Environ. Sci. Technol. 2008, 42, 1761–1765
Cytotoxicity and Inflammatory
Potential of Soot Particles of
Low-Emission Diesel Engines
D A N G S H E N G S U , * ,†
ANNALUCIA SERAFINO,‡
JENS-OLIVER MÜLLER,†
R O L F E . J E N T O F T , † R O B E R T S C H L Ö G L , * ,†
AND SILVANA FIORITO‡
Fritz Haber Institute of the Max Planck Society, Faradayweg
4-6, D-14195 Berlin, Germany and Institute of Neurobiology
and Molecular Medicine, National Research Council (CNR),
Via Fosso del Cavaliere 100, 00133 Rome, Italy
Received July 5, 2007. Revised manuscript received
November 27, 2007. Accepted December 3, 2007.
We evaluated, in vitro, the inflammatory and cytotoxic
potential of soot particles from current low-emission (Euro IV)
diesel engines toward human peripheral blood monocytederived macrophage cells. The result is surprising. At the same
mass concentration, soot particles produced under lowemission conditions exhibit a much higher toxic and inflammatory
potential than particles from an old diesel engine operating
under black smoke conditions. This effect is assigned to the
defective surface structure of Euro IV diesel soot, rendering it
highly active. Our findings indicate that the reduction of soot
emission in terms of mass does not automatically lead to a
reduction of the toxic effects toward humans when the structure
and functionality of the soot is changed, and thereby the
biological accessibility and inflammatory potential of soot is
increased.
1. Introduction
Since the implementation of the 1970 Clean Air Act in the
United States of America, progress has been made in the
reduction of exhaust gas and soot emissions of light-duty
and heavy-duty vehicles (passenger cars and trucks). Particulate standards for diesel engines were introduced in 1982
and were tightened in 1991, 1994, and 1998 (1). The European
Union followed with emission standards for heavy-duty diesel
engines in 1992 (Euro I), and in stiffer form in 1998 (Euro II),
2000 (Euro III), and in October 2005 (Euro IV) (1). All major
automobile companies have developed low-emission engines
as well as filters for soot particles. Research and development
strategies have focused on the reduction of soot emission
yet have neglected the question of how changes in soot quality
may change its effect on human health. Hence, the question
is: does the low-emission engine Euro IV soot pose the same
health risk per unit mass as the soot produced from old
engines?
The cytotoxicity and inflammatory potential of soot
nanoparticles (NPs) can be assessed by in vitro studies.
Macrophages constitute the primary cellular effectors of the
immune response, playing a pivotal role in the detection of
* Address correspondence to either author. E-mail: dangsheng@
fhi-berlin.mpg.de (D.S.S.) and [email protected] (R.S.).
†
Fritz Haber Institute of the Max Planck Society.
‡
Institute of Neurobiology and Molecular Medicine.
10.1021/es0716554 CCC: $40.75
Published on Web 01/25/2008
 2008 American Chemical Society
all foreign bodies. These cells are ubiquitously present in the
mucosal and submucosal tissues (especially in the bronchial
and alveolar membrane), and human macrophage primary
cultures in vitro can provide a model of potential effects
upon in vivo inhalation of the soot NPs. When these cells
come in contact with particles or pathogens, they become
activated and secrete a variety of chemical mediators of
inflammation, very aggressive against foreign molecules or
particles. Currently, the toxicity of NPs is a hot research topic
because the increasing production of nanomaterials is likely
to significantly enhance the exposure of humans to NPs (2–4).
However, the research in the field of nanotoxicology is still
at its infancy. The parameters that determine the toxicity of
NPs are not known in any detail, as one can tell from the
large number of review articles published recently on the
topic (5). The parameter most frequently used as a measure
of dose is the surface area. However, lung inflammation
studies involving instillation of different types of carbon NPs
in mice have revealed a much more complex situation:
particles prepared by different techniques exhibit significant
differences in surface toxicity (5).
