Extract of Motorcycle Exhaust Particles Induced Macrophages

damage to respiratory tract. In this study, we reported that the extract of motorcycle exhaust particles (MEP) might affect the immune system by induc...
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Extract of Motorcycle Exhaust Particles Induced Macrophages Apoptosis by Calcium-Dependent Manner Chen-Chen Lee and Jaw-Jou Kang* Institute of Toxicology, College of Medicine, National Taiwan University, 1 Jen-Ai Road, Section 1, Taipei, Taiwan, R.O.C. Received June 11, 2002

Large survey and experiments have reported that environment pollutants from fossil fuel combustion would cause immune system deleterious by enhancement of allergic reaction and damage to respiratory tract. In this study, we reported that the extract of motorcycle exhaust particles (MEP) might affect the immune system by inducing cell apoptosis on macrophages. The motorcycle exhaust particles were collected from a two-stoke engine and their cytotoxic effect on macrophages was investigated. We found MEP is cytotoxic and induced apoptosis in RAW 264.7 cells, murine peritoneal macrophage, and rat alveolar macrophage. Pretreatment with mitochondria permeability transition inhibitor (cyclosporin A), intracellular (BAPTAAM) and extracellular (EGTA) Ca2+ chelator, and antioxidants (NAC, GSH, catalase, SOD) attenuated the MEP-induced cell apoptosis, and BAPTA-AM was the most effective one. Utilized Fura-2/AM loaded RAW 264.7 cells to directly detect the change of intracellular Ca2+ concentration ([Ca2+]i), we found that MEP could induce a sustained increase of [Ca2+]i. The raise of [Ca2+]i induced by MEP could be completely blocked by the intracellular Ca2+ chelator, BAPTA-AM, however, only partially inhibited by the extracellular Ca2+ chelator, EGTA. These results suggested that both influx of extracellular Ca2+ and release of Ca2+ from the internal storage were involved. We also found that MEP caused a decrease of mitochondria membrane potential and an increase of oxidative stress in RAW 264.7 cells. In conclusion, we found that the particles, collected from the motorcycle exhaust, contain chemicals that will induce apoptosis of macrophage in calcium-dependent manner.

Introduction Large surveys have documented a continuing increase in nasal and pulmonary allergic disease over the course of last century (1-5), and there is growing evidence that environmental pollutants can affect the immunologic function that is involved in asthma and chronic obstructive pulmonary disease (6-10). This trend seems to be in concert with the increase in fossil fuel combustion and emission of particulate pollutants (8). Indeed, the air pollution from fuel and motor vehicle has been a serious problem since the industrial revolution, especially in urban areas, and vehicles are now regarded as the main source of variety of pollutants (11). For example, the sulfate and particle matter, which are primarily from fossil fuel combustion would cause enhancement of allergic reaction and deleterious effect to respiratory tract (12). In addition, a large number of experiments have been reported that the particulate matter in the exhaust of diesel engine (DEP)1 had adverse effects of induction and propagation of allergic inflammatory, including increase IgE production, Th2 cytokine production, and mucosal inflammation in human and animals challenged by allergens (13-16). * To whom correspondence should be addressed. E-mail: jjkang@ ha.mc.ntu.edu.tw. Telephone: 886-2-23123456 ext 8603. Fax: 886-223410217. 1 Abbreviations: MEP, motorcycle exhaust particles; ROI, reactive oxygen intermediates; MMP, mitochondria membrane potential; NAC, N-acetylcysteine; GSH, glutathione; SOD, superoxide dismutas; DMSO, dimethyl sulfoxide; BAPTA-AM, 1,2-bis (2-aminophenoxy)ethaneN,N,N′,N′-tetraacetic acid; EGTA, N,N,N′,N′-tetraacetic acid.

