TY - JOUR
AU1 - Shima,, Hirotoshi
AU2 - Koike,, Eiko
AU3 - Shinohara,, Ritsuko
AU4 - Kobayashi,, Takahiro
AB - Abstract Diesel exhaust particles (DEP) are known to induce adverse biological responses such as inflammation of the airway. However, the relationship between the chemical characteristics of organic compounds adsorbed on DEP and their biological effects is not yet fully understood. In this study, the dichloromethane-soluble fraction (DMSF) from DEP was fractionated into its n-hexane-soluble fraction (n-HSF) and n-hexane-insoluble fraction (n-HISF). Using these DEP fractions, we designed the present studies to elucidate (1) chemical characteristics, (2) biological characteristics, and (3) the relationship between the chemical and the biological characteristics of these DEP fractions. Dithiothreitol (DTT) assay, Fourier transform-infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance (1H-NMR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS) were used to characterize their chemical properties. Heme oxygenase-1 (HO-1) protein expression, viability of rat alveolar type II epithelial cell line (SV40T2), and inflammatory cell infiltration into the peritoneal cavity of BALB/c mice were evaluated as markers of oxidative stress, cytotoxicity, and inflammatory response, respectively. The oxidative ability of the DEP fractions was n-HISF > DMSF > n-HSF. IR, 1H-NMR, and GC-MS spectra showed that n-HISF was mainly composed of compounds having many functional groups related to oxygenation, such as hydroxyl and carbonyl groups. The relative strength of HO-1 protein expression, cytotoxicity, and inflammatory responses was also n-HISF > DMSF > n-HSF. All of the n-HISF-induced biological activities were decreased by reduction with N-acetyl-L-cysteine (NAC). These results suggest that n-HISF has high oxidative ability and many functional groups related to oxygenation and that this ability strongly contributes to the induction of oxidative stress, cytotoxicity, and inflammatory response. diesel exhaust particles, n-hexane-insoluble fraction (n-HISF), oxidative ability, oxidative stress, cytotoxicity, inflammation Epidemiological studies have demonstrated that PM 2.5 (diameter of particulate matters < 2.5μm) exposure leads to increased morbidities and mortalities from pulmonary/respiratory disorders such as asthma and chronic bronchitis (Dockery et al., 1993; Pope et al., 1995, 2002; Seaton et al., 1995; Von Klot et al., 2002). One of the major sources of these atmospheric particles is assumed to be diesel exhaust particles (DEP), which become deposited mainly in the alveolar region of the lungs (Snipes, 1989). DEP are composed of carbonaceous cores onto which thousands of organic compounds such as aliphatic and aromatic hydrocarbons, heterocyclics, quinones, aldehydes, and unknown compounds, are adsorbed (Bayona et al., 1988; Draper, 1986; Schuetzle et al., 1981). These adsorbed organic compounds may be dissolved in the alveolar surface-active materials such as dipalmitoyl-phosphatidyl choline. Therefore, organic compounds adsorbed on DEP may have adverse effects on the human lung. Oxidative stress is assumed to be one of the factors contributing to the adverse effects of DEP. In previous studies we showed that organic extracts of DEP provoke oxidative stress on alveolar macrophages, alveolar type II cells (Koike et al., 2002, 2004), and endothelial cells (Hirano et al., 2003). Reactive oxygen species (ROS) such as peroxide, which induces oxidative stress, are known to induce oxidation of various molecules comprising the living body, such as proteins, lipids, and DNA. Oxidation of these molecules is known to be associated with damage to cells, which damage induces various biological responses such as inflammation. Moreover, oxidative stress has been shown to activate redox-responsive signaling pathways including those involving activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB), which are related to inflammation (Donaldson et al., 2003; Nel et al., 2001). Therefore, ROS in DEP extracts might be involved in lung diseases. Carbonaceous cores may also have biological effects. Hesterberg et al. (2005) reviewed that the carcinogenic effects in rats exposed by inhalation appear to be caused by lung-overload with the solid particles. Taken together, the toxicological effects of DEP are likely to be associated with both the particle itself and the adsorbed chemicals. Koike and Kobayashi (2005), however, reported that DEP and organic extract in DEP induced oxidative stress, but the residual particles only had very small ability. So, in this study, we regarded oxidative stress as the source of the toxicity of DEP and focused on the DEP-adsorbed chemicals rather than particle itself. Recently, quinones and PAH, were contained in PM and DEP (Cho et al., 2004; Schuetzle et al., 1981), have been reported as substances to produce