The purpose of this study was to compare the cytotoxicity
and the inflammatory response, in vitro, of human monocytederived macrophage cells (MDMs) to a Euro IV test heavyduty diesel engine soot and to soot from an old diesel engine
and to relate the results to the microstructure of these
particles, previously determined in detail by means of highresolution transmission electron microscopy and other
methods of NP characterization.
2. Experimental Section
In the following, the soot from a Euro IV test heavy-duty
diesel engine will be referred to as EuroIV soot; the soot from
an old diesel engine operating at black smoke conditions
will be referred to as BS soot. The methods of soot production
and collection have been described elsewhere (6). Briefly,
the EuroIV soot originated from a modified MAN D0836 LF4V six cylinder engine (6.9 L displacement, 228 kW), with
two-stage controlled turbocharging, an externally controlled
cooled exhaust gas recirculation, and a common rail injection
system. The engine was developed to fulfill the Euro IV
emission standard. The engine was set for a NOx emission
of 3.3 g/kWh and a PM emission of 50 mg/kWh (European
stationary cycle, ESC). The BS soot originated from a D2876
CR engine, operated at 30% load, extra-low rail pressure,
and air throttling (blackening number 5). The emission rate
of the BS engine is 200–600 mg/kWh. The diesel fuel used
for both engines was a standard low-sulfur type, containing
78% paraffin and 22% aromatic hydrocarbons (European
Norm 590). All samples were collected directly from the
exhaust gas of the engine using a special particle collector
that was heated to the exhaust gas temperature at the
collection position (200 °C).
Transmission electron microscopy, energy-dispersive
X-ray spectroscopy, and temperature programmed oxidation
studies revealed that EuroIV soot contained about 10% ash
from the combusted engine lubricant oil (7). This kind of ash
was not found in BS soot. For the in vitro studies, the EuroIV
and BS soot was sterilized by heating to 180 °C, washed three
times in distilled water, then suspended in PBS at a stock
concentration of 1 mg/mL and sonicated for 48 h before the
use.
Human peripheral blood monocytes were isolated from
buffy coats of healthy donors by density gradient centrifugation using lympholyte-H (Cederlane, Hornby, Ontario,
Canada). The lymphocytic/monocytic fraction was then
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resuspended in RPMI 1640 medium (Hyclone Laboratories
Inc. Logan, Utah) supplemented with 10% (v/v) heatinactivated fetal calf serum (FCS) (Hyclone), L-glutamine (2
mM), penicillin (100 IU/mL), and streptomycin (100 mg/
mL). Cells were seeded on 175 cm2 flasks and were maintained
at 37 °C in 5% CO2 to generate adhering macrophages
(MDMs). After 1 h of culture, nonadhering cells were removed,
and the residual adhering MDMs were maintained in culture
for 7 days to obtain partially differentiated macrophages.
For in vitro testing, soot particles were added to the MDM
culture medium to obtain the desired treatment concentrations. In a preliminary dose–response experiment, performed
using the Trypan blue dye exclusion method (8), cells were
treated with soot particles at concentrations ranging from
15 to 60 µg/mL. The concentration of 30 µg/mL was found
to be at the upper end of the linear dose–response curve (see
below) and was selected for all further tests in which the
cytotoxicity of the soot particles on MDM cultures was
evaluated: (1) by observing the nuclear morphology under
a confocal laser scanning microscope (CLSM) and counting
the number of apoptotic/necrotic cells stained with propidium iodide (Sigma-Aldrich Co., St. Louis, MO), and (2) by
a two-color fluorescence cell viability assay that distinguishes
metabolically active cells from injured and dead cells (Live/
Dead Cell Vitality Assay, Molecular Probes, Eugene, OR). The
analysis of particles uptake was carried out by CLSM on cells
fixed with paraformaldehyde and counter-stained with 1 µg/
mL propidium iodide (PI).