In Taiwan, motorcycles are widely used, and more than 11 million motorcycles were registered in 2000 (17). The use of motorcycles, especially the two-stoke engine, introduced about 16 000 and 15 000 tons of total suspended particle (TSP) and the particulate matter of 10 µm (PM10), respectively, per year in Taiwan (18). The impact of the motorcycle exhaust to the environment and their biological effects are relatively unknown. Studies have indicated that motorcycle exhaust particles (MEP) were mutagenic (19) and genotoxic in vitro (20). It was also shown that MEP could induce impairment effect on rat aorta (11) and several metabolic enzymes in rat tissues (21). However, no study has been taken to examine the effects of MEP on immune systems. Macrophages have many important biological functions such as being phagocytic cells in providing a first line of defense against infection, being antigen presenting cells for activation by armed effectors T cells, controlling tumor growth, excluding damaged or senescent cells, and participating in wound healing and tissue repair (22). In addition, they are in charge of removing pollutants from bronchoalveolar region of the lung (23). However, macrophages are also the target of many environment pollutants, including PAHs, polychlorinated biphenyls, halogenated PAHs, and DEP in the respiratory tract, and would be activated and participated in metabolism of these compounds (24-30). Damage to macrophages would not only exacerbate microbial infection (31) and tissue damage (32) but the death of macrophages would also, resulting in spread toxic compounds that accumulated by phagocytosis to damage neighbor cells (23).

10.1021/tx0255727 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/05/2002

MEP-Induced Macrophage Cells Apoptosis

In this study, we have investigated the motorcycle exhaust particle (MEP) on macrophage. Our data showed that MEP-induced apoptosis of macrophages in calciumdependent manner.

Materials and Methods Chemicals. Ascorbic acid (VitC) was purchased from Merck (Darmstadt, Germany). Fura-2/AM, pluronic F127, 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)], and 5,6-carboxy-2′,7′-dichlorofluorescein-diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene, OR). Brewer’s thioglycollate medium was purchased from Difco (Detroit, MI). DMEM medium, FBS (fetal bovine serum), sodium pyruvate was purchased from Gibco (Grand island, NY). Antibiotic was purchased from Irvine Scientific (Santa Ana, CA). All other chemicals were purchased from Sigma (St. Louis, MI). Collection and Preparation of Motorcycle Exhaust Particles. The motorcycle exhaust particles (MEP) were collected, on a 0.5-µm quartz fiber filter, from a 50-cm3 Yamaha two-stroke engine using 95% octane unleaded gasoline. The sampling apparatus consisted of in sequence a 40-cm long by 2.2-cm diameter stainless dilute tube, a filter holder, and a vacuum pump. The engine was running at 150 rpm on an empty load, and the pump was set at a flow rate of 20 L/min to collect particles for 1 h of four times daily. The filters with particles matters were left to dry and repeatedly extracted with methanol of four times under sonication. The methanol fractions were pooled and the methanol was removed by a vacuum evaporator. The final residues, the motorcycle exhaust particles (MEP), were collected and kept desiccated at -20 °C. Basically, 32.7 µg of final residue (MEP) could be derived from 1 L of motorcycle exhaust. For preparing the particles free extract, the MEP was filtered with organic solvent resistant 0.2 µm filter (Advantec MFS, Inc. CA) to remove the particle, the filtrate was then concentrated as MEP to yield motorcycle exhaust particulate extract (MEPE). Cells. Mouse peritoneal macrophages were elicited by intraperitoneal injection of 1 mL of 4% Brewer’s thioglycollate medium into BALB/c mice (7-12 weeks) (33). Peritoneal exudate cells were obtained 4 days after injection by peritoneal lavage with ice cold phosphate-buffered saline (PBS) contained of 145 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4. Peritoneal exudate cells were incubation with RBC lysis buffer (17 mM Tris, 140 mM ammonium chloride, pH 7.2) for 3 min and centrifuged at 1500 rpm for 5 min at 4 °C to remove RBC. The cell pellet was washed twice with PBS and resuspended in DMEM medium supplemented with 10% heat-inactivated FBS containing 1 mM sodium pyruvate, 2 mM glutamine, 100 units/ mL penicillin, and 100 µg/mL streptomycin (DMEM complete medium). Purity and viability (trypan blue exclusion) were >95% under light microscopy. Rat alveolar macrophage cells were collected by bronchoalveolar lavage (34). Briefly, the rats were sacrificed by fully anesthesia of ether. A 18-gauge angiocatheter was inserted through a tracheostomy and the lungs were lavaged eight times with 5 mL of cold PBS. The lavage fluid was kept on ice and centrifuged at 1000 rpm for 5 min at 4 °C, and the cell pellet was resuspended in DMEM complete medium. Purity and viability (trypan blue exclusion) were >95% under light microscopy. RAW 264.7 mouse macrophage cells (ATCC) were grown in DMEM complete medium, passage 1:10 (v/v) twice a week and incubated at 37 °C under a CO2/95% air atmosphere. Determination of Cell Viability by MTT Assay. Viability of cells was assessed using the 3-(4,5-dimethylthylthiazol-2-yl)2,5 -diphenyltetrazolium bromide (MTT) assay that measures mitochondrial dehydrogenase activity (35). Viable cells with active mitochondria caused cleavage of the tetrazolium ring into a visible dark blue formazan reaction product. MTT was dissolved in DMEM complete medium, and stock solution was 2 mg/mL.

