Induction of Macrophage Apoptosis by an Organochlorine Insecticide

Jan 23, 2009 - Macrophages, which play a key role in inflammation, host defense, and reactions against a spectrum of autologous and foreign invaders, ...
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Chem. Res. Toxicol. 2009, 22, 504–510

Induction of Macrophage Apoptosis by an Organochlorine Insecticide Acetofenate Meirong Zhao,† Ying Zhang,‡ Cui Wang,† Zhengwei Fu,† Weiping Liu,*,† and Jay Gan§ Research Center of Green Chirality, College of Biological and EnVironmental Engineering, Zhejiang UniVersity of Technology, Hangzhou 310032, People’s Republic of China, Institute of EnVironmental Science, College of EnVironmental and Resource Sciences, Zhejiang UniVersity, Hangzhou 210027, People’s Republic of China, and Department of EnVironmental Sciences, UniVersity of California, RiVerside, California 92521 ReceiVed September 29, 2008

Acetofenate (AF) is a widely used insecticide in China and other regions of southeastern Asia. A previous study showed that AF caused adverse developmental effects in zebrafish. Macrophages, which play a key role in inflammation, host defense, and reactions against a spectrum of autologous and foreign invaders, are crucial for innate immunity. However, cytotoxicity and apoptosis of macrophages caused by organochlorine pesticides (OCPs) have so far received little attention. In this study, we used AF as a model chemical to investigate the cytotoxic effects of OCPs on mouse macrophage cell line RAW264.7. Results from cell viability and apoptosis assays showed that AF induced apparent apoptosis in RAW 264.7 cells. Furthermore, AF induced intracellular reactive oxygen species (ROS) generation and DNA damage and resulted in the alteration of a series of signaling molecules including up-regulation of p53 and cytochrome c protein levels, decline of the Bcl-2/Bax protein ratio, and activation of the caspases cascade through caspase-9 and caspase-3. These results, for the first time, revealed that the increase of endogenous ROS and DNA damage comediating OCP-induced apoptosis in macrophages may be by the mitochondria and p53 signal pathway. Our results suggested that macrophages are involved in AF-induced adverse immune effects. Considering the ubiquitous environmental presence of OCPs, this study provided new information on the potential long-term physiological and immunological effects due to chronic exposures to OCPs. Introduction Although the use of most organochlorine pesticides (OCPs), such as 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT), hexachlorocyclohexane (HCH), dieldrin, and chlordane, has been discontinued as a result of their environmental persistence, exposure to OCPs will continue during the coming years (1, 2). As a consequence, many OCPs may induce chronic toxicities through long-term exposure even if doses are relatively low (3). Previous studies using laboratory animals and wildlife species have demonstrated chronic toxicities of OCPs, and the observed toxicities include immunotoxicity (4), reproductive toxicity (5), developmental toxicity, and genotoxicity (6). The immune system consists of complex and highly specialized cells, tissues, and organs and of innate and inducible immune functions to protect organisms from invaders. The immune system plays a critical role in maintaining homeostasis. However, the balance can be potentially broken when exposed to certain chemicals, which may result in an enhancement of the immune response that may lead to allergy, autoimmunity, or immunosuppression that may increase cancer susceptibility and the risk of infections (7). Constant exposure to a variety of environmental pollutants like pesticides is known to have a deleterious influence on immune functions (8, 9). For example, widespread use of DDT was associated with potential health hazards, and subchronic DDT exposure suppressed cell-mediated * To whom correspondence should be addressed. Tel: +86-571-88320666. Fax: +86-571-8832-0884. E-mail: [email protected]. † Zhejiang University of Technology. ‡ Zhejiang University. § University of California.

immunity in experimental animals (10). Other studies in this regard showed immunotoxicity by other OCPs, such as PCB (11, 12), HCB (13), and lindane (14). In the immune system, macrophages, which play a key role in inflammation, host defense, and reactions against a spectrum of autologous and foreign invaders, are crucial in innate immunity (15, 16). However, when the control mechanisms go awry, the inflammatory response of macrophages may result in persistent swelling, pain, and eventually tissue injury. The functional aspects of macrophages have been proposed for use as an important biomarker for immunotoxic chemicals (17). Some studies have shown that the exposure to OCPs can affect the immune system through macrophages. For example, DDT inhibited the functional activation of murine macrophages (18); macrophages were involved in HCB-induced adverse immune effects (19); dieldrin/carbofuran mixture had an antagonistic effect on the macrophage phagocytic activity (20). However, in these studies, there was little understanding of cytotoxic effects and cell damages by OCPs in macrophages, such as apoptosis, oxidative damage, and genotoxicity. The investigation of cytotoxicity of OCPs in macrophages will improve the mechanistic understanding of immunotoxicity from OCPs. Acetofenate (AF), also known as 7504, plifenate, and benzethazet, was developed as an analogue of DDT. Because of its low toxicity, biodegradability, and high efficacy against pests that have developed resistance to DDT and HCH (21), AF is considered a good replacement to these prohibited OCPs and is widely used to control mosquitoes and flies both indoors and outdoors in China and other regions of the southeastern Asia (21, 22). Although there are no data on environmental levels of AF, there are numerous reports on the environmental

