TOXICOLOGICAL SCIENCES 91(1), 218–226 (2006)
doi:10.1093/toxsci/kfj119
Advance Access publication January 27, 2006
Oxidative Ability and Toxicity of n-Hexane Insoluble Fraction of
Diesel Exhaust Particles
Hirotoshi Shima,* Eiko Koike,* Ritsuko Shinohara,† and Takahiro Kobayashi‡,1
*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
Received November 14, 2005; accepted January 14, 2006
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-hexanesoluble 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.
Key Words: diesel exhaust particles; n-hexane-insoluble fraction
(n-HISF); oxidative ability; oxidative stress; cytotoxicity;
inflammation.
1
To whom correspondence should be addressed at Environmental Health
Sciences Division, National Institute for Environmental Studies, Onogawa
16-2, Tsukuba, Ibaraki 305-8506, Japan. Tel/Fax: þ81-298-50-2353. E-mail:
[email protected].
Epidemiological studies have demonstrated that PM 2.5
(diameter of particulate matters < 2.5lm) 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-jB), 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
The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For Permissions, please email: [email protected]
OXIDATIVE ABILITY CONTRIBUTES TO TOXICITY
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,
219
France), was used to evaluate oxidative stress and cytotoxicity. SV40T2 cells
were grown to confluence in DMEM containing 100 U/ml penicillin, 100 lg/ml
streptomycin, and 10% heat-inactivated FBS in a humidified incubator at 37C
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 d 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 3 254 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 80C 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 lg/ml in 250 mM Tris-HCl
buffer (pH 8.9).
Briefly, 20 ll 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 37C in a water bath.
Then 40 ll of 16 mM DTNB was added to this mixture to develop the yellow
color. After color development, samples (200 ll) 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).
1
H-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 3 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 lg/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 ll 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
220
SHIMA ET AL.
(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 3 105 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 lg/ml in
FBS-free DMEM containing 0.1% DMSO. Control cells were exposed to FBSfree 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 4C. 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 lg/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 lmol 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.
FIG. 1. 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 ¼ 3).
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.
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
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 lg/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
FIG. 2. IR spectra of DMSF (a), n-HSF (b), n-HISF (c), engine oil (d),
and fuel (e).
OXIDATIVE ABILITY CONTRIBUTES TO TOXICITY
3000 cm1 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 cm1, 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 cm1,
whereas that of n-HSF showed far weaker adsorption over this
range. The absorption between 1600 and 1800 cm1 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.
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.
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
TABLE 1
Distribution of Percentage of Five Chemical Shift Ranges
in DMSF, n-HSF, n-HISF, Engine Oil, and Fuel Based on
1
H-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
n-HSF
n-HISF
Engine oil
Fuel
26.2
25.9
23.6
25.3
29.8
71.4
70.6
60.0
70.5
61.1
1.0
1.9
8.0
2.3
6.1
0.1
0.1
7.5
0.2
0
1.3
1.5
0.9
1.7
3.0
221
10 to 25 min, suggesting that this pattern might be a result of
pyrolysis and recombination of reactive intermediates during
the combustion process.
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 lg/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.
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.
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.
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.
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.
Figure 9 shows the effects of NAC on the n-HISF-induced
inflammatory response. Pretreatment with 150 and 300 lmol
NAC decreased the percentage of n-HISF-infiltrated neutrophils that infiltrated in response to n-HISF by 43 and 67%,
respectively.
222
SHIMA ET AL.
FIG. 3. Total ion chromatograms of DMSF (a), n-HSF (b), n-HISF (c), engine oil (d), and fuel (e).
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.
223
OXIDATIVE ABILITY CONTRIBUTES TO TOXICITY
TABLE 2
Number of Cells in Peritoneal Cavity Lavage Fluid from
Mice 24 h after Administration
Animals (n)
Vehicle (control)
DMSF
n-HSF
n-HISF
Engine oil
Fuel
5
5
5
5
5
5
Total cells (3106)
2.53
2.50
1.99
2.62
2.51
1.42
±
±
±
±
±
±
0.89
0.34
0.70
0.86
0.61
0.31
Neutrophils (3105)
0.73
2.39
0.85
7.11
1.94
0.57
±
±
±
±
±
±
0.37
0.32*
0.32
2.46*
0.74
0.16
Note. Values are shown as mean ± SEM (n ¼ 5).
Significantly different from control: *p < 0.05.
FIG. 4. Expression of HO-1 protein per 1 3 105 viable cells 24 h after
exposure of SV490T2 cells to various concentrations (10, 30, and 100 lg/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 ¼ 2–4). Significantly
different from control: ***p < 0.001.
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
FIG. 5. Viability changes of SV40T2 cells exposed to various concentrations (10, 30, and 100 lg/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 ¼ 3). Significantly different from the control: *p < 0.05;
**p < 0.01; ***p < 0.001.
FIG. 6. 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 ¼ 5). Significantly
different from the control: *p < 0.05; ***p < 0.001.