Chem. Res. Toxicol., Vol. 15, No. 12, 2002 1535 Mouse peritoneal macrophages, rat alveolar macrophages, and RAW 264.7 cells were plated in 96-well microtiter plates at 5 × 105/mL of cells/well in a final volume of 200 µL in DMEM complete medium at 37 °C for 24 h. Cells were treated with different concentrations of MEP for another 24 h. After incubation, 50 µL MTT was added and incubated at 37 °C for 3 h, and then the medium was gently moved. Cells and dye crystals were dissolved in 100 µL of DMSO and absorption measured at 570 nm in an ELISA reader (MRX-TC; Dynex Technology, Chantilly, VA). Measurement of Intracellular Calcium ([Ca2+]i) by Fura-2/AM. Intracellular calcium ([Ca2+]i) was measured according to Lawrie et al. (36). Cells were grown on 22-mm diameter coverslips and loaded with 2 µM Fura-2/AM in PBS containing 0.1% FBS and 0.0125% (v/v) pluronic F127 at 37 °C for 45 min. After washing out the loading buffer, coverslips were placed in a chamber of microscope and changes in fluorescence were monitored using a PTI M-series spectrofluorometer (PTI, Monmouth Junction, NJ) with dual excitation wavelength of 340 and 380 nm and an emission wavelength of 510 nm. The increase of [Ca2+]i was calculated from background subtracted 340/380 nm ratio signals using the formula described by Grynkiewicz et al. (37). Rmax and Rmin were derived with addition of 5 µM ionomycin and 20 mM EGTA, respectively. Identification and Quantification of Apoptotic Cells by Flow Cytometry. Two milliliters of 3 × 105/mL of cells was seeded in 6-well culture dish, and different concentrations of MEP were added. Different antioxidants and calcium chelators were added at 30 and 10 min, respectively, before treatment of MEP. After incubation at the time point indicated in each experiment, both floating and trypsinized adherent cells were collected, washed twice with ice cold PBS, and fixed in 100% methanol. After fixation, cells were washed with PBS, added to 10 µg/mL RNAase A for 30 min, and then stained with propidium iodide 40 µg/mL for 20 min in the dark. Stained cells were analyzed flow cytometrically by FACScalibur (Becton Dickinson, San Jose, CA). DNA Fragmentation Assay on Agarose Gel. Cells were treated with MEP 300 µg/mL for 6, 12, 18, 24 h, then harvested and washed twice with ice cold PBS, resuspended in lysis buffer containing 10 mM EDTA, 50 mM Tris-HCl, 0.5% Sarkosyl and proteinase K 1 mg/mL (pH 8.0) at 56 °C for 3 h. Cell lysates were then treated with RNAase A 2 mg/mL for further 1.5 h at 56 °C. DNA was extracted with phenol/chloroform/IAA (25:24: 1) and centrifuged 14 500 rpm for 10 min at 25 °C. Electrophoresis was carried out for 0.5 h at 100 V in 2% agarose gel prepared in TBE buffer (80 mM Tris-Borate, 2 mM EDTA, pH 8.0). After electrophoresis, gels were stained 0.1 µg/mL ethidium bromide and examined under ultraviolet light and photographed (38). Analysis of Mitochondrial Membrane Potential (MMP) and ROI Production by Flow Cytometry. Mitochondrial membrane potential (MMP) was estimated by staining with DiOC6(3), a cationic lipophilic dye (39). The production of ROI was estimated by staining with oxidation-sensitivity fluorescent dye DCFH-DA (40). Basically, 2 × 106 cells were stained with 40 nM DiOC6(3) or 20 µg/mL DCFH-DA for 15 and 30 min, respectively, at 37 °C in the dark and analyzed by FACScalibur (Becton Dickinson, San Jose, CA) at excitation wavelength of 488 nm and emission wavelength of 525 nm. Glutathione Levels. Cellular glutathione levels were detected by colorimetric assay with GSH-400 kit (OXIS, Portland, OR) (41). Briefly, 1 × 107 cells were treated with chemicals for different time intervals and then harvested by centrifugation 2000 rpm for 5 min at 4 °C. The cell pellets were homogenized with 1 mL of 33% metaphosphoric acid and centrifugation 3000g for 10 min at 4 °C. The supernatants were collected for GSH determination, and the pellets were collected for protein determination. For GSH determination, 250 µL of supernatant in 650 µL of buffer [200 mM potassium phosphate, pH 7.8, containing 0.2 mM diethylenetriamine pentaacetic acid (DTPA) and 0.025% (w/v) lubrol] was added with 50 µL of 12 mM chromogenic