10.1021/tx800358b CCC: $40.75  2009 American Chemical Society Published on Web 01/23/2009

Induction of Apoptosis in Macrophage by Acetofenate

occurrence of DDT and derivatives. For example, the mean DDT concentrations followed the order fish > crab > shrimp in southern China, suggesting food-web biomagnification. DDT concentrations in fish from Daya Bay ranged from 1.7 to 462 ng g-1 (median ) 40) (23), and DDT concentrations (ng g-1) in the brown trout diet in Lake Redon were 1.72-18.76 ng g-1 (24). These findings would translate into DDT concentrations in the range of 10-7-10-6 M in some aquatic organisms. Our group recently reported that AF exhibited significant development toxicities in zebrafish embryo and caused various nonlethal malformations even at 1.8 ppm (21). The results suggest that despite its low acute toxicity, AF may have other potential toxic effects. In this study, we used AF as a model OCP and mouse macrophage cell line RAW264.7 as an in vitro model to evaluate the possible effects of AF on innate immune functions through cytotoxicity of macrophages. Evaluations focused on cell viability, oxidative damage and genetic damage, and the mechanism of macrophage apoptosis.

Materials and Methods Chemicals and Reagents. AF [97.3% purity, 2,2,2-trichloro-1(3,4-dichlorophenyl)ethyl acetate] was obtained from Xinhuo Technology Institute (Baoding, China). HPLC grade n-hexane and ethanol were purchased from Tedia (Fairfield, OH) and used as solvents. AF was dissolved in n-hexane at 2000 mg L-1 and kept at 4 °C in the dark as a stock solution. Test solutions at a range of concentrations were prepared in ethanol, with the final solvent content at 0.1% by volume. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, Utah, UT). Other chemicals or solvents used in this study were of cell culture, HPLC, or analytical grade. Cell Culture and Treatments. RAW 264.7 cells, obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China), were cultured in the DMEM medium supplemented with 10% FBS in 25 cm2 flasks, at 37 °C in a humidified CO2 incubator (Thermo Electron, Marietta, OH) consisting of 5% CO2 and 95% air. The culture media was refreshed every 2-3 days, and passage at a ratio of 1:3 was performed with routine trypsinization every 5-6 days. Before treatment, the culture medium was replaced with the experimental medium (DMEM containing 2% FBS) for 1 day to reduce the effect of serum. On the basis of the results of pretests and our previous study (21), the cells were treated with dosing medium (the experimental medium along with the test compound) at concentrations of 10-9-10-5 mol L-1 for 3 days for the cell viability assay, 10-7 or 10-6 mol L-1 for 6 h for generation of the reactive oxygen species (ROS) assay, 1 day for the comet assay, and 2 days for apoptosis and Western blotting analysis. Ethanol (0.1% v/v) was used as the negative control. Assessment of Cell Viability. Cell viability is an important end point for the assessment of cytotoxicity. The cell viability of RAW 264.7 cells was determined by the MTT [(3-(4,5-dimethylthiazol2-yl)-2,5- diphenyltetrazolium bromide (Amresco, Solon, OH)] assay based upon our previous study (25). Cells were seeded in 96 well plates at an initial concentration of 1000 cells per well. After 24 h of attachment, the medium was changed to dosing medium containing five concentrations of the test compound for 3 days. To determine the influence of exposure time on cell proliferation, the cells were detected at a concentration of 10-7 mol L-1 by the same method at every 24 h interval until reaching 72 h. After the exposure of AF, MTT solution [5 mg mL-1 in phosphate-buffered saline (PBS)] was added into wells and then incubated at 37 °C for 4 h. After the medium was removed from the wells, 150 µL of dimethyl sulfoxide (DMSO) per well was added into the wells. The absorbance was measured at 490 nm with a Bio-Rad model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA) after 10 min of shaking. Results were expressed as O.D. 490 of each exposure group and the vehicle control.