ROS through redox cycling directly or indirectly (Kumagai et al., 2002; Nel et al., 2001; Pening et al., 1999). However, adsorbed organic compounds are thought to consist of a wide variety of compounds that include unburned fuel, engine oil, compounds generated from combustion and pyrolysis process of fuel, engine oil, and reaction products. Generally, compounds generated from combustion and pyrolysis process include very complex oxygenated chemicals. Therefore, some of them might be closely related to the toxicity of DEP. On getting information about unknown toxic compounds in DEP, it is important to investigate not only biological characteristic but chemical properties of them. Although there are plentiful reports about biological effects of DEP, the relationship between the chemical characteristics of various fractions of organic compounds adsorbed on DEP and their biological effects are not yet fully known. In this study, we fractionated organic compounds adsorbed on DEP into non-polar and polar fractions to elucidate (1) the chemical characteristics of these DEP fractions by dithiothreitol (DTT) assay, Fourier transform-infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance (1H-NMR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS); (2) their biological effects in terms of heme oxygenase-1 (HO-1) protein expression, viability of lung epithelial cells, and inflammatory response (neutrophils infiltration); and (3) the relationship between their oxidative ability and biological effects. MATERIALS AND METHODS Reagents. Dichloromethane, n-hexane, chloroform-d, dimethylsulfoxide (DMSO), and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Dithiothreitol (DTT), N-acetyl-L-cysteine (NAC), penicillin, streptomycin, protease inhibitor cocktail, HEPES, and CHAPS were obtained from Sigma Chemical Co. (St. Louis, MO). Dulbecco's Modified Eagle Medium (DMEM) and typan blue stain were purchased from Invitrogen Corp. (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Dainippon Pharmaceutical Co. (Osaka, Japan); and ELISA starter accessory kit, from Bethyl Laboratories, Inc. (Montgomery, TX, USA). Engine oil (CASTLE NEW SPECIAL II) was purchased from Toyota Motor Corp. (Aichi, Japan); and fuel (ENEOS light oil, sulfur content; 20 ppm), from Nippon Oil Corp. (Tokyo, Japan). We used the same fuel and engine oil in all of our experiments. Cell line. The rat pulmonary type II epithelial cell line (SV40T2), which was generously provided by Prof. A. Clement (Hospital Armand Trousseau, France), was used to evaluate oxidative stress and cytotoxicity. SV40T2 cells were grown to confluence in DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated FBS in a humidified incubator at 37°C and having an atmosphere of 5% CO2. Animals. This study proceeded after the approval of the Ethics Committee for Experimental Animals at the National Institute for Environmental Studies. Male BALB/c mice (five weeks old) were purchased from Japan CLEA Inc (Tokyo, Japan). Food and water were available ad libitum. Six-week old mice (weighing 21–25 g) were used in all experiments. Collection of diesel exhaust particles and preparation of the three extracts. DEP were generated by a 4JB1-type diesel engine (Isuzu Automobile Company, Tokyo, Japan), the specifications of which were light-duty (2740 cc exhaust volume) and 4 cylinders, operated at a speed of 1500 rpm under the load of 10 torque (kg • m). At a flow rate of 300 l/min, approximate 1 g (1.08 ± 0.01 g) of DEP was collected on a glass-fiber filter (203 mm × 254 mm) per 12 h in a constant-volume sampler system fitted to the end of a stainless steel dilution tunnel and was then extracted with 100 ml of dichloromethane in a soxhlet apparatus for 6 h. The dichloromethane solution was evaporated to dryness in a rotary evaporator and then a vacuum pump. The residue was designated as the dichloromethane-soluble fraction (DMSF). A 0.5-ml volume of n-hexane was added to the DMSF, and the n-hexane-soluble component was recovered. This process was repeated three times. The soluble component and the residue were designated as the n-hexane-soluble fraction (n-HSF) and n-hexane-insoluble fraction (n-HISF), respectively. These fractions were evaporated to dryness by rotary evaporation as in the case of the DMSF. These extracts were dissolved in DMSO and stored in a glass vial at −80°C until tested. Measurement of oxidative ability. Oxidative ability was determined in triplicate by conducting the DTT assay, which is used for the quantitative measurement of ROS formation in vitro (Kumagai et al., 2002). All samples were prepared at concentrations of 10, 30, and 100 μg/ml in 250 mM Tris-HCl buffer (pH 8.9). Briefly, 20 μl of 16 mM DTT solution and 2 ml of a test sample including a blank (250 mM Tris-HCl buffer only), containing DMSO (final concentration 0.1%), were mixed in tubes and incubated for 10 min at 37°C in a water bath. Then 40 μl of 16 mM DTNB was added to this mixture to develop the yellow color. After color development, samples (200 μl) were placed in microtiter wells, and the absorbance was measured at 414 nm with a microplate reader (Immuno Reader NJ-2000, Inter Med., Tokyo, Japan). Spectral analysis of DEP fractions. IR spectra were obtained by using a Fourier Transform Infrared Spectrometer (JASCO Corp., Tokyo, Japan). 