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reagent (4-chloro-1-methyl-7-trifluoromethyl-quinolinium methyl sulfate, dissolved in 0.2 N HCl) and 30% NaOH separately. After incubation at 25 ( 3 °C for 15 min in the dark, GSH concentrations were quantitated photometrically at 400 nm with GSH as a standard and were normalized per microgram of protein. Protein concentrations were determined by the method of Bradford assay (42). Oxygen Consumption Measurement. Cells respiration and O2 content of the incubation mixture were measured with a Clark-type oxygen electrode (model 5300; YSI Inc., Yellow Springs, OH) in a 3 mL incubation chamber maintained at 37 °C (43). A total of 2 × 106 cells/mL of RAW 264.7 cells was kept at 37 °C in Krebs-Henseleit buffer containing HEPES (25 mM), pH 7.4, during oxygen consumption measurement, after obtaining the basal rate of oxygen consumption, and 10 mM succinate was added to measure the activity of mitochondria. Statistical Analysis. All values refer to mean ( SEM of at least three separate experiments. Statistically significant difference between two groups was using Student’s t-test. The minimal level of significance was p < 0.05.

Results Induction of Cytotoxicity and Apoptosis by MEP in Macrophages. The cytotoxicity effect of MEP in macrophages was examined by using the MTT assay. MEP could dose- (Figure 1A) and time-dependently (Figure 1B) decrease the cell viability in RAW 264.7 cells, murine peritoneal macrophages, and rat alveolar macrophages with LD50 values of 29.5, 66.1, and 85.1 µg/mL, respectively, at 24 h. The cytotoxicity induced by MEP was further characterized by propidium iodide staining and flow cytometric analysis. As shown in Figure 2A, MEP at 300 µg/mL, induced apoptosis in all three macrophage cells as judged by the significant increase in subG1 (M1) content. The subG1 fractions of the MEP-treated RAW 264.7 cells were 42.0 ( 2.4 and 77.8 ( 4.6% at concentrations of 100 and 300 µg/mL, respectively. The MEP-induced apoptosis was also time dependent (Figure 2B). Agarose gel electrophoretic analysis of RAW 264.7 cell chromosomal DNA treated by MEP showed a ladder-like pattern of DNA fragments consisting of multiples of approximal 180-200 base pairs (Figure 2C), a specific characteristic of apoptotic cells. In addition, we found that the particle-free MEP, the MEPE, induced apoptosis of macrophages to the same degree as MEP (data not shown). Inhibition of MEP-Induced Cells Apoptosis in RAW 264.7 Cells by Calcium Chelators and Antioxidants. Since there is increasing evidence showing that increase of [Ca2+]i, induction of ROI production, and alteration of mitochondria function are linked to cell apoptosis (44). The mitochondria permeability transition (MPT) inhibitor (cyclosporin A) (45), intracellular (BAPTAAM), and extracellular (EGTA) calcium chelators and different antioxidants (NAC, GSH, catalase, SOD) were used to study factors