All optical observations were carried out using the confocal
microscope LEICA TCS SP5 (Leica Instruments, Heidelberg,
Germany). The excitation/emission wavelengths employed
were 568/590 nm for PI labeling, respectively, whereas the
soot particles were visualized by recording the reflected
intensity of the laser beam. The cell morphology was
visualized by differential interference contrast (DIC). The
merged images of the three signals (PI/Refl/DIC) were
recorded. To distinguish signals stemming from particles
internalized or adhering on the surface of the cells, vertical
sections xzy (xz-planes along y-axes) were also acquired for
each sample in addition to the horizontal confocal sections
xyz (xy-planes along z-axes, see the definition in the schematic
diagram in Figure 2c). Morphological changes in MDM
culture, indicative of macrophage activation, were examined
by scanning electron microscopy (SEM). For SEM observation, MDMs were fixed with 2.5% glutharaldehyde in 0.1 M
Millonig’s phosphate buffer (MPB) at 4 °C for 1 h. After
washing in MPB, cells were postfixed with 1% OsO4 in the
same buffer for another 1 h at 4 °C and then dehydrated
using acetone with increasing concentrations. The specimens
were then critical-point dried using liquid CO2 and were
sputter-coated with gold before examination on a Stereoscan
240 scanning electron microscope (Cambridge Instruments,
Cambridge, United Kingdom). The amount of the proinflammatory (IL-1β and IL-6) and anti-inflammatory cytokines (IL-10) secreted by MDMs challenged with the soot
particles was assessed by an ELISA testing (TEMA Ricerca)
in the supernatants of the cell cultures. Student’s t-test was
used for statistical analysis. For each variable, at least three
independent experiments were carried out.
3. Results
The morphological changes induced by soot NP treatment
of human MDM cultures are illustrated in Figure 1. A
comparison of representative images of untreated and soot
particle-treated MDM cultures obtained by phase contrast
and fluorescence microscopy is given in Figure 1a. The images
show that both EuroIV and BS soot particles were able to
induce macrophage activation, an early phase of the inflammatory reaction. This is revealed by the presence of more
developed microvillous structures on the cell surface and by
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FIGURE 1. Comparison of representative images of untreated
(control, Ctr) and particle-treated MDM cultures (30 µg/ml BS
or EuroIV soot). (a) Phase contrast and fluorescence microscopy
after PI staining of nuclei (red hue). A higher number of
apoptotic/necrotic cells (black arrows) is seen to be present in
EuroIV soot treated cultures as compared to BS treated cells.
Panels (b), (c), and (d) show scanning electron microscopy
views of cells. The examples in panel (c) show, at high
magnification, that BS and EuroIV soot treatment can induce
pronounced morphological changes indicative of macrophage
activation. The images in panel (d) show, also at high
magnification, a necrotic cell (left) and an apoptotic cell (right);
the open arrow points to an apoptotic body detaching from the
cell surface. Scale bars: 20 µm.
the size of the activated cells, which are typically two times
larger than the nonactivated ones (Figure 1, panels b and d).
While BS soot particles did not induce significant signs of
necrosis or apoptosis, EuroIV soot particles produced
extensive damage of cells, as revealed by the appearance of
numerous apoptotic and necrotic cells (Figure 1, panels b
and d). The analysis of particles uptake by confocal microscopy clearly showed that EuroIV soot particles were more
uniformly distributed than the BS soot particles, which were
found to aggregate into big clusters (Figure 2, panels a and
b). The images also suggest that the EuroIV soot particles
were internalized by MDMs in a much larger number than
the bigger aggregates of BS NPs. The internalization of a
higher amount of EuroIV soot particles may produce more
cytotoxic effects in MDMs and may stimulate a more intensive
inflammatory reaction as compared to BS soot.