1H-NMR spectra were obtained with a NMR spectrometer (JNM-A500, JEOL, Tokyo, Japan); and GC-MS data, with a gas chromatograph (Hewlett-Packard, HP-5890 series II)/high resolution mass spectrometer (JMS-700, Mstation, JEOL) equipped with a fused silica gel column (0.25 mm × 30 m; DB-1MS, J&W Scientific, CA). Measurement of heme oxygenase-1 (HO-1) protein. After SV40T2 cells had been grown to confluence in 60-mm petri dishes (Becton Dickinson, Co., NJ), the cells were exposed for 24 h in duplicate to the samples prepared at concentrations of 10, 30, and 100 μg/ml in FBS-free DMEM containing 0.1 % DMSO. Control cells were exposed to FBS-free DMEM containing 0.1% DMSO. One of the duplicate cultures was used to estimate the number of viable cells by the trypan blue dye exclusion method. The other was washed with phosphate-buffered saline (PBS) and lysed in 200 μl of 50 mM HEPES buffer containing 150 mM NaCl, 2% CHAPS, and 2% protease inhibitor cocktail and used to determine, in duplicate, the expression of HO-1 protein by ELISA. Briefly, after each microtiter well had been coated with mouse anti HO-1 antibody (Stressgen Biotechnologies Corp., BC, Canada) for 60 min, it was blocked with blocking solution for 30 min. Subsequently, the samples were added to each well, and incubation was carried out for 60 min. Recombinant rat HO-1 protein (Stressgen Biotechnologies Corp.), used as a standard, was applied to other wells. Following incubation, rabbit anti HO-1 antibody (Stressgen Biotechnologies Corp.) was added to each well; and after a 60-min incubation, HRP-conjugated secondary antibody (Stressgen Biotechnologies Corp.) was added to each well. Following another 60-min incubation, TMB was added to each well as a substrate; and the samples were then incubated for 20 min in the dark. After the enzyme reaction had been stopped by the addition of 2 M H2SO4, the absorbance at 450 nm was measured. The results were evaluated in terms of the amount of HO-1 protein induced per 1 × 105 viable cells. Measurement of cell viability. SV40T2 cells were exposed for 24 h in triplicate to the samples prepared at concentrations of 10, 30, and 100 μg/ml in FBS-free DMEM containing 0.1% DMSO. Control cells were exposed to FBS-free DMEM containing 0.1% DMSO. The cell monolayer was trypsinized and then suspended with FBS-free DMEM. Viability was calculated by counting viable (trypan blue dye-excluding) cells in each suspension. Measurement of inflammatory response. As a marker for inflammatory response, we investigated the number of inflammatory cells infiltrating into the peritoneal cavity of BALB/c mice. Briefly, each group of five mice received 30 mg of DMSF, n-HSF, n-HISF, engine oil or fuel in PBS containing 0.1% DMSO per body weight (kg) by ip injection. Twenty-four hours after the administration, the peritoneal cavities were lavaged five times with 1 ml of PBS each time. The cells were recovered from the lavage fluid by centrifugation at 1500 rpm for 10 min at 4°C. Viable cells were determined by trypan blue dye exclusion. Differential cell counts were performed on the cytocentrifuged smears stained with Diff Quik solution (International Reagents, Co., Ltd., Kobe, Japan). Assessment of contribution of oxidative ability of HISF to the biological effects. SV40T2 cells were exposed to 10 and/or 30 μg/ml n-HISF for 24 h in presence or absence of 5 or 10 mM NAC, after which HO-1 protein expression and viability were measured. BALB/c mice were administered 30 mg n-HISF/kg into their peritoneal cavity with or without 150 or 300 μmol NAC/kg. The number of neutrophils that infiltrated into the peritoneal cavity 24 h after administration was counted. Details of the methods used for measurements were described above. Statistical analysis. The results were expressed as the mean ± SEM. The significance of differences between means was assessed by using Student's unpaired t-test, whereby values of p < 0.05 were considered to be significant. RESULTS Weight Ratio of n-HSF and n-HISF to DMSF The average weight of DMSF was 0.74 ± 0.01 g per 1.08 ± 0.01 g DEP and the weight ratio (%) of DMSF to DEP was 69.2 ± 1.1%. The weight ratio (%) of n-HISF and n-HSF to DMSF was 10.5 ± 2.9 % and 89.5 ± 2.9 %, respectively. In other words, nearly 70% of DEP, by weight, consists of adsorbed organic compounds, but 10% of these compounds are polar compounds. Oxidative Ability of DEP Fractions The oxidative ability of DMSF and the two fractions was evaluated by