INERIS-DRC-03-26193-TOXI-GLc-n°03CR097
Toxicology of diesel exhaust particles
Ghislaine Lacroix
Institut National de l'Environnement Industriel et des Risques (INERIS)
Verneuil-en-Halatte (France)
The material emitted by diesel engines is a complex mixture that includes more than several
hundred different organic and inorganic particulate and gaseous compounds (Kagawa, 2002).
The particles are all respirable (mainly in the 0.02 – 1 µm range) and fall into two general
chemical classes: (1) "soot" or elemental carbon particles coated with condensed organic and
inorganic compounds, and (2) ultrafine particles of condensed organic material and sulfur
compounds having little or no elemental carbon content (Mauderly, 2001).
As in many pollutant mixtures, the composition of diesel exhaust varies considerably with the
conditions under which it is produced, i.e. condition and type of diesel engine, fuel and oil
used, time during the driving period, load on the engine, etc (Zelikoff, 2000). Those last years,
improvement of fuel quality (i.e. low sulfur fuels) together with new after-treatment systems,
have changed dramatically the composition of diesel exhaust. Continuously regenerating traps
with oxydative catalysts significantly reduce or eliminate particulate matter, carbon monoxide
and volatile hydrocarbons (Bunn et al., 2002). Since the chemical nature of a compound
determines for a great part its biological effects, this variability and evolution is of particular
interest regarding toxicological features of diesel exhaust.
Estimates of diesel exhaust concentrations to which human populations are exposed range
from 1-10 µg/m3 in the general urban environment to > 1 mg/m3 in some underground mining
operations (Zelikoff, 2000).
The toxicity of inhaled diesel exhaust has been studied by numerous investigators worldwide
and a number of excellent reviews currently exist on the topic (Cohen and Nikula, 1999;
Grigg, 2002; Kagawa, 2002; Pandya et al., 2002; Sydbom et al., 2001). The first part of this
paper deals with interaction between particles and the lung. The second part describes health
effects of diesel exhaust (mainly particulate matter), from the most classical and oldest
endpoints studied (pulmonary effects and cancer) to the most recent ones (effects on allergy,
reproduction and cardiovascular system). The third part deals with volatile nanoparticles
which are a specific part of the diesel exhaust. The fourth part will try to answer the following
question: which properties of particles are important for their toxicity?
I.
Interactions between particles and lung
I.1. The respiratory system (Harkema et al., 2000; McClellan, 2000)
The human respiratory system is a structurally complex arrangement of organs designed
principally for the intake of oxygen and the elimination of carbon dioxide (i.e. respiration).
Though its main function is gas exchange, the respiratory system is composed of specialized
tissues and cells that have other important functions such as olfaction, control of acid-base
balance of the blood and body as a whole, contribution to thermal regulation of the body, the
production of proteins and lipids, the activation and inactivation of hormones and the
metabolism of xenobiotic compounds entering the body through inhalation and other routes.
Another important function of the respiratory system is defense against inhaled infectious
(e.g., bacteria, viruses, fungi) and non-infectious agents (e.g., respirable dusts and gaseous air
pollutants).
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The respiratory tract is optimized for gas exchange. It comprises the largest mucosal surface
of the body with an internal surface area that is 25 times greater than the external surface of
the body covered by skin. In contrast to the other mucosa-lined organs of the body (e.g.,
alimentary and reproductive), that are only periodically exposed to the external environment,
the respiratory organs are constantly being exposed to large amounts of inhaled air. An adult
human at rest takes in 10,000 – 15,000 L of ambient air through the nasal passages each day.
Therefore, the respiratory tract serves as an important interface between the environment and
the host and plays a crucial role in maintaining the immune status of the body.
The respiratory tract can be divided into three major compartments (figure 1) based on gross
anatomy and physiology:
1) the nasopharyngeal compartment begins at the nose and mouth, where air enters the body,
and extends to the larynx. In this compartment, the temperature of the air is equilibrated to
approximate that of the body and the air is humidified. Here olfaction occurs by olfactory
sensory cells. Inspired airborne particles are also removed in the nasopharynx.
2) The tracheobronchial compartment extends from the larynx to the terminal bronchioles.
This set of conducting airways of decreasing cross-sectional diameter delivers gases to
and collects them from the pulmonary region. The tracheobronchial airways are lined by
ciliated cells, with an overlying blanket of mucus that serves an important role in
transported macrophages and particulate material from the pulmonary region to the
nasopharyngeal compartment.