Figure 3 shows the results of the dose–response experiment for human MDMs treated with increasing concentrations of EuroIV and BS soot particles, assessed by Trypan
blue dye exclusion methods. Linear responses were obtained
for both soot particles up to a dose rate of 30 µg/mL, where
higher doses result in the saturation of the response, especially
for BS soot particles. However, in all doses tested, EuroIV
soot induces a higher percentage of dead cells than the BS
soot. Figure 4a displays the evaluation of dead cells by live/
dead cell vitality assay and of apoptotic cells in untreated
(Ctr) and particles-treated (30 µg/mL BS or EuroIV soot)
MDMs. After 24 h of treatment, the EuroIV soot particles
induced a significantly higher percentage (p < 0.001) of
apoptotic and necrotic cells as compared to the particles
from the BS soot. Moreover, EuroIV soot particles were able
to stimulate human MDMs to secrete the pro-inflammatory
FIGURE 2. Confocal microscopy images showing the uptake of (a) BS and (b) EuroIV soot particles by human MDMs. The presence
of soot particles is visualized by differential interference contrast (DIC), as well as by the intensity of the reflected laser beam (Refl).
The cell cytoplasm and nucleus are visualized by the fluorescence signal of PI staining; merged images of the three signals (PI/Refl/
DIC) are also shown. For each cell, the horizontal confocal section along the xyz-axes and the vertical sections along the xzy-axes,
obtained as schematized in panel (c), are also reported.
FIGURE 3. Dose–response data testing the cytotoxicity of BS
and EuroIV particles on human MDMs, assessed by Trypan blue
dye exclusion methods. The solid lines reflect a linear dose
response.
cytokines IL-1β and IL-6, (Figure 4a), whereas the BS soot
particles from the old diesel engine did not induce a
significant secretion of these pro-inflammatory cytokines
(Figure 4b). At present we cannot explain the reason why the
BS soot particles seem to inhibit the secretion of proinflammatory cytokines. This topic will be the aim of future
studies.
We suspect that the difference in the inflammatory
potential of soot particles is due to their pronounced
dissimilarity in microstructure and reactivity. These aspects
have previously been studied in detail by high-resolution
TEM (HRTEM) and electron energy loss spectrometry
(7, 9, 10). Briefly, the following results were obtained. The
mean size of EuroIV soot particles (small nuclei, 10–15 nm;
spherical particles, 18 nm) is much smaller than that of BS
soot (35 nm). BS soot (Figure 5a) reveals the common
morphology of a spherical secondary structure made from
homogeneously sized flat basic structural units (11). EuroIV
soot particles exhibit distinctly rough surfaces and strongly
bent graphene sheets, many of them having irregular forms
(Figure 5b). HRTEM images reveal that the surface of EuroIV
soot is often decorated by abundant, very small soot
structures (Figure 5c). A recent study by means of quantitative
HRTEM analysis (12) has shown that EuroIV soot exhibits a
high degree of distortion due to defects such as non-sixmembered rings in the graphitic network. This reactive
structure can be expected to have significant consequences
for its function in the environment and in technical processes.
The olefinic electronic structure and the excess presence of
chemically reactive edges tend to destabilize the observed
carbon structures, as revealed by temperature-programmed
oxidation studies (7, 9). For instance, the combustion rates,
an indicator for the chemical reactivity and surface functionalization, are quite different for BS and EuroIV soot. In
an oxidative atmosphere (5% O2 in N2), the onset temperature
and the temperature of maximum reaction rate are 200 and
70 °C, respectively, lower for EuroIV than for BS soot (7, 9).
The large abundance of reactive structural elements in
the EuroIV soot facilitates the anchoring of heteroatoms such
as hydroxyl groups. X-ray photoelectron spectroscopic
measurements (12) revealed that the surface concentration
of oxygen in EuroIV soot is as high as 12%, significantly higher
than that for BS soot (7.4%). This is in accordance with the
results of infrared studies showing that EuroIV soot contains
a high concentration of OH groups (7). Such soot particles
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FIGURE 4. (a) Evaluation of dead cells by live/dead cell vitality assay and of apoptotic MDM cells (30 µg/mL BS or EuroIV soot). (b)
Effect of BS and EuroIV soot particle treatment on IL-1beta and IL-6 proinflammatory cytokines production by MDMs (30 µg/mL).