conducting the DTT assay (Fig. 1). At any concentration tested, n-HISF had stronger ability to consume DTT than DMSF or n-HSF (n-HISF > DMSF > n-HSF). Especially, DTT consumption rate of DMSF vs. n-HISF at 100 μg/ml was approximate 30 vs. 300. Taken into consideration that 10% of DMSF consists of n-HISF, and by weight, DMSF should have 10% of consumption of n-HISF, this result indicates that all/most of the oxidative ability of DMSF are a result of its n-HISF content. In addition, we investigated the effects of the engine oil and fuel used in operating the diesel engine. Their oxidative abilities were almost the same as that ability of n-HSF. FIG. 1. Open in new tabDownload slide The DTT consumption of DMSF, n-HSF, n-HISF, engine oil, and fuel after a 10-min incubation. Oxidative ability was measured in triplicate by use of the DTT assay. DTT consumption of the blank (8.8 ± 2.3 nmol) was subtracted. Values are shown as the mean ± SEM (n = 3). FIG. 1. Open in new tabDownload slide The DTT consumption of DMSF, n-HSF, n-HISF, engine oil, and fuel after a 10-min incubation. Oxidative ability was measured in triplicate by use of the DTT assay. DTT consumption of the blank (8.8 ± 2.3 nmol) was subtracted. Values are shown as the mean ± SEM (n = 3). Spectral Characteristics of DEP Fractions IR spectra of all samples are shown in Figure 2. The most intensive absorption was observed over the range from 2800 to 3000 cm−1 for all samples, and it was mainly attributed to aliphatic C-H vibration. The IR spectrum of n-HISF (Fig. 2c) showed strong absorption attributed to O-H groups over the range from 2500 to 3500 cm−1, whereas that of n-HSF (Fig. 2b) did not show any in this range. The spectrum of n-HISF also showed intensive broad absorption from 1600 to 1800 cm−1, whereas that of n-HSF showed far weaker adsorption over this range. The absorption between 1600 and 1800 cm−1 was mainly due to oxygenated functional groups such as carbonyl and NO2 groups. DMSF (Fig. 2a) had almost the additive spectrum of n-HISF and n-HSF. The spectra of engine oil and fuel (Figs. 2d and 2e) were almost the same as the n-HSF spectrum. These results suggest that n-HISF has many functional groups related to oxygenation. FIG. 2. Open in new tabDownload slide IR spectra of DMSF (a), n-HSF (b), n-HISF (c), engine oil (d), and fuel (e). FIG. 2. Open in new tabDownload slide IR spectra of DMSF (a), n-HSF (b), n-HISF (c), engine oil (d), and fuel (e). The 1H-NMR data were divided into five chemical shift ranges (0 ∼ 1.0, 1.0 ∼ 2.0, 2.0 ∼ 3.5, 3.5 ∼ 6.0, 6.0 ∼ 10.0 ppm) and are displayed by their relative integrated intensity in Table 1. The 1H-NMR spectra of all samples showed major peaks at 0 ∼ 2 ppm, indicating the presence of aliphatic protons. The relative integrated intensity (%) of n-HISF at 2.0 ∼ 3.5 ppm and 3.5 ∼ 6.0 ppm was higher than that of DMSF or n-HSF. In contrast, this value at both 2.0 ∼ 3.5 ppm and 3.5 ∼ 6.0 ppm was similar to those values for DMSF and engine oil. This finding suggests that n-HISF has many functional groups related to oxygenation and pyrolysis during the combustion process, such as hydroxyl and carbonyl groups, as well as double bonds, as compared with n-HSF. These results were supported by the IR spectra. TABLE 1 Distribution of Percentage of Five Chemical Shift Ranges in DMSF, n-HSF, n-HISF, Engine Oil, and Fuel Based on 1H-NMR Integration . Chemical shift (ppm) . . . . . Sample . <1.0 . 1.0 ∼ 2.0 . 2.0 ∼ 3.5 . 3.5 ∼ 6.0 . 6.0 ∼ 9.0 . DMSF 26.2 71.4 1.0 0.1 1.3 n-HSF 25.9 70.6 1.9 0.1 1.5 n-HISF 23.6 60.0 8.0 7.5 0.9 Engine oil 25.3 70.5 2.3 0.2 1.7 Fuel 29.8 61.1 6.1 0 3.0 . Chemical shift (ppm) . . . . . Sample . <1.0 . 1.0 ∼ 2.0 . 2.0 ∼ 3.5 . 3.5 ∼ 6.0 . 6.0 ∼ 9.0 . DMSF 26.2 71.4 1.0 0.1 1.3 n-HSF 25.9 70.6 1.9 0.1 1.5 n-HISF 23.6 60.0 8.0 7.5 0.9 Engine oil 25.3 70.5 2.3 0.2 1.7 Fuel 29.8 61.1 6.1 0 3.0 Open in new tab TABLE 1 Distribution of Percentage of Five Chemical Shift Ranges in DMSF, n-HSF, n-HISF, Engine Oil, and Fuel Based on 1H-NMR Integration . Chemical shift (ppm) . . . . . Sample . <1.0 . 1.0 ∼ 2.0 . 2.0 ∼ 3.5 . 3.5 ∼ 6.0 . 6.0 ∼ 9.0 . DMSF 26.2 71.4 1.0 0.1 1.3 n-HSF 25.9 70.6 1.9 0.1 1.5 n-HISF 23.6 60.0 8.0 7.5 0.9 Engine oil 25.3 70.5 2.3 0.2 1.7 Fuel 29.8 61.1 6.1 0 3.0 . Chemical shift (ppm) . . . . . Sample . <1.0 . 1.0 ∼ 2.0 . 2.0 ∼ 3.5 . 3.5 ∼ 6.0 . 