that might affect the MEP-induced apoptosis of RAW 264.7 cells (Figure 3). The degree of MEP-induced apoptosis was reduced by around 10% in the presence of cyclosporin A. Treatment with extracellular Ca2+ chelator, EGTA, greatly inhibited the apoptosis induced by MEP in RAW 264.7 cells, however, not as effectively as the intracellular Ca2+ chelator, BAPTA-AM. The apoptosis induced by MEP was almost completely inhibited by BAPTA-AM. Pretreatment of antioxidants and antioxidative enzymes also shows protective effect on MEP-induced apoptosis.

Figure 1. Cytotoxicity of MEP on macrophages. (A) Different types of macrophage cells were treated with different concentrations of MEP for 24 h. (B) RAW 264.7 cells were treated with MEP 300 µg/mL for different time periods. Cell viability was determined by MTT assay as described in Materials and Methods and compared to the vehicle control. Data points were expressed as mean ( SEM (n g 3).

MEP-Induced Increase of [Ca2+]i in RAW 264.7 Cells. The results suggested that Ca2+ might play an important role in MEP-induced apoptosis, therefore, the effect of MEP on Ca2+ homeostasis was further studied. The change of intracellular Ca2+ concentration ([Ca2+]i) in RAW 264.7 cells was measured with the fluorescent probe, Fura-2/AM, and a typical trace was shown in Figure 4A. MEP-induced a sustained increase of [Ca2+]i as evident by the increase of fluorescence ratio value. When cells were incubated in Ca2+-free HBSS containing EGTA, a small increase of [Ca2+]i was observed, and the [Ca2+]i level was quickly return to basal level. However, when cells were preincubated with intracellular Ca2+ chelator, BAPTA-AM, no change of [Ca2+]i was observed (Figure 4A). The average value of elevation of [Ca2+]i induced by MEP 300 µg/mL was 1388.9 ( 248.3 nM and dropped to 325 ( 75 nM in the presence of EGTA (Figure 4B). In a separate experiment, we have found that the change of [Ca2+]i induced by MEP and MEPE were to

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Figure 2. Induction of apoptosis by MEP on macrophage cells. (A) Cells were treated with MEP 300 µg/mL for 24 h and then stained with propidium iodide for detection of cell apoptosis. Propidium iodide staining of nuclear DNA was analyzed in FACcan flow cytometry as described in the Materials and Methods. (1) RAW 264.7 cells. (2) Murine peritoneal macrophages. (3) Rat alveolar macrophage. M1: apoptosis region. M2: normal region. (B) RAW 264.7 cells were treated with MEP 300 µg/mL for different time periods and then stained with propidium iodide for detection of cell apoptosis. Data points were expressed as mean ( SEM (n g 3) (*) P < 0.05, (***) P < 0.001 as compared to vehicle control. (C) RAW 264.7 cells were treated with MEP 300 µg/mL for different time periods as indicated. DNA were extracted for gel electrophoresis and visualized with ethidium bromide as described in the Materials and Methods. M: marker