3) The pulmonary region consists of the respiratory bronchioles, alveolar ducts, alveolar sacs
and alveoli. The relatively small diameter of the alveoli maximizes the surface area of the
pulmonary region, thereby optimizing the exchange of gases between the alveolar air sacs
and the blood circulating through the large network of capillaries lying between the
alveoli. The alveoli are lined by a thin layer of surfactant material that is essential for
maintaining alveolar structure as the gas volume changes in a cyclic manner.
Gaseous exchange between air and blood is restricted to the latter portion located in the lung
parenchyma.
Régions
Nasopharynx
Tracheobronchial
Pulmonary
Figure 1. Diagrammatic overview of the human lung and upper respiratory tract. The lung lobe on the left
side of the diagram illustrates the branching pattern of the intrapulmonary airways, from the bronchi to the
alveolar sacs. The pattern of the pulmonary vasculature is illustrated in the lung lobe on the right (Harkema et
al., 2000).
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I.2. Mechanisms of deposition of inhaled particles (McClellan, 2000; Schulz et
al., 2000)
During inhalation, particles are transported with the inspired air through the extrathoracic
airways and the bifurcating tracheobronchiolar system to the gas-exchanging region of the
lung. A certain number of these particles are caught in the respiratory system by touching the
wet airspace surfaces, a phenomenon generally referred to as particle deposition. Therefore,
with exhalation, not all particles are recovered. Figure 2A shows the fraction of particles
deposited in the respiratory system (total deposition) during quiet mouth breathing as a
function of the particle diameter.
Figure 2. Schematic overview of (A) total deposition fraction and of deposition fraction in (B) the extrathoracic,
(C) the tracheobronchial and (D) the alveolar region of the human respiratory system for unit-density spheres
during mouth breathing (Schulz et al., 2000).
This fraction is small for particles in the size range between 0.1 and 1 µm, but it becomes
larger for smaller and larger particles, reaching almost 100% for 0.01- or 10-µm particles.
However, the particle size determines not only how many particles are deposited, but also in
which region of the respiratory tract these particles are deposited (regional deposition, see
figures 1B-D).
The three most significant mechanisms of deposition are sedimentation, impaction and
diffusion. In some cases, interception, and electrostatic precipitation also occur (figure 3).
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Figure 3. Primary mechanisms of deposition of inhaled particles in the respiratory tract (McClellan, 2000).
Sedimentation. This phenomenon is due to gravity and becomes significant for particles
larger than 0.5 µm. The distance a particle settles within a given time increases with its mass.
the longer a particle remains in the respiratory system, the larger is the settling distance the
particle will cover and, hence, the probability that the particle will touch airspace walls.
Therefore, the relative long residence time in the small conducting airways and in the gasexchanging region of the lung will favor particle deposition by gravitational sedimentation.
Impaction. Impaction is due to inertia, which is the inherent property of a moving mass
to resist accelerations. Inertia may cause particles to continue to move to their original
direction and not follow airflow streamlines, so that they deposit on airway walls by
impaction. The inertia of a particle depends not only on the particle density and diameter, but
also on the airflow velocity. Inertial impaction will most likely occur in the extrathoracic
airways and in the large conducting airways of the lung, where flow velocities are high and
rapid changes in airflow direction occur.
Diffusion. It concerns mainly particles with a diameter less than 0.5 µm and is due to
collisions between gas molecules and a particle, which causes numerous very small random
displacements of that particle. The distance a particle will travel by diffusion increases with
time and with decreasing particle diameter. Hence, the probability of particles to hit airspace
surfaces by diffusional transport is larger the smaller the particles are and the longer they
remain in the respiratory system. Consequently, the lung periphery, with its small airway
dimensions, favors deposition by diffusion. Residence time is long, and the distance a particle
has to travel before it hits an airspace wall is short.
Interception. It takes place when one of the edges of a particle touches the surface of the
respiratory tract. Interception is usually important only for fibrous particles because
deposition by interception requires that the particle size is a significant fraction of the airway
diameter.
Electrostatic forces.
Deposition of particles in the respiratory tract by electrostatic
precipitation is usually negligible because most ambient particles become neutralized
naturally by air ions. However, many freshly generated particles are electrically charged and
may have an enhanced deposition.
The biological characteristics of the individual inhaling the particles also influence deposition.
The two major determinants are lung geometry, with age- and gender- specific differences,
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and the volume of air inhaled (as determined by respiratory rate and tidal volume), which is
increased during exercise according to the higher oxygen demand of the body.
I.3. Lung defense mechanisms (McClellan, 2000)
The first line of defense of the respiratory tract to protect the body from toxic effects of the
deposited particles is particle clearance. The response of the respiratory tract will vary
depending on where the particles are deposited, extending from the nostrils to the alveolar
spaces.