6.0 ∼ 9.0 . DMSF 26.2 71.4 1.0 0.1 1.3 n-HSF 25.9 70.6 1.9 0.1 1.5 n-HISF 23.6 60.0 8.0 7.5 0.9 Engine oil 25.3 70.5 2.3 0.2 1.7 Fuel 29.8 61.1 6.1 0 3.0 Open in new tab The GC-MS chromatograms of all samples are shown in Figure 3. The total ion chromatogram (TIC) of n-HSF (Fig. 3b) was similar to that of DMSF (Fig. 3a). n-HSF had similar peaks as engine oil at retention times from 30 to 40 min (Fig. 3d) and fuel at retention times from 14 to 25 min (Fig. 3e). Also for n-HSF, aliphatic peaks of low-molecular mass (C9∼C16), as observed for fuel at 3–14 min, were almost disappeared. These compounds may be burned during the combustion process or not be condensed on DEP. n-HISF (Fig. 3c) showed a different pattern of peaks, which could not be observed in TICs of unburned engine oil and fuel at shorter retention times from 10 to 25 min, suggesting that this pattern might be a result of pyrolysis and recombination of reactive intermediates during the combustion process. FIG. 3. Open in new tabDownload slide Total ion chromatograms of DMSF (a), n-HSF (b), n-HISF (c), engine oil (d), and fuel (e). FIG. 3. Open in new tabDownload slide Total ion chromatograms of DMSF (a), n-HSF (b), n-HISF (c), engine oil (d), and fuel (e). Effects of DEP Fractions on the Expression of HO-1 Protein in Epithelial Cells Figure 4 shows the effects of DEP fractions on HO-1 protein expression in cells of the SV40T2 alveolar epithelial cell line. At any concentration tested, n-HISF induced stronger expression of HO-1 protein than found in the control; and DMSF at the concentration of 100 μg/ml gave a value significantly higher than the control one. The relative strength of the induction of HO-1 protein was n-HISF > DMSF > n-HSF. The values obtained for the engine oil and fuel were almost same as that value for n-HSF. These results suggest that n-HISF induces strong oxidative stress in SV40T2 cells. FIG. 4. Open in new tabDownload slide Expression of HO-1 protein per 1 × 105 viable cells 24 h after exposure of SV490T2 cells to various concentrations (10, 30, and 100 μg/ml) of DMSF, n-HSF, n-HISF, engine oil. Control cells were exposed to FBS-free DMEM containing 0.1% DMSO. The HO-1 protein expression was measured by ELISA. Values are shown as the mean ± SEM (n = 2–4). Significantly different from control: ***p < 0.001. FIG. 4. Open in new tabDownload slide Expression of HO-1 protein per 1 × 105 viable cells 24 h after exposure of SV490T2 cells to various concentrations (10, 30, and 100 μg/ml) of DMSF, n-HSF, n-HISF, engine oil. Control cells were exposed to FBS-free DMEM containing 0.1% DMSO. The HO-1 protein expression was measured by ELISA. Values are shown as the mean ± SEM (n = 2–4). Significantly different from control: ***p < 0.001. Effects of DEP Fractions on the Viability of the Epithelial Cells Figure 5 shows the effects of DEP fractions on the viability of SV40T2 cells. The effects of all samples on viability decreased concentration-dependently. n-HISF had the most potent cytotoxicity, followed by DMSF and n-HSF. Effects of engine oil and fuel on viability were almost same as the effect of n-HSF. FIG. 5. Open in new tabDownload slide Viability changes of SV40T2 cells exposed to various concentrations (10, 30, and 100 μg/ml) of DMSF, n-HSF, n-HISF, engine oil, and fuel for 24 h. Control cells were exposed to FBS-free DMEM containing 0.1% DMSO. Viability was estimated by trypan blue dye exclusion. Values are shown as the mean ± SEM (n = 3). Significantly different from the control: *p < 0.05; **p < 0.01; ***p < 0.001. FIG. 5. Open in new tabDownload slide Viability changes of SV40T2 cells exposed to various concentrations (10, 30, and 100 μg/ml) of DMSF, n-HSF, n-HISF, engine oil, and fuel for 24 h. Control cells were exposed to FBS-free DMEM containing 0.1% DMSO. Viability was estimated by trypan blue dye exclusion. Values are shown as the mean ± SEM (n = 3). Significantly different from the control: *p < 0.05; **p < 0.01; ***p < 0.001. Effects of DEP Fractions on Inflammatory Response Table 2 and Figure 6 show the effects of DEP fractions on the inflammatory response in vivo. Table 2 shows no significant difference in the total cell number in the peritoneal lavage fluid between any fraction and the control. However, the number of neutrophils in mice treated by DMSF and n-HISF; and the percentage of neutrophils to total cells were significantly increased as compared with the control values (Fig. 6). The induction of the inflammatory response by n-HISF was stronger than that by DMSF or n-HSF (n-HISF > DMSF > n-HSF). Engine oil showed an increase in the percentage of neutrophils, but fuel did not. These results suggest that n-HISF induces strong inflammatory responses in vivo. FIG. 6. Open in new tabDownload slide Percentage of neutrophils to total cells that infiltrated into the peritoneal cavity of BALB/c mice 24 h after administration of DMSF, n-HSF, n-HISF, engine oil, or fuel. Control group was administered PBS containing 0.1% DMSO. Values are shown as the mean ± SEM (n = 5). Significantly different from the control: *p < 0.05; ***p < 0.001. FIG. 6. Open in new tabDownload slide Percentage of neutrophils to total cells that infiltrated into the peritoneal cavity of BALB/c mice 24 h after administration of DMSF, n-HSF, n-HISF, engine oil, or fuel. Control group was administered PBS containing 0.1% DMSO. Values are shown as the mean ± SEM (n = 5). Significantly different from the control: *p < 0.05; ***p < 0.001. TABLE 2 Number of Cells in Peritoneal Cavity Lavage Fluid from Mice 24 h after Administration . Animals (n) . Total cells (×106) . Neutrophils (×105) . Vehicle (control) 5 2.53 ± 0.89 0.73 ± 0.37 DMSF 5 2.50 ± 0.34 2.39 ± 0.32* n-HSF 5 1.99 ± 0.70 0.85 ± 0.32 n-HISF 5 2.62 ± 0.86 7.11 ± 2.46* Engine oil 5 2.51 ± 0.61 1.94 ± 0.74 Fuel 5 1.42 ± 0.31 0.57 ± 0.16 . Animals (n) . Total cells (×106) . Neutrophils (×105) . Vehicle (control) 5 2.53 ± 0.89 0.73 ± 0.37 DMSF 5 2.50 ± 0.34 2.39 ± 0.32* n-HSF 5 1.99 ± 0.70 0.85 ± 0.32 n-HISF 5 2.62 ± 0.86 7.11 ± 2.46* Engine oil 5 2.51 ± 0.61 1.94 ± 0.74 Fuel 5 1.42 ± 0.31 0.57 ± 0.16 Note. Values are shown as mean ± SEM (n = 5). Significantly different from control: *p < 0.05. Open in new tab TABLE 2 Number of Cells in Peritoneal Cavity Lavage Fluid from Mice 24 h after Administration . Animals (n) . Total cells (×106) . Neutrophils (×105) . Vehicle (control) 5 2.53 ± 0.89 0.73 ± 0.37 DMSF 5 2.50 ± 0.34 2.39 ± 0.32* n-HSF 5 1.99 ± 0.70 0.85 ± 0.32 n-HISF 5 2.62 ± 0.86 7.11 ± 2.46* Engine oil 5 2.51 ± 0.61 1.94 ± 0.74 Fuel 5 1.42 ± 0.31 0.57 ± 0.16 . Animals (n) . Total cells (×106) . Neutrophils (×105) . Vehicle (control) 5 2.53 ± 0.89 0.73 ± 0.37 DMSF 5 2.50 ± 0.34 2.39 ± 0.32* n-HSF 5 1.99 ± 0.70 0.85 ± 0.32 n-HISF 5 2.62 ± 0.86 7.11 ± 2.46* Engine oil 5 2.51 ± 0.61 1.94 ± 0.74 Fuel 5 1.42 ± 0.31 0.57 ± 0.16 Note. Values are shown as mean ± SEM (n = 5). Significantly different from control: *p < 0.05. Open in new tab Relationship between Oxidative Ability and Toxicity Figure 7 shows that treatment with 5 or 10 mM NAC decreased n-HISF-induced HO-1 protein expression 46 and 62%, respectively, in a concentration-dependent fashion. FIG. 7. Open in new tabDownload slide Effects of NAC on n-HISF-induced HO-1 protein expression in SV40T2 cells. The cell monolayer was exposed to 30 μg/ml of n-HISF in the absence (open column) or presence of 5 mM (cross-hatched column) or 10 mM NAC (black column) for 24 h. HO-1 protein was measured by ELISA, and the results are expressed as the amount (ng) of HO-1 protein induced per 1 × 105 viable cells. Values are shown as the mean ± SEM (n = 4). Significantly different from n-HISF (30 μg/ml) in the absence of NAC: **p < 0.01 FIG. 7. Open in new tabDownload slide Effects of NAC on n-HISF-induced HO-1 protein expression in SV40T2 cells. The cell monolayer was exposed to 30 μg/ml of n-HISF in the absence (open column) or presence of 5 mM (cross-hatched column) or 10 mM NAC (black column) for 24 h. HO-1 protein was measured by ELISA, and the results are expressed as the amount (ng) of HO-1 protein induced per 1 × 105 viable cells. Values are shown as the mean ± SEM (n = 4). Significantly different from n-HISF (30 μg/ml) in the absence of NAC: **p < 0.01 The effects of NAC on n-HISF-induced cytotoxicity are shown in Figure 8. Pretreatment with NAC ameliorated the drop in the viability induced by n-HISF, concentration-dependently. FIG. 8. Open in new tabDownload slide Effects of NAC on n-HISF-induced cytotoxicity toward SV40T2 cells. The cell monolayer was exposed to 10 or 30 μg/ml of n-HISF in the absence (open column) or presence of 5 mM (cross-hatched column) or 10 mM NAC (black column) for 24 h. Viability was estimated by counting the number of viable (trypan blue dye-excluding) cells. Values are shown as the mean ± SEM (n = 3). **p < 0.01 compared with n-HISF (10 μg/ml) in the absence of NAC; ###p < 0.001 compared with n-HISF (30 μg/ml) in the absence of NAC. ++p < 0.01 compared with n-HISF (30 μg/ml) in the presence of 5 mM NAC. FIG. 8. Open in new tabDownload slide Effects of NAC on n-HISF-induced cytotoxicity toward SV40T2 cells. The cell monolayer was exposed to 10 or 30 μg/ml of n-HISF in the absence (open column) or presence of 5 mM (cross-hatched column) or 10 mM NAC (black column) for 24 h. Viability was estimated by counting the number of viable (trypan blue dye-excluding) cells. Values are shown as the mean ± SEM (n = 3). **p < 0.01 compared with n-HISF (10 μg/ml) in the absence of NAC; ###p < 0.001 compared with n-HISF (30 μg/ml) in the absence of NAC. ++p < 0.01 compared with n-HISF (30 μg/ml) in the presence of 5 mM NAC. Figure 9 shows the effects of NAC on the n-HISF-induced inflammatory response. Pretreatment with 150 and 300 μmol NAC decreased the percentage of n-HISF-infiltrated neutrophils that infiltrated in response to n-HISF by 43 and 67%, respectively. FIG. 9. Open in new tabDownload slide Effects of NAC on n-HISF-induced inflammatory response. Each group of 5 BALB/c mice received an intraperitoneal injection of 30 mg of n-HISF without (open column) or with 150 μmol (cross-hatched column) or 300 μmol of NAC (black column) in PBS containing 0.1 % DMSO per weight (kg). The results are expressed as the percentage of neutrophils to total cells that infiltrated into the peritoneal cavity of the mice 24 h after administration. Values