Chem. Res. Toxicol., Vol. 22, No. 3, 2009 505 Hoechest 33324 Staining. To examine the nucleus condensation in the apoptotic cells, the RAW264.7 cells were stained with hoechest 33324. Cells were incubated with vehicle or 10-7 and 10-6 mol L-1 of AF for 48 h and washed with cold PBS twice. Cultured cells were then incubated with hoechest 33324 for 20 min at room temperature. After mounting, the cells were observed under a Leica fluorescence microscope (Leica, Wetzlar, Germany). Annexin-V/Propidium Iodide (PI) Staining. The annexin-V/ PI assay provides a simple and effective method to detect apoptosis at a very early stage. The AF-induced cell apoptosis was determined by Annexin-V fluorescent kit according to the manufacturer’s instructions (Roche, Rotkreuz, Switzerland). RAW264.7 cells incubated with 10-7 and 10-6 mol L-1 of AF for 48 h were harvested and washed with cold PBS twice. Cells were resuspended in binding buffer at a concentration of 1 × 106 cells mL-1, and a 200 µL cell suspension was subsequently transferred to a 1.5 mL tube and treated with 5 µL of Annexin-V and 5 µL of PI at 37 °C for 15 min in the dark. Then, cells were diluted with 200 µL of binding buffer, and 10000 cells in each group were analyzed on FACS Calibur (Becton Dickinson, Franklin Lakes, NJ) equipped with a single laser emitting excitation light at 488 nm. Measurement of Intercellular ROS. The intracellular ROS generation was assayed using a fluorescence probe, 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma, St. Louis, MO). The nonpolar, nonionic DCFH-DA is known to cross cell membranes and to be enzymatically hydrolyzed by intracellular esterases to nonfluorescent DCFH. In the presence of ROS, such as H2O2 or low molecular weight peroxyl radicals, DCFH is rapidly oxidized to highly fluorescent 2′,7′-dichlorofluorescein (DCF) (26). In brief, RAW264.7 cells were treated with vehicle or AF at 10-7 or 10-6 mol L-1 for 6 h and then washed three times with ice-cold PBS. The cells were incubated with 10 µmol L-1 DCFH-DA (prepared in DMSO at100 mmol L-1) for 30 min at 37 °C. At the end of incubation, the fluorescence intensity was measured at 485 nm excitation and 535 nm emission using a fluorescent spectrophotometer (Infinite M200, Tecan, Switzerland). The relative levels of ROS were expressed as the fluorescence intensity of AF-treated groups and negative control. Comet Assay. The single cell gel electrophoresis assay (also known as comet assay) is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. The protocol used for the comet assay followed the guidelines of Singh et al. (27). Observations were made at a final magnification of 400× using an epifluorescence microscope (Zeiss, Oberkochen, Germany) equipped with filters suitable for ethidium bromide. A number of 50 randomly selected cells per sample (25 from each slide) were measured to evaluate tail length. Comet assay analyses were made by use of a computerized image analysis system (Comet Assay II, Perceptive Instruments, Suffolk, United Kingdom). Western Blot Analysis. The protocol used for the Western blot analysis was similar to that given in a previous study (28). The cultured cells were lysed with lysis buffer (20 m mol L-1 tris-HCl, pH 8.0, 1 mmol L-1 DL-dithiothreitol, 0.2% NP40, 100 µmol L-1 phenylmethanesulfonyl fluoride, 5 mg mL-1 aprotinin, chymostatin, leupeptin, pristine, and trypsin inhibitor) on ice for 20 min. The lysates were centrifuged at 13000 rpm, and the supernatants were collected. After the protein concentration was measured by Bradford assay (29), 25 µg of protein was subjected to 10% SDSpolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% defatted milk in PBS containing 0.1% Tween20, then incubated with mouse antimouse p53, Bcl-2, Bax, cytochrom C, β-actin IgG (1:000, Santa Cruz, CA) and antimouse caspase-3, caspase-9, and anti-Apaf-1 (1:000, Cell Signaling Technology, Danvers, MA), and finally incubated with horseradish peroxidase (HRP)-conjugated goat antimouse IgG (1:2000; Santa Cruz). Final visualization was achieved by the ECL Western Blotting Analysis System (Pierce, Rockford, IL). The membrane was exposed to X-ray film (Fuji, Shizuoka, Japan) and analyzed by the Gel-Pro Analyzer (United Bio, San Francisco, CA).