Clearance at low-exposure concentrations involves interaction between mechanical and
biological mechanisms. In all regions of the respiratory tract, macrophages are present and
begin engulfing particles as soon as they are deposited. Both free and engulfed particles are
available for mechanical removal.
Particulate matter deposited in the nasopharyngeal portions of the respiratory tract can trigger
mucus or serous secretion and flow. The fluid moves either anteriorly to the nares, where it is
removed by blowing or dripping, or posteriorly into the pharynx, where it may be swallowed.
Particulate mater deposited in the trachea and conducting airways encounters a blanket of
mucus moving on top of beating cilia in normal persons (fig. 4). The particles entrapped
within macrophages or directly within the mucus are carried up to the mucociliary escalator to
the pharynx, where the material is swallowed. The time period for tracheobronchial clearance
for most of the particulate matter is on the order of hours. A small fraction may be cleared
more slowly, and there is evidence that a very small fraction may actually be carried into the
epithelial cells and the underlying tissue.
Particulate matter that reaches the alveolar spaces has a high probability of being ingested by
macrophages (fig.4). If the particles are nontoxic, they reside in individual macrophages until
they die, and then the particulate matter and debris are engulfed by other macrophages. Over
time, some portion of the particles, presumably largely within macrophages, reaches the
terminal bronchioles, gains access to the mucociliary escalator, and is removed from the body.
Other particles may be carried to the interstitial spaces by macrophages or other inflammatory
cells or by direct penetration. Some particulate matter is transported to the regional lymph
nodes through the lymphatics, and some particulate matter may gain access directly to the
bloodstream.
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Figure 4. Schematic rendering of the mechanism of clearance particles deposited in the respiratory tract
(McClellan, 2000).
Simulation modeling of the deposition and retention in the lungs of inhaled relatively
insoluble particles (such as diesel exhaust particles) shows that the lung burdens always
slowly increase over a period of months to ultimately reach lung burdens that are at
equilibrium with the concentration of particles in the air. Occasionally, this equilibrium may
be perturbed by pathological changes associated with the rate of particle deposition, the
accumulated lung burden of particles, or both. At high concentrations (for example
3.5 mg/m3), some experimental studies in animals have shown that lung burdens are much
greater than expected based on the kinetics observed at a lower exposure concentration
(0.35 mg/m3). This is the so-called overload phenomenon, due to an impairment of alveolar
macrophages-mediated lung clearance (Oberdorster, 1995). However, this phenomenon is
unlikely to occur in environmental conditions.
I.4. Pathological process (McClellan, 2000)
The respiratory tract has a limited number of ways it can respond to inhaled particles. The
initial responses are intended to clear the particles from the respiratory tract and, indeed, from
the body. However, responses that begin as physiologically adaptive can also progress and
become pathological.
The initial responses of coughing and sneezing are generally viewed as being physiological,
but even they can continue to the extent that the afflicted person may begin to wonder if they
are more than a nuisance. Production of serous fluid and mucus is initially increased by
discharging of intracellular stores and may then progress to hypertrophy of the existing cells
and, at the extreme, hyperplasia of these cells.
The continuous injury of cells lining the nasal cavity and trachea, bronchi, and lower airways
can be sufficient to result in metaplastic transformations. Sometimes, this may progress to
sheets of squamous cells lining portions of the conducting airways.
With prolonged exposure to particulate matter at sufficiently high concentrations, the particles
continuously deposited in the alveolar spaces can trigger a sustained inflammatory reaction.
The inflammation can alter particle clearance, increasing initially the rate of clearance, and
then causing inhibition of clearance. This, in turn, intensifies the inflammatory reaction,
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which further impairs clearance and enhances the rate of particle accumulation in the alveolar
region. Some of these particles are found in the interstitial areas and others in the alveolar
spaces, where aggregates of macrophages, particles, and proteinaceous material may be
observed. Adjacent epithelial cells become hypertrophic, hyperplastic, and occasionally,
metaplastic. Frequently, bronchiolar epithelium may appear to be extending down into the
alveoli. This pattern of particle-induced overload disease has been described in detail in rats
chronically exposed to diesel exhaust or carbon black particles (Mauderly, 1996). The extent
to which lesions are produced in humans exposed to similar levels of diesel soot or carbon
black is not well understood.
II.
Health effects of diesel exhaust particles
II.1. Non cancer pulmonary effects
Laboratory studies using human subjects
Experimental human exposures studies have mainly been carried out using exposure chamber
set-ups with controlled diesel exhaust emissions. It is critical to ensure that the method is
designed so as to maintain a certain relationship between the particulate and gaseous
components and to obtain particles of the same size and chemical properties throughout the
exposure series (Sydbom et al., 2001).