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Figure 3. Factors affecting cell apoptosis induced by MEP in RAW264.7 cells. Cells were pretreated with cyclosporin A and calcium chelators for 10 min and antioxidants for 0.5 h, then treated with MEP 300 µg/mL for 6 h and stained with propidium iodide. Propidium iodide staining of nuclear DNA was analyzed in FACcan flow cytometry as described in the Materials and Methods. Data were expressed as mean ( SEM (n g 3) (a*) p < 0.05, (a**) p < 0.01, (a***) p < 0.001, compared with DMSO 0.1% group, (b***) p < 0.001,compared to the control group treated with MEP 300 µg/mL only.

the same degree, with the average value of 1250 ( 226 nM and 1150 ( 119 nM, respectively. Induction of Oxidative Stress by MEP in RAW 264.7 Cells. The induction of reactive oxygen intermediates (ROI) production by MEP was measured with the fluorescent dye, DCFH-DA. We found that MEP induced an increase of ROI production in time-dependent manner reached maximum at 2 h and sustained for at least 24 h (Figure 5A). The degree of ROI production was greatly attenuated by the calcium chelators and antioxidants (Figure 5B) Glutathione (GSH) is an important antioxidative molecule in cell, and the drop of GSH level was often an indication of increase of oxidative stress (46). By direct measurement of GSH content, we found that the content of GSH was significantly reduced upon MEP treatment in RAW264.7 cells (Table 1). MEP Treatment Caused a Decrease of Mitochondria Membrane Potential in RAW 264.7 Cells. The effect of MEP on mitochondria was assessed by monitoring the change of mitochondria membrane potential (MMP) of RAW 264.7 cells and data shown in Figure 6. A 0.4-fold decrease in the mean of fluorescence intensity was observed in RAW 264.7 cells treated with prontonophore CCCP 50 µM for 6 h (Figure 6A, trace b), as compared to vehicle treated control cells (Figure 6A, trace a), indicating the collapse of MMP. After treatment with MEP 100 µg/mL (Figure 6B, trace b) and MEP 300 µg/ mL (Figure 6B, trace c), there was a 0.3- and 0.5-fold decrease in the mean fluorescence intensity, respectively, as compared to vehicle treated control cells (Figure 6B, trace a). In the presence of Ca2+ chelators and antioxidants, the MEP-induced decrease of MMP was partially reversed but never completely recovered (Figure 7). The direct effect of MEP on mitochondria was assessed by measuring the rate of oxygen consumption in RAW 264.7 cells. We found that MEP, at concentrations greater than

Figure 4. Induction of increase of [Ca2+]i by MEP in RAW 264.7 cells. (A) Cells were stained with 5 µM Fura-2/AM and [Ca2+]i is measured as described in the Materials and Methods at different condition in (b) normal HBSS containing 1 mM CaCl2 (O) HBSS with 2.5 mM EGTA (1) cells were pretreated with 20 µM BAPTA-AM. (B) Quantitative values of increase of [Ca2+]i induced by MEP 300 µg/mL in different condition as described above. The increase of [Ca2+]i was calculated by subtracting the basal level of [Ca2+]i. Data were expressed as mean ( SEM (n g 3) (*) p < 0.05, (***) p < 0.001, compared to the control group treated with MEP 300 µg/mL only.

100 µg/mL could significantly inhibit the rate of mitochondria oxygen consumption by at least 50% (data not shown).