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Figure 1. Effect of AF on the viability of murine RAW264.7 cell lines. RAW264.7 cells were incubated with (A) different concentrations of AF for 72 h and (B) 10-7 mol L-1 of AF for 24, 48, and 72 h followed by the MTT assay (* indicates p < 0.05, n ) 5).

Statistical Analysis. All of the experiments were repeated at least three times. Unless otherwise stated, all data were expressed as means ( standard deviations. Comparison of the values between groups was performed by ANOVA, and p < 0.05 was considered statistically significant.

Results Assessment of Cell Viability. The cytotoxicity assay is a type of in vitro assay to examine the toxic effects of environmental chemicals by analyzing cell growth and apoptosis on target cells. Our results showed a clear dose-response of cell growth inhibition after the treatment of AF. The growth of RAW 264.7 cells was significantly reduced by AF within the range of 10-9 to 10-5 mol L-1, and the EC50 of AF was about 10-6.8 mol L-1 (Figure 1A). Data from RAW 264.7 cells exposed to 10-7 mol L-1 of AF for different durations of time showed that AF inhibited the cell growth in a time-dependent manner. Exposure for 24 h to 10-7 mol L-1 of AF almost had no effect on cell viability, but longer exposures (more than 48 h) decreased cell viability by 35% or more (Figure 1B). These results suggested that AF possessed apparent cytotoxicity to macrophage cells and could be potentially immunotoxic through macrophage damage, especially at low concentrations and with long time exposures. Induction of Apoptosis. Cytotoxicity of the cells may be caused by effects on cell cycles and induction of apoptosis. Our results from Annexin-V/PI staining indicated that 10-7 and 10-6 mol L-1 of AF induced a significant increase in Annexin-V-

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positive, PI-negative cells with dose dependence (Figure 2A). The cells treated with negative control and 10-7 and 10-6 mol L-1 of AF had values of 1.28, 6.73, and 16.97% early apoptotic cells, respectively. AF also induced RAW 264.7 cell necrosis (Annexin-V-positive, PI-positive) with dose dependence, with 10-6 mol L-1 of AF treatment causing 8% necrosis. Meanwhile, the results of hoechst 33324 staining also suggested a significant increase of the ratio of condensation of chromatin in RAW264.7 cells after 48 h of AF treatment (Figure 2B). These data revealed that AF caused intensive macrophage apoptosis, which may lead to alteration of innate immune functions. Induction of ROS Generation. Oxidative damage and ROS generation caused by some chemicals play an important role in cytotoxicity in a variety of cell models. ROS are thought to be involved in many forms of apoptosis (30). As shown in Figure 3, the intracellular ROS was enhanced by 2.0- and 3.8-fold after treatment of AF at 10-7 and 10-6 mol L-1, respectively. This observation was consistent with the cytotoxicity induced by AF. These results therefore indicated that ROS generation may be a required step for AF-induced macrophage apoptosis. Induction of Genotoxicity. DNA damage represented by DNA single strand breaks was reflected by an increase in tail moments. The DNA damage was evaluated using the comet assay, in which tail length is an important quantitative parameter. Considering tail length (Figure 4B), significant dose-dependent induction was observed after the treatment of 10-7 or 10-6 mol L-1 of AF for 24 h. Meanwhile, from the comet assay (Figure 4A), the integrity of the nucleolus decreased in cells incubated with 10-7 or 10-6 mol L-1 of AF, exhibiting obvious DNA damage with dose dependence. Alteration of Apoptosis-Related Molecules. The effects of stimuli on macrophages apotosis are usually connected to specific alterations in gene and protein expression after a series of intracellular signals initiated by ligand-receptor interactions including p53 (31), Bcl-2 (32), Bax, cytochrome c (33), apoptotic protease-activating factor-1 (Apaf-1) (34), and caspases (35). As shown in Figure 5, there was an apparent increase in p53 protein expression after the AF treatment; the protein level of Bcl-2 was reduced by more than 60% after 10-6 mol L-1 of AF exposure (Figure 5). However, there were no notable changes in the Bax protein expression. Nevertheless, the ratio of antiapoptotic Bcl-2 to pro-apoptotic Bax dropped. Meanwhile, we investigated the possibility that AF-induced apoptosis might be related to mitochondria, especially cytochrome c. The results showed that the cytochrome c was released after AF treatment with dose dependence. From previous studies, mitochondrial damage allows cytochrome c to escape from the mitochondrial to cytosol and to combine with Apaf-1 and activate caspase-9 (36). Our results showed that with the release of cytochrome c, two other intracellular proteins involved in mitochondrialmediated apoptosis, Apaf-1, a major component of apoptosome, and caspsse-9, were also up-regulated in a dose-dependent manner. Protein expression of effector caspase, caspase-3, was increased. All of the above observations indicated that p53 and the proteins of components of the intracellular molecules may be involved in the process of AF-induced macrophage apoptosis.

