoptotic cell treated with (−)-ICP was twofold higher than that of (+)-ICP at the concentration of 20 mg L−1 . It is well-known that the Bcl-2 protein is a suppressor of apoptosis that homodimerizes with itself and forms heterodimers with a homologous protein Bax, a promoter of apoptosis (Chen et al., 2005; Fletcher and Huang, 2008). These two proteins are critical mediators of apoptosis and their expression ratio are changed by apoptosis inducer. In this study, we found that (−)-ICP and rac-ICP up-regulated Bax protein expression and down-regulated Bcl-2 expression levels, which resulted in an increase in Bax/Bcl-2 ratio with the apoptosis co-ordination. However, (+)-ICP had no significant effect on
119
Bax/Bcl-2 expression compared to control cells. These data suggest that the isomers of ICP enantioselectively caused hepatic apoptosis resulting, at least in part, from their different actions on Bax/Bcl-2 ratio. Our previous observation indicated that MAPK signaling pathway was involved in the enantioselectivity of chiral pesticides in cytotoxicity (Liu et al., 2009). It is of importance to investigate the roles of MAPK signaling molecules in ICP-induced enantioselective hepatocyte toxicity. MAPK family includes the extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 kinase, which are involved in cell survival, proliferation and apoptosis in response to various growth or stress stimuli. The activation of ERK has been implicated in cell proliferation and cell cycle progression (Mebratu and Tesfaigzi, 2009), while JNK and p38 are more commonly activated in response to stress and toxicants which induce cell apoptosis (Xia et al., 1995). Importantly, a number of studies supported the concept that a sustained activation of JNK leads to apoptosis (Roos and Kaina, 2006; Wullaert et al., 2006). This idea was also proved by the present study, as evidenced by the fact that treatment with racemates of ICP caused a sustained JNK activation and the specific inhibitor of JNK significantly blocked (−)-ICP-induced apoptosis of Hep G2 cells. Notably, the activation of JNK was enantioselectively stimulated by (−)-ICP but not (+)-ICP. Although ERK1/2 was also activated by ICP, the inhibition of ERK activity only partially reversed (−)-ICP-induced hepatocyte toxicity. These results demonstrated that ICP enantioselectively induced hepatocyte toxicity dominantly through the sustained activation of JNK pathway, but only partially via ERK cascade. Interestingly, this observation is also consistent with our previous report that JNK mediated the enantioselectivity of bifenthrin in cytotoxicity of Hep G2 cells (Liu et al., 2009). It is conceivable that JNK is a common signaling mediator of enantioselective toxicity of chiral chemicals in mammalian cells or tissues. Oxidative stress has been implicated as the mechanism of hepatocytic toxicity from numerous toxicants (Czaja, 2007; Singh and Czaja, 2007). In the present study, (−)-ICP and rac-ICP induced ROS production, while (+)-ICP had no effect on ROS generation. It suggests that the ROS production induced by rac-ICP could be dominantly attributed to (−)-ICP but not (+)-ICP. This enantioselectivity of ICP in oxidative stress further confirmed the enantioselective toxicity of (−)-ICP enantiomer. Recent studies have demonstrated the mechanistic involvement of alterations in signal transduction cascades in response to ROS generation (Czaja, 2007; Singh and Czaja, 2007). We also found in this study that antioxidant MnTBAP attenuated (−)-ICP induced activation of JNK and ERK, indicating that the outcome from challenge with (−)-ICP enantiomer dependents on the oxidative stress-induced activation of a series of signaling cascades that promote hepatocyte apoptosis. As summarized in Fig. 9, we hypothesize the intracellular response in Hep G2 cells following exposure to individual enantiomers of ICP: (−)-ICP enantioselectively causes the change of Bax/Bcl-2 ratio, triggers the generation of intracellular ROS and sequentially induces sustainable activation of JNK, which in turn, results in a decrease in cell viability and an increase in cell apoptosis. This is the first report that estimates possible risks of the enantioselectivity of ICP for mammalian cells at the cytotoxic and molecular levels. Our observations provide further insight into enantiomers toxicity pathways which is able to differentiate between enantiomer activities at molecular level. Our enantiomer-specific researches may develop a predictive capability for enantioselectivity, so that these evidences may guide manufacturers toward the production of more single- or enrichedenantiomer pesticides, which are environmentally friendly and simultaneously efficient.