FIG. 7. Effects of NAC on n-HISF-induced HO-1 protein expression in
SV40T2 cells. The cell monolayer was exposed to 30 lg/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 3 105
viable cells. Values are shown as the mean ± SEM (n ¼ 4). Significantly
different from n-HISF (30 lg/ml) in the absence of NAC: **p < 0.01
224
SHIMA ET AL.
FIG. 8. Effects of NAC on n-HISF-induced cytotoxicity toward SV40T2
cells. The cell monolayer was exposed to 10 or 30 lg/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 ¼ 3). **p < 0.01 compared with n-HISF (10 lg/ml) in the absence of
NAC; ###p < 0.001 compared with n-HISF (30 lg/ml) in the absence of NAC.
þþp < 0.01 compared with n-HISF (30 lg/ml) in the presence of 5 mM NAC.
expression by exposure to DMSF and n-HISF (10, 30, and 100
lg/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 lg/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
FIG. 9. 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 lmol (cross-hatched column) or
300 lmol 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 ¼ 4–5). **p < 0.01 compared with
n-HISF (10 lg/ml) in the absence of NAC; #p < 0.05 compared with n-HISF
(30 lg/ml) in the presence of 150 lmol NAC.
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-a 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-jB, which are activated by ROS (Donaldson et al., 2003).
ROS in n-HISF may induce activation of AP-1 and NF-jB 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
225
OXIDATIVE ABILITY CONTRIBUTES TO TOXICITY
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 concentrationdependent 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. Also remaining to be
elucidated is the nature of the major compounds that are the
source of the oxidative ability of n-HISF. On this point, our data
might be the valuable basic data on exploring the substances
responsible for the toxicity of DEP.
In conclusion, (1) n-HISF but not n-HSF, had strong
oxidative ability and many functional groups related to oxygenation; (2) n-HISF but not n-HSF induced oxidative stress,
cytotoxicity, and an inflammatory response; and (3) the high
oxidative ability of n-HISF contributed to the induction of
oxidative stress, cytotoxicity, and the inflammatory response.
These facts suggest that the oxidative property of n-HISF might
play an important role in various diseases including airway
diseases.
REFERENCES
Bayona, J. M., Markides, K. E., and Lee, M. L. (1988). Characterization of
polar polycyclic aromatic compounds in a heavy-duty diesel exhaust
particulate by capillary column gas chromatography and high-resolution
mass spectrometry. Environ. Sci. Technol. 22, 1440–1447.
Bonvallot, V., Baeza-Squiban, A., Baulig, A., Brulant, S., Boland, S., Muzeau, F.,
Barouki, R., and Marano, F. (2001). Organic compounds from diesel exhaust
particles elicit a proinflammatory response in human airway epithelial cells
and induce cytochrome p450 1A1 expression. Am. J. Respir. Cell Mol. Biol.
25, 515–521.
Cho, A. K., Di Stefano, E., You, Y., Rodriguez, C. E., Schmitz, D. A., Kumagai,
Y., Miguel, A. H., Eiguren-Fernandez, A., Kobayashi, T., Avol, E., et al.
(2004). Determination of four quinones in diesel exhaust particles, SRM
1649a, an atmospheric PM2.5. Aerosol Sci. Technol. 38, 68–81.
Dockery, D. W., Pope III, C. A., Xu, X., Spengler, J. D., Ware, J. H., Fay, M.
E., Ferris, B. G., and Speizer, F. E. (1993). An association between airpollution and mortality in 6 united-states cities. N. Engl. J. Med. 329,
1753–1759.
Donaldson, K., Stone, V., Borm, P. J., Jimenez, L. A., Gilmour, P. S., Schins,
R. P., Knaapen, A. M., Rahman, I., Faux, S. P., Brown, D. M., et al. (2003).
Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM10). Free Radic. Biol. Med. 34, 1369–1382.
Draper, W. M. (1986). Quantitation of nitropolycyclic and dinitropolycyclic
aromatic hydrocarbons in diesel exhaust particulate matter. Chemosphere 15,
437–447.
Hesterberg, T. W., Bunn, W. B., McClellan, R. O., Hart, G. A., and Lapin, C. A.
(2005). Carcinogenicity studies of diesel engine exhausts in laboratory
animals: A review of past studies and a discussion of future research needs.
Crit. Rev. Toxicol. 35, 379–411.
Hirano, S., Furuyama, A., Koike, E., and Kobayashi, T. (2003). Oxidative-stress
potency of organic extracts of diesel exhaust and urban fine particles in rat
heart microvessel endothelial cells. Toxicology 187, 161–170.
Hiura, T. S., Kaszubowski, M. P., Li, N., and Nel, A. E. (1999). Chemicals in
diesel exhaust particles generate reactive oxygen radicals and induce
apoptosis. J. Immunol. 163, 5582–5591.
Koike, E., Hirano, S., Shimojo, N., and Kobayashi, T. (2002). cDNA microarray
analysis of gene expression in rat alveolar macrophages in response to
organic extract of diesel exhaust particles. Toxicol. Sci. 67, 241–246.
Koike, E., Hirano, S., Furuyama, A., and Kobayashi, T. (2004). cDNA
microarray analysis of rat alveolar epithelial cells following exposure to
organic extract of diesel exhaust particles. Toxicol. Appl. Pharmmacol. 201,
178–185.