are shown as the mean ± SEM (n = 4–5). **p < 0.01 compared with n-HISF (10 μg/ml) in the absence of NAC; #p < 0.05 compared with n-HISF (30 μg/ml) in the presence of 150 μmol NAC. FIG. 9. Open in new tabDownload slide Effects of NAC on n-HISF-induced inflammatory response. Each group of 5 BALB/c mice received an intraperitoneal injection of 30 mg of n-HISF without (open column) or with 150 μmol (cross-hatched column) or 300 μmol of NAC (black column) in PBS containing 0.1 % DMSO per weight (kg). The results are expressed as the percentage of neutrophils to total cells that infiltrated into the peritoneal cavity of the mice 24 h after administration. Values are shown as the mean ± SEM (n = 4–5). **p < 0.01 compared with n-HISF (10 μg/ml) in the absence of NAC; #p < 0.05 compared with n-HISF (30 μg/ml) in the presence of 150 μmol NAC. DISCUSSION In the present study, we focused on organic compounds adsorbed on DEP, and designed experiments to elucidate the chemical and biological characteristics of DEP fractions and the relationship between the two. The results of the DTT assay showed that n-HISF had the strongest oxidative ability as compared with DMSF and n-HSF (Fig. 1) and that taken the weight rate of the DEP fractions into consideration, all/most of oxidative ability of DMSF were due to that of n-HISF. The IR and 1H-NMR data suggest that n-HISF has many functional groups related to oxygenation (Fig. 2 and Table 1). Retention times of the n-HISF-GC-MS pattern were shifted to shorter ones (Fig. 3). This result suggests that many substances having short carbon chains with polar functional groups might be contained in HISF and/or that unstable reactive intermediates such as peroxide in n-HISF were pyrolyzed at the injection port of GC and/or ionization chamber of MS and changed into low-molecular-weight compounds. Therefore, the shift of GC-MS pattern also may suggest that n-HISF could have oxygenation-related compounds. In contrast, n-HSF treatment resulted in very low consumption of DTT. The IR and 1H-NMR data showed that n-HSF has few functional groups related to oxygenation. Moreover, the GC-MS data indicated that n-HSF mainly consists of aliphatic hydrocarbons that originated from unburned engine oil and fuel. This finding suggests that aliphatic hydrocarbons have no oxidative ability. In fact, the consumption of DTT by engine oil or fuel was very low, as in the case of n-HSF. HO-1, an antioxidant enzyme, is known to be a marker for acute oxidative stress and to be activated when various cells are exposed to DEP. In previous studies, we confirmed that HO-1 was induced at both gene and protein levels when SV40T2 cells were exposed to DEP extracts (Koike et al., 2004). The present study also showed that the induction of HO-1 protein expression by exposure to DMSF and n-HISF (10, 30, and 100 μg/ml) occurred and that n-HISF was the strongest inducer of the three extracts (Fig. 4). Taking into consideration that n-HSF-induction of HO-1 protein expression at 100 μg/ml was equivalent to the control level, we can conclude that all of the DMSF-triggered oxidative stress was due to n-HISF. Generally, the chemical property of oxidative ability is considered to be associated with oxidative stress. In fact, DEP- or organic compound-triggered antioxidants enzymes such as HO-1 are diminished by NAC treatment of different cell types (Hirano et al., 2003; Hiura et al., 1999; Koike et al., 2004; Whitekus et al., 2002; Xiao et al., 2003). Our data also show that n-HISF-induced HO-1 protein was decreased by NAC in a concentration-dependent manner (Fig. 7), thus suggesting that induction of HO-1 protein depended on the oxidative property of n-HISF. The cell viability data (Fig. 5) also showed that n-HISF was the most cytotoxic. This cytotoxicity of n-HISF might have mainly resulted from protein oxidation, lipid peroxidation, and/or DNA damage due to the strong oxidative ability of this fraction. In support of this possibility, Fig. 8 showed that the cytotoxicity of n-HISF was reduced by NAC. A previous study of ours (Hirano et al., 2003) also showed that the viability of rat heart microvessel endothelial cells exposed to DEP extracts was improved by NAC. These results suggest that strong oxidative ability is associated with cytotoxicity. In this study, we used the rat alveolar type II epithelial cell lines (SV40T2). The advantage to use the cell line derived from rat is to be able to compare it with previous results that we found by using same cell lines in vitro (Koike et al., 2004) and rat in vivo. However, it is necessary to compare data from our cell lines with those from human cells, for it might be the possibility that the results from them are not completely coincided with those from human cells. On the other hand, n-HSF, engine oil, and fuel had little cytotoxic ability. The IR data for them showed that their compositions were