Discussion AF is an OCP commonly used in China and other regions of Asia. In this study, we explored the role of AF in inducing apoptosis of macrophages and delineated the underlying molecular mechanisms. To the best of our knowledge, this study was the first case in which the role of ROS generation and oxidative damage has been considered in OCP-induced macrophage apoptosis.

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Figure 2. Evaluation of apoptotic cells by the Annexin-V staining assay and hochest 33324 staining. (A) Apoptosis quantified by Annexin V-PI staining after RAW264.7 cells were stained by Annexin-V and PI and then analyzed by flow cytometer. (B) Evaluation of apoptotic cells by hoechst 33324 staining after treatment of AF. The red arrows point out several apoptotic cells with typical chromatin condensation. Natural cells are pointed out with white arrows. Letters A, B, and C represent proteins derived from the RAW264.7 cells treated with the negative control, 10-7, and 10-6 mol L-1 of AF, respectively.

Figure 3. Effect of AF on intracellular ROS production. RAW264.7 cells were exposed to 10-7 and 10-6 mol L-1 of AF for 6 h, followed by ROS determination (* indicates p < 0.05, n ) 5).

The cytotoxic effect of AF was determined by MTT and the cell apoptosis assay. Our data indicated clear cytotoxicity of AF on RAW 264.7 cells. Cytotoxicity studies with several OCPs in vitro and in vivo have been reported. For example, DDT affected macrophage viability in an in vivo model (37), and endosulfan caused apoptotic and necrotic cytotoxicity in murine thymocytes (38). However, the molecular mechanisms of the macrophage cytotoxicity of OCPs are poorly understood and require further studies. Cytotoxicity of the cells may be partly caused by effects on cell cycles and induction of apoptosis. In the immune system apoptosis is not restricted to immune cells, which exhibits the morphology of apoptosis as shrinkage and zeiosis, nucleus collapse, and condensation of the chromatin into nucleosomal fragments (39). Our results revealed that AF at 10-7 or 10-6 mol L-1 displayed an increasing dose-dependent cellular apoptotic effect. ROS have been shown to mediate apoptosis in a variety of cell models, and ROS generation is considered an important apoptotic signal. In this study, we monitored ROS after AF exposure, and the results suggested a likely role of endogenous

ROS in apoptotic cells induced by AF. In previous studies, OCPinduced ROS generation was investigated in some other cell line models. For instance, dieldrin was found to induce hepatotoxicity via oxidative stress in mice (40). DDT induced oxidative damage in human blood mononuclear cells (41). However, our results were the first to confirm the role of ROS in OCP-induced macrophage apoptosis. Results from this and previous studies together suggested that ROS generation is a required process for OCP-induced apoptosis. It is well-established that there are close relationships between ROS generation and DNA damage (42). Our data clearly showed that AF induced DNA strand breaks in RAW 264.7 cells with the highest level of damage. The breakage of DNA involves several mechanisms, such as the formation of DNA adducts, intercalating of coplanar molecules in DNA, and oxidative damage of bases (43). Although the clear mechanism of ROS production and DNA damage by AF is unknown, some results from other OCPs including DDT provide probable explanations. There were two possible pathways that could have been involved in the DNA damage: the activation of cytochromes P450 (44) and the interaction with the mitochondrial chain complexes (45). It is likely that the RAW264.7 cells were susceptible to ROS generation and oxidative damage due to the fact that AF shared a similar mechanism to DDT. Most apoptotic signaling processes are related to alterations of apoptosis-related molecules, such as p53, Bcl-2/Bax, and cytochrome c, among others. The p53 protein is an important regulator of induction of apoptosis (45). The Bcl-2 family of intracellular proteins is the central regulator of caspase activation, and its opposing factions of anti- and pro-apoptotic members arbitrate the life-or-death decision (46). Additionally, the Bcl-2 family is a number of potent regulators of apoptosis that can influence the permeability of outer mitochondrial membrane and the release of cytochrome c (47), with mitochondria playing a pivotal role in apoptosis. In the present study, p53 protein expression was up-regulated after AF treatment, indicating that p53 might be involved in AF-induced apoptosis in macrophages. The results also revealed that the ratio of

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Figure 4. Effect of AF on DNA damage by comet assay. (A) Comet assay pictures and (B) tail length data of RAW264.7 cells. RAW264.7 cells were exposed to AF at 10-7 or 10-6 mol L-1 for 24 h, followed by the comet assay (* indicates p < 0.05, n ) 5). Letters A, B, and C represent proteins derived from the RAW264.7 cells treated with the negative control, 10-7, and 10-6 mol L-1 of AF, respectively.