4. Discussion
FIGURE 5. High-resolution TEM images of (a) BS soot showing
almost spherical soot particles, (b) EuroIV soot with core–
shelled primary particles showing defective bulk and surface
structure, and (c) an expanded section of the image of an
EuroIV soot showing small soot structures at the periphery.
are hydrophilic and may disperse in aqueous media, whereas
particles of industrial carbon black are largely hydrophobic
and thus aggregate in water.
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Our experiments show that EuroIV soot particles are more
cytotoxic and have a higher inflammatory potential than soot
particles from an old diesel engine. Various parameters such
as surface area, number of particles, and joint length have
been examined to interpret or quantify the lung inflammatory
response to nanoparticle exposure (5). The results presented
in this work may suggest that the particle size is a proper
dose metric for nanotoxicity because the mean particle size
of EuroIV soot is much smaller than that of BS soot. However,
we strengthen that the reported effects are more likely due
to the strongly defective structure, the high abundance of
chemically reactive structural elements (edges), and the
presence of surface functional groups on the EuroIV soot
particles that enabled them to be phagocytized more readily
by MDMs than the larger BS particles. The expected
hydrophilic surface chemistry due to attached OH groups
should also be considered. These properties allow facile
chemical and morphological contact with hydrophilic biomolecules. The OH and olefinic surface chemistry is a novel
property of the EuroIV soot as opposed to the BS soot with
its smooth, inert surface and its hydrophobic character. All
theses aspects need to be taken into account in future health
risk assessment.
The present results are generally in accordance with the
results of recent studies involving C60-fullerenes that were
found to exhibit a rather low acute toxicity against human
and animal cells in vitro and animal tissues in vivo (13, 14).
However, after surface derivatization or functionalization they
became cytotoxic (15).
Low-emission diesel engines emit a comparatively small
amount of soot particulate matter. Our study, however, has
shown that, by mass, soot nanoparticles produced under
low-emission conditions have a higher cytotoxic and inflammatory potential against human peripheral blood monocytederived macrophage cells than particles from an old diesel
engine. The macrophages exposed to soot particles of lowemission engines showed characteristic features of necrosis
and degeneration. A high apoptotic cell death rate was
observed. This effect is assigned to the functionalized
defective surface structure of the low-emission diesel engine
soot, rendering it highly active. Moreover, the particles size
of EuroIV soot is smaller than that of BS soot that tends to
aggregate in bigger clusters. This makes the internalization
of a higher amount of EuroIV particles possible, leading to
more cytotoxic effects and stimulating a more intensive
inflammatory reaction, as compared to BS soot.
Our findings imply that a reduction of the emission rate
of soot particulates does not automatically lead to a reduction
of the toxic effects toward humans if, concurrently, the
structure and functionality of the soot changes and therefore
the biological accessibility and inflammatory potential of
the soot increases. Fortunately, the microstructural features
that aggravate the health risk also lead to a more effective
oxidation of soot particles to CO2, provided suitable filtering
techniques are applied (16). Hence, the development of
filtering technology must be directed toward the removal of
ultrasmall particles that, per unit mass, pose a higher risk to
the biosphere than the more conventional forms of largeparticle soot.
Acknowledgments
This work was part of the project “Katalytisches System zur
filterlosen kontinuierlichen Rußpartikelverminderung für
Fahrzeugdieselmotoren” supported by the Bayerische Forschungsstiftung, Munich. We are indebted to E. Jacob and
D. Rothe, Nürnberg, for access to the motor test equipment
and for helpful discussions. We acknowledge multiple
discussions with T. Velden. We also acknowledge F. Andreola
for the technical assistance in preparing cell cultures and
biological tests. We are very grateful to the anonymous
reviewer for helpful contributions to the manuscript and the
data presentation.
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