mainly alphatic hydrocarbons without functional groups related to oxygenation. These results suggest that the cytotoxic ability of aliphatic hydrocarbon originating from unburned engine oil and fuel or non-polar by-product is weak. Figure 6 showed that n-HISF elicited the most potent inflammation, followed by DMSF and n-HSF, in mice. This result suggests that n-HISF in DMSF plays a key role in causing the inflammatory response. It has been reported that organic compounds from DEP induce proinflammatory response (Bonvallot et al., 2001; Nel et al., 1998). It is known that many mediators such as cytokines (e.g., tumor necrosis factor-α and interleukin [IL]-1), chemoattractants (e.g., leukotriene B4, arachidonic acid metabolites), chemokines (e.g., IL-8 and macrophage inflammatory protein-2) induce neutrophil migration (Wagner and Roth, 2000). Some of the proinflammatory mediators are released by activation of AP-1 and NF-κB, which are activated by ROS (Donaldson et al., 2003). ROS in n-HISF may induce activation of AP-1 and NF-κB by causing a change in the thiol status, resulting in inflammation. Figure 9 revealed that the inflammation elicited in response to exposure to n-HISF was decreased by NAC treatment. Therefore, the oxidative ability of n-HISF is closely associated with the inflammatory response. On the other hand, the inflammatory cells such as macrophages and neutrophils can produce ROS by the action of NADPH oxidase in the phagocytes (Hiura et al., 1999; Roos et al., 1996). Epithelial cells also can produce ROS (Martin et al., 1997). These facts suggest that not only oxidative stress caused by oxidants in DEP extracts, but also secondary production of ROS from various cells may aggravate the inflammatory response. We selected the number of neutrophils in the peritoneal cavities of mice to evaluate the ability to induce inflammation, because it is easy to examine whether the inflammation is induced by DEP fractions. The results are valid and suggestive to evaluate the ability to induce inflammation in human lungs. However, it might be the limitations of transferring results from them to mice lungs or human lungs exposed by inhalation as respect the difference between the cells constituted in peritoneal cavities and those in lungs, and the sensitivity of cells in mice and human to oxidative stress. Thus, it remained to be elucidated the inflammatory response of the lungs exposed by DEP fractions. The present study showed that all of the n-HISF-induced biological effects were decreased largely in a NAC concentration-dependent manner. These results suggest that oxidative ability of n-HISF is closely associated with its biological effects. Compounds contained in this fraction might contribute to airway diseases such as asthma, bronchitis, and pollenosis. However, the blocking effects of NAC treatment were not complete. This fact suggests that other mechanisms might exist and also play a role in the induction of oxidative stress, cytotoxicity, and the inflammatory response. Thousands of organic compounds are adsorbed onto DEP. Quinones and PAH in them have been reported as substances to produce ROS through redox cycling directly or indirectly (Kumagai et al., 2002; Nel et al., 2001; Pening et al., 1999). However, we investigated the ability of some quinones (e.g., 1,2-naphthoquinone, 1,4-naphthoquinone, 9,10-anthraquinone, and 9,10-phenanthraquinone) to induce HO-1 protein, but any of them could not reproduce the HO-1 protein expression which was induced by n-HISF (data not shown). It is also reported that 9,10-phenanthraquinone, a major quinone included in DEP, downregulated HO-1 in human pulmonary epithelial cells (Sugimoto et al., 2005). These findings suggest that the induction of HO-1 protein with n-HISF might be not associated with quinones. In the present studies, we demonstrated that n-HISF had many functional groups related to oxygenation, and were mainly composed of compounds generated from combustion and pyrolysis process of fuel, engine oil, and reaction products. Therefore, not only quinones and PAH but also some of them might be closely related to the oxidative ability and toxicity of DEP. 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Author notes *PM2.5 and DEP Research Project, National Institute for Environmental Studies, Onogawa 16-2, Tsukuba, Ibaraki 305-8506, Japan; †Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Chiba 274-8510, Japan; and ‡Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan © The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
TI - Oxidative Ability and Toxicity of n-Hexane Insoluble Fraction of Diesel Exhaust Particles
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfj119
DA - 2006-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/oxidative-ability-and-toxicity-of-n-hexane-insoluble-fraction-of-dPGAD0N0a2
SP - 218
EP - 226
VL - 91
IS - 1
DP - DeepDyve
ER -