120
H. Liu et al. / Toxicology 276 (2010) 115–121
Fig. 9. A hypothetical model of the intracellular response in Hep G2 cells following exposure to individual enantiomers of ICP.
Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was supported by the National Natural Science Foundations of China (Nos. 20837002, 40973077), the National Basic Research Program of China (Nos. 2009CB421603, 2010CB126101). References Aprea, C., Strambi, M., Novelli, M.T., Lunghini, L., Bozzi, N., 2000. Biologic monitoring of exposure to organophosphorus pesticides in 195 Italian children. Environ. Health Perspect. 108, 521–525. Barr, D.B., Bravo, R., Weerasekera, G., Caltabiano, L.M., Whitehead Jr., R.D., Olsson, A.O., Caudill, S.P., Schober, S.E., Pirkle, J.L., Sampson, E.J., Jackson, R.J., Needham, L.L., 2004. Concentrations of dialkyl phosphate metabolites of organophosphorus pesticides in the U.S. population. Environ. Health Perspect. 112, 186–200. Barr, D.B., Allen, R., Olsson, A.O., Bravo, R., Caltabiano, L.M., Montesano, A., Nguyen, J., Udunka, S., Walden, D., Walker, R.D., Weerasekera, G., Whitehead Jr., R.D., Schober, S.E., Needham, L.L., 2005. Concentrations of selective metabolites of organophosphorus pesticides in the United States population. Environ. Res. 99, 314–326. Chen, L., Willis, S.N., Wei, A., Smith, B.J., Fletcher, J.I., Hinds, M.G., Colman, P.M., Day, C.L., Adams, J.M., Huang, D.C., 2005. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol. Cell. 17, 393–403. Chishti, M.A., Rotkiewicz, T., 1992. Ultrastructural alterations produced in cockerels after mercuric chloride toxicity and subsequent interaction with an organophosphate insecticide. Arch. Environ. Contam. Toxicol. 22, 445–451. Chishti, M.A., Rotkiewicz, T., 1993. Hepatic and renal ultrastructural changes in cockerels exposed to cadmium chloride and subsequent interaction with organophosphate insecticide. J. Environ. Pathol. Toxicol. Oncol. 12, 35–45. Circu, M.L., Aw, T.Y., 2010. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic. Biol. Med. 48, 749–762. Curl, C.L., Fenske, R.A., Elgethun, K., 2003. Organophosphorus pesticide exposure of urban and suburban preschool children with organic and conventional diets. Environ. Health Perspect. 111, 377–382. Czaja, M.J., 2007. Cell signaling in oxidative stress-induced liver injury. Semin. Liver Dis. 27, 378–389. Fletcher, J.I., Huang, D.C., 2008. Controlling the cell death mediators Bax and Bak: puzzles and conundrums. Cell Cycle 7, 39–44.