Koike, E., and Kobayashi, T. (2005). Organic extract of diesel exhaust particles
stimulates expression of Ia and costimulatory molecules associated with
antigen presentation in rat peripheral blood monocytes but not in alveolar
macrophages. Toxicol. Appl. Pharmacol. 209, 277–285.
Kumagai, Y., Koide, S., Taguchi, K., Endo, A., Nakai, Y., Yoshikawa, T., and,
Shimojo, N. (2002). Oxidation of proximal protein sulfhydryls by phananthraquinone, a component of diesel exhaust particles. Chem. Res. Toxicol. 15,
483–489.
Martin, L. D., Krunkosky, T. M., Dye, J. A., Fischer, B. M., Jiang, N. F.,
Rochelle, L. G., Akley, N. J., Dreher, K. L., and Adler, K. B. (1997). The role
of reactive oxygen and nitrogen species in the response of airway epithelium
to particulates. Environ. Health. Perspect. 105, 1301–1307.
226
SHIMA ET AL.
Nel, A. E., Diaz-Sanchez, D., Ng, D., Hiura, T., and Saxon, A. (1998).
Enhancement of allergic inflammation by the interaction between diesel
exhaust particles and the immune system. J. Allergy Clin. Immunol. 102, 539.
fractions of diesel particulate extracts. Int. J. Environ. Anal. Chem. 9,
93–144.
Seaton, A., MacNee, W., Donaldson, K., and Godden, D. (1995). Particulate
air pollution and acute health effects. Lancet 345, 176–178.
Nel, A. E., Diaz-Sanchez, D., and Li, N. (2001). The role of particulate
pollutants in pulmonary inflammation and asthma: Evidence for the
involvement of organic chemicals and oxidative stress. Curr. Opin. Pulm.
Med. 7, 20–26.
Pening, T. M., Burczynski, M. E., Hung, C. F., McCoull, K. D., Palackal, N. T.,
and Tsuruda, L. S. (1999). Dihydrodiol dehydrogenases and polycyclic
aromatic hydrocarbon activation: generation of reactive and redox active
o-quinones. Chem. Res. Toxicol. 12, 1–18.
Sugimoto, R., Kumagai, Y., Nakai, Y., and Ishii, T. (2005). 9,10-Phenanthraquinone in diesel exhaust particles downregulates Cu,Zn-SOD and HO-1 in
human pulmonary epithelial cells: Intracellular iron scavenger 1,10phenanthroline affords protection against apoptosis. Free Radic. Biol. Med.
38, 388–395.
Pope III, C. A., Thun, M. J., Namboodiri, M. M., Dockery, D. W., Evans, J. S.,
Speizer, F. E., and Heath, C. W., Jr. (1995). Particulate air pollution as
a predictor of mortality in a prospective study of U.S. adults. Am. J. Respir.
151, 669–674.
Von Klot, S., Wolke, G., Tuch, T., Heinrich, J., Dockery, D. W., Schwartz, J.,
Kreyling, W. G., Wichmann, H. E., and Peters, A. (2002). Increased asthma
medication use in association with ambient fine and ultrafine particles. Eur.
Respir. J. 20, 691–702.
Pope III, C. A., Burnett, R. T., Thun, M. J., Calle, E. E., Krewski, D., Ito, K.,
and Thurston, G. D. (2002). Lung cancer, cardiopulmonary mortality, and
long-term exposure to fine particulate air pollution. J. Am. Med. Assoc. 287,
1132–1141.
Wagner, J. G., and Roth, R. A. (2000). Neutrophil migration mechanisms, with
an emphasis on the pulmonary vasculature. Pharmacol. Rev. 52, 349–374.
Whitekus, M. J., Li, N., Zhang, M., Wang, M., Horwitz, M. A., Nelson, S. K.,
Horwitz, L. D., Brechun, N., Diaz-Sanchez, D., and Nel, A. E. (2002). Thiol
antioxidants inhibit the adjuvant effects of aerosol diesel exhaust in amurine
model for ovalhumin sensitization. J. Immunol. 168, 2560–2567.
Roos, D., de Boer, M., Kuribayashi, F., Meischl, C., Weening, R. S., Segal,
A. W., Ahlin, A., Nemet, K., Hossle, J. P., Bernatowska-Matuszkiewicz, E.,
et al. (1996). Mutations in the X-linked and autosomal recessive forms of
chronic granulomatous disease. Blood 87, 1663–1681.
Schuetzle, D., Lee, F. S., and Prater, T. J. (1981). The identification of
polynuclear aromatic hydrocarbon (PAH) derivatives in mutagenic
Snipes, M. B. (1989). Long-term retention and clearance of particles inhaled
by mammalian species. Crit. Rev. Toxicol. 20, 175–211.
Xiao, G. G., Wang, M., Li, N., Loo, J. A., and Nel, A. E. (2003). Use of
protemics to demonstrate a hierarchical oxidative stress response to diesel
exhaust particle chemicals in a macrophage cell line. J. Biol. Chem. 278,
50781–50790.