Figure 5. Western blot analysis to manifest the effect of p53, Bcl-2, Bax and cytochrome c, Apaf-1, caspase-9, and caspase-3 protein expression by AF. (A) Typical results of Western blotting. Letters A, B, and C represent proteins derived from the RAW264.7 cells treated by the negative control, 10-7, and 10-6 mol L-1 of AF, respectively. Panels B and C show statistical analysis by ANOVA for Western blotting analyses. The values of p53, Bcl-2, Bax, Apaf-1, caspase-9, caspase-3, and cytochrome c were normalized by that of β-actin, and the relative amount is presented as the mean ( SD (* indicates p < 0.05, n ) 3).

antiapoptotic Bcl-2 to pro-apoptotic Bax decreased. During the apoptotic stimulation, the alteration of Bcl-2/Bax protein ratio may affect the mitochondria and cytochrome c release (48). From previous studies, mitochondrial damage allows cytochrome c to escape from the mitochondrial to cytosol and to

combine with Apaf-1 and activate caspase-9 (34). The activated caspase-9 initiated a death program in the cells by cleaving the executioner caspases, such as caspase-3 and caspase-7 (34). Hence, we investigated the possibility that AF-induced apoptosis might be related to mitochondria, especially cytochrome c, Apaf-

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the ubiquitous presence of other OCPs in the environment, it would be worthwhile to carry out more comprehensive studies and epidemic investigations to understand the potential immunotoxicity of OCPs. Acknowledgment. This study was supported by the National Natural Science Foundations of China (nos. 20837002 and 20877071), the National Basic Research Program of China (no. 2009CB421603), the Natural Science Foundation of Zhejiang Province, China (no. Y507214), and the Program for Changjiang Scholars and Innovative Research Team in Chinese Universities (no. IRT 0653).

References

Figure 6. Hypothetical model for AF-induced apoptotic pathway in macrophages.

1, “initiator” caspases-9, and effector caspases-3. The results showed that cytochrome c was released after treatment of AF. With the release of cytochrome c, the protein level of Apaf-1 and caspases-9 increased. It is likely that caspases-9 was activated and interacted with Apaf-1 to form the apoptosome complex after cytochrome c was released from damaged mitochondria. The apoptosome complex appeared to be central to the activation of the caspase cascade. Existing knowledge on the potential mechanisms for OCP-induced immune cell toxicity is rather limited. A previous study showed that exposure to DDT suppressed Bcl-2 protein and mRNA transcript levels but up-regulated p53 protein and mRNA transcripts in T lymphocytes (49). To the best of our knowledge, this study represented the first instance suggesting the involvement of mitochondria, cytochrome c, caspases, and p53 in OCP-induced macrophage apoptosis. On the basis of our experimental results, we propose a hypothetical model of pathway on the apoptotic effect of AF in macrophages (Figure 6). In this pathway, AF first triggered intrinsic ROS generation, which subsequently initiated oxidative damage. The increased ROS levels and genotoxicity upregulated p53 protein levels and attenuated Bcl-2 protein, leading to a declined ratio of Bcl-2/Bax. The alteration of Bcl-2/Bax ratio resulted in the release of cytochrome c. Finally, with the release of cytochrome c, Apaf-1 and caspase-9 formed the apoptosome complex that activated caspase-3 and eventually led to macrophage apoptosis. However, the molecular mechanisms of the proposed pathway merit further investigations.

Conclusions AF is a current-use organochlorine insecticide that possesses effects on induction of macrophage apoptosis. Through this study, we explored for the first time the role of OCPs in inducing p53 and mitochondrial-mediated apoptosis of macrophages. Our findings not only suggested that AF possesses potential immunotoxicity but also advanced the understanding on the probable molecular mechanisms of OCPs-induced macrophage cell apoptosis. Considering the widespread use of AF in China and

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