Garrison, A.W., 2006. Probing the enantioselectivity of chiral pesticides. Environ. Sci. Technol. 40, 16–23. Hreljac, I., Zajc, I., Lah, T., Filipic, M., 2008. Effects of model organophosphorous pesticides on DNA damage and proliferation of HepG2 cells. Environ. Mol. Mutagen. 49, 360–367. Hu, F., Li, L., Wang, C., Zhang, Q., Zhang, X.F., Zhao, M.R., 2010. Enantioselective induction of oxidative stress by permethrin in rat adrenal pheochromocytoma (PC12) cells. Environ. Toxicol. Chem. 29, 683–690. Jia, Z.Q., Li, Y., Lu, S., Peng, H.Z., Ge, J.Y., Chen, S.D., 2006. Treatment of organophosphate-contaminated wastewater by acidic hydrolysis and precipitation. J. Hazard. Mater. 129, 234–238. Jin, M.Q., Zhang, Y., Ye, J., Huang, C.J., Zhao, M.R., Liu, W.P., 2010. Dual enantioselective effect of the insecticide bifenthrin on locomotor behavior and development in embryo-larval zebrafish. Environ. Toxicol. Chem. 29, 1561–1567. Lin, K., Liu, W., Li, L., Gan, J., 2008. Single and joint acute toxicity of isocarbophos enantiomers to Daphnia magna. J. Agric. Food Chem. 56, 4273–4277. Liu, H., Zhao, M., Zhang, C., Ma, Y., Liu, W., 2008. Enantioselective cytotoxicity of the insecticide bifenthrin on a human amnion epithelial (FL) cell line. Toxicology 253, 89–96. Liu, H., Xu, L., Zhao, M., Liu, W., Zhang, C., Zhou, S., 2009. Enantiomer-specific, bifenthrin-induced apoptosis mediated by MAPK signalling pathway in Hep G2 cells. Toxicology 261, 119–125. Lu, C., Knutson, D.E., Fisker-Andersen, J., Fenske, R.A., 2001. Biological monitoring survey of organophosphorus pesticide exposure among pre-school children in the Seattle metropolitan area. Environ. Health Perspect. 109, 299–303. Lu, X., Hu, F., Ma, Y., Wang, C., Zhang, Y., Zhao, M., 2009. The role of oxidative stress in enantiomer-specific, bifenthrin-induced cytotoxicity in PC12 cells. Environ. Toxicol. (Epub ahead of print). Mebratu, Y., Tesfaigzi, Y., 2009. How ERK1/2 activation controls cell proliferation and cell death: Is subcellular localization the answer? Cell Cycle 8, 1168– 1175. Roos, W.P., Kaina, B., 2006. DNA damage-induced cell death by apoptosis. Trends Mol. Med. 12, 440–450. Singh, R., Czaja, M.J., 2007. Regulation of hepatocyte apoptosis by oxidative stress. J. Gastroenterol. Hepatol. 22 (Suppl. 1), S45–S48. Wang, L., Ye, W., Zhou, S., Lin, K., Zhao, M., Liu, W., 2009a. Acute and chronic toxicity of organophosphate monocrotophos to Daphnia magna. J. Environ. Sci. Health B 44, 38–43. Wang, L., Zhou, S., Lin, K., Zhao, M., Liu, W., Gan, J., 2009b. Enantioselective estrogenicity of o,p -dichlorodiphenyltrichloroethane in the MCF-7 human breast carcinoma cell line. Environ. Toxicol. Chem. 28, 1–8. Wullaert, A., Heyninck, K., Beyaert, R., 2006. Mechanisms of crosstalk between TNFinduced NF-kappaB and JNK activation in hepatocytes. Biochem. Pharmacol. 72, 1090–1101. Xia, Z., Dickens, M., Raingeaud, J., Davis, R.J., Greenberg, M.E., 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326–1331.
H. Liu et al. / Toxicology 276 (2010) 115–121 Xu, W.N., Liu, W.B., Liu, Z.P., 2009. Trichlorfon-induced apoptosis in hepatocyte primary cultures of Carassius auratus gibelio. Chemosphere 77, 895–901. Zhao, M., Liu, W., 2009. Enantioselectivity in the immunotoxicity of the insecticide acetofenate in an in vitro model. Environ. Toxicol. Chem. 28, 578–585. Zhao, M., Zhang, Y., Liu, W., Xu, C., Wang, L., Gan, J., 2008. Estrogenic activity of lambda-cyhalothrin in the MCF-7 human breast carcinoma cell line. Environ. Toxicol. Chem. 27, 1194–1200.
121
Zhao, M., Wang, C., Liu, K.K., Sun, L., Li, L., Liu, W., 2009. Enantioselectivity in chronic toxicology and accumulation of the synthetic pyrethroid insecticide bifenthrin in Daphnia magna. Environ. Toxicol. Chem. 28, 1475– 1479. Zhao, M., Chen, F., Wang, C., Zhang, Q., Gan, J., Liu, W., 2010. Integrative assessment of enantioselectivity in endocrine disruption and immunotoxicity of synthetic pyrethroids. Environ. Pollut. 158, 1968–1973.