Discussion The major goal of this study is to determine whether the particulate fraction of the motorcycle exhaust could adversely affect the immune system, particularly macrophage cells. Apoptosis of macrophage could affect the tissue responses by three ways (47). First, the shedding of apoptotic bodies may spread toxic chemicals to neighbor cells, which engulf the apoptotic remnants from dying cells. Second, damage to macrophages may decrease tissue defenses to infection, for example, increased multiplication of respiratory viruses in the lungs of mice exposed to DEP (31). Third, macrophages take charge of clearance of death granulocytes and their fragments and inhibit the release of proinflammatory cytokines to accelerate resolution of lung inflammation (48), while apoptosis of macrophage might attenuate the resolution of inflammation.

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Figure 5. Induction of increase of ROI production by MEP in RAW 264.7 cells. (A) Cells were treated with MEP 300 µg/mL for different time periods as indicated and ROI production was analyzed by flow cytometry as described in the Materials and Methods. Data were expressed as mean ( SEM (n g 3) (**) p < 0.01, (***) p < 0.001, compared to respective control. (B) Cells were pretreated with calcium chelators for 10 min and antioxidants for 0.5 h, then treated with MEP 300 µg/mL for 2 h and then analyzed by flow cytometry. Data were expressed as mean ( SEM (n g 3) (a***) p < 0.001, compared to groups of respective vehicle control, (b***) p < 0.001, compared to the group of only treated of MEP 300 µg/mL. Table 1. Effect of MEP on Cellular Glutathione Content in RAW 264.7 Cellsa cellular glutathione content (pmol/µg of protein) vehicle control 46.9 ( 1.0 BSO 24 h 17.2 ( 2.32* MEP 300 µg/mL (h) 0.5 h 1h 2h 4h

40.6 ( 0.7* 40.8 ( 2.2* 38.9 ( 2.0* 37.2 ( 1.5*

a RAW 264.7 cells were treated with L-buthionine-[S,R]-sulfoximin (BSO) for 24 h or MEP 300 µg/mL for different time periods. Intracellular glutathione content was measured by the colorimetic assay as described in the Materials and Methods. Data were expressed as mean ( SEM. (*) p < 0.05, compared to vehicle control.

The results from this study have shown that motorcycle exhaust particles (MEP) are cytotoxic to macrophage cell line RAW 264.7, murine peritoneal macrophage, and rat alveolar macrophage, with the RAW 264.7 being the most sensitive one. The flow cytometric analysis suggested that macrophage underwent apoptosis upon treated with MEP. This is further supported that the DNA from the MEP-treated macrophage was fragmented, which is a characteristic of apoptosis (49-51). Several conditions were used to assess the underlying mechanisms of MEP-induced apoptosis on macrophage cells. The MEP-induced apoptosis was attenuated by pretreatment with Ca2+ chelators, antioxidants, and mitochondria transition pore blocker, with the Ca2+ chelators being the most effective one. These results suggested that Ca2+ might play an important role in MEP-induced apoptosis in macrophage.

By direct measurement of [Ca2+]i in macrophage cells using the fluorescent indicator, Fura 2/AM, we have found that MEP induced a transient increase followed by a sustained raise of [Ca2+]i. The increase of [Ca2+]i was completely inhibited when the cells were pretreated with the intracellular Ca2+ chelator, BAPTA-AM. However, only the sustained part was abolished when extracellular Ca2+ was chelated with EGTA. This result agrees with EGTA only partially inhibiting the MEP-induced apoptosis while BAPTA-AM can block the apoptosis to greater extent. These data indicated that MEP could indeed affect the Ca2+ homeostatsis and cause the raise of [Ca2+]i by induction of Ca2+ release from the internal Ca2+ store and influx of extracellular Ca2+. Recently, several studies have shown that mitochondria acts as regulators of apoptosis (52). Before classical signs of apoptosis become manifest, mitochondria membrane potential will be disrupted, as well as release of intermembrane proteins through the outer membrane (53). The mitochondria membrane potential (MMP) is the driving force of mitochondria ATP synthesis and declines during apoptosis, and restoration of MMP prevents apoptosis (54). When MMP is irreversibly lost, the intermembrane mitochondria protein cytochrome c and apoptosis inducing factor would release, consequently activating a series of caspases and ultimately inducing cell apoptosis (55-57). In our experiments, we have found that the MMP of the MEP-treated macrophage declines as seen by Timothy et al. with DEP (23). Increase of [Ca2+]i has been shown to cause mitochondrial damage (44) and a drop of MMP due to the transition of inner membrane permeability. However, our data has shown that pretreatment of calcium chelators was unable to

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Figure 6. Effect of MEP on mitochondrial membrane potential in RAW 264.7 cells. Cells were treated first with chemical compounds at dose and time indicated and then stained with 40 nM mitochondria membrane potential sensitive dye DiOC6(3). The changes of mitochondrial membrane potential were analysis by FACcan flow cytometry as described in the Materials and Methods. (A) Cells were treated with vehicle control (a) or CCCP 50 µM (b) for 6 h. (B) Cells were treated with vehicle control (a), MEP 100 µg/mL for 4 h (b), or MEP 300 µg/mL for 2 h (c).

restore completely the MMP, suggesting that MEP might also affect the function of mitochondria directly. This speculation was supported by the fact that the rate of oxygen consumption in MEP-treated macrophage was greatly inhibited. Damage of mitochondria will not only result in the loss of ATP production but also the loss of its ability to regulate cell [Ca2+]i (44), both of which result in the irreversible raise of [Ca2+]i. In addition to the change of [Ca2+]i and MMP, we have also found that the oxidative stress in MEP-treated macrophage was increased as evident by the increase of ROI production and decrease of GSH content. It has been shown that sustained increase of [Ca2+]i might lead to the ROI production through activation of xanthane oxidase or membrane NADPH oxidase. However, chelation of intracellular Ca2+ was unable to completely inhibit the ROI formation induced by MEP treatment. Therefore, it is possible that MEP, by itself, might produce ROI as seen with DEP (47). Further experiments are needed to clarify this uncertainty. In conclusion, the present experiment demonstrates that MEP might cause the adverse effect of the immune system due to its cytotoxic effect on macrophage. The results indicated that alteration of [Ca2+]i homeostasis, increase of oxidative stress, and damage of mitochondria are involved in the induction of apoptosis by MEP. Among all the factors involved, [Ca2+]i seems to play the major role. We have found that the MEP and the particle-free

Lee and Kang

Figure 7. Calcium chelators and antioxidant partly attenuate the decrease of MMP caused by MEP in RAW 264.7 cells. Cells were pretreated with calcium chelators for 10 min and antioxidants for 0.5 h, then treated with MEP 300 µg/mL for 1 h and stained with 40 nM mitochondria membrane potential sensitive dye DiOC6(3) and analysis by FACcan flow cytometry as described in the Materials and Methods. Data were expressed as mean ( SEM (n g 3) (a*) p < 0.05, (a**) p < 0.01, (a***) p < 0.001, compared to groups of respective vehicle control, (b*) p < 0.05, (b**) p < 0.01, (b***) p < 0.001, compared to the group of only treated of MEP 300 µg/mL.

MEPE induced apoptosis and change of [Ca2+]i in macrophage to similar degrees, suggesting that the effects seen in this study were caused by the chemical absorbed by the motorcycle exhaust particles but not particle itself. This agrees with the finding with DEP by Hiura et al. (47). Motorcycle exhaust particles are crude mixture, which contain >110 different organic compounds including polycyclic aromatic hydrocarbons (PAHs) (58). Along with their carcinogenic (59, 60), mutagenic (61), and genotoxic (62) effects, some PAHs have been shown to induce apoptosis (63) via Ca2+-dependent mechanism (64) in macrophage as well as other cells, including epithelial cell (65, 66) and T cell (67, 68). Whether the effects observed in this study were caused by PAHs in MEP or other unidentified compounds is still awaiting further investigation.

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