Rudell and colleagues conducted several exposure studies. They exposed healthy volunteers
in an exposure chamber to diesel exhaust and examined changes in lung function with
dynamic spirometry. All the exposed subjects reported an unpleasant smell and eye irritation
but there was no alteration in the lung function tests measured as forced expiratory volume in
one second (FEV1) (Rudell et al., 1994). They subsequently used more sensitive whole-body
plethysmography to measure changes in lung function in healthy volunteers in an exposure
chamber for one hour during light work (Rudell et al., 1996). These volunteers were exposed
to whole diesel exhaust or to diesel exhaust with a particle trap at the tail pipe, which reduced
the number of particles by 46% without affecting other exhaust components. The main
symptoms during exposure were eye and nose irritation and an unpleasant smell. Both airway
resistance and airway inflammation were noted during exposures. Macrophage phagocytosis
was also reduced. The particle trap did not reduce the symptoms, lung function or
inflammation caused by diesel exhaust exposure. Therefore, for these effects, the relative
importance of diesel particles and other components of the exhaust has yet to be established.
In a more recent study, filters intended for use in the air intake into the passenger
compartment of vehicles were tested to prevent diesel exhaust effects (Rudell et al., 1999).
Healthy non-smoking subjects were exposed six time for one hour in a specially designed
exposure chamber, once to air and once to unfiltered diesel exhaust and subsequently to diesel
exhaust filtered with four different air intake filters. Particle concentrations during exposure to
unfiltered diesel exhaust were kept at 300 µg/m3. The study included measurements of lung
function, symptoms and nasal responses. While no acute effects were seen on nasal lavage,
rhinometry and lung function, there were major effects on subjective symptoms (irritation,
detection of unpleasant smell…). The use of a particle filter in combination with an active
charcoal filter (that might reduce certain gaseous components) was demonstrated to reduce
the symptoms and discomfort caused by the exhaust.
Salvi and colleagues exposed 15 healthy volunteers (age 21-28) to diluted diesel exhaust
(PM10 = 300 µg/m3, NO2 = 1.6 ppm) and air for 1 hour with moderate exercise (Salvi et al.,
1999a). Lung function was measured immediately before and after the exposure and
bronchoscopy was performed and peripheral blood samples obtained six hours following
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exposure. No exposure-related changes in pulmonary function were observed, but a pattern of
inflammatory response, both pulmonary and systemic, was observed. The investigators
reported increases in neutrophils, mast cells, CD4+ and CD8+ lymphocytes, up regulation of
endothelial adhesion molecules, and increased LFA-1 in bronchial tissue cells. The
inflammatory response was an order of magnitude greater than the effects documented after
allergen challenge in atopic asthmatics (Montefort, cited in (Sydbom et al., 2001), indicating a
pronounced signal for inflammatory cell recruitment as a response to diesel exhaust exposure.
In contrast, lung function parameters were found unaffected following exposure to diesel
exhaust (Salvi et al., 1999a). Consequently, lung function measurements alone cannot be used
to exclude adverse air-pollution-associated airway responses (Sydbom et al., 2001).
Studies of induced sputum have also been used to evaluate diesel exhaust effects on the
human airways (Nordenhall et al., 2000). Sixteen healthy nonsmoking subjects were exposed
to air and diesel exhaust at a particle concentration of 300 µg/m3 for 1 hour. Six hours after
exposure to diesel exhaust, a significant increase was found in neutrophil percentage of total
cells in sputum, together with an increase in the concentration of IL-6 and methyl-histamine,
compared to control air exposures.
Laboratory studies using animals
Some general points must be raised regarding animal studies. Apart from the possibility that
mechanisms may be very different from those in man, studies using radioactive particles have
also demonstrated that there is a large difference in the dosimetry of the small airways of
rodents compared to humans. Therefore, care must be taken when extrapolating animal data to
humans. In addition, in many studies on animals, the doses of diesel exhaust are much higher
than those humans are exposed to in daily life (Sydbom et al., 2001). A clear lack of
understanding of the bioavailability and bioactivity of diesel exhaust components at realistic
doses exists. Measurements in the Scandinavian countries have shown that the average 24-h
particulate matter concentration varied from 30-150 µg/m3 total suspended particles.
However, in certain industrial areas, concentrations of up to 1,500 µg/m3 have been measured
(Sydbom et al., 2001).
Several animal studies have shown that diesel exhaust, even at high concentrations (up to
6,000 µg/m3 particles for 9 weeks), has a limited effect on mortality (White and Garg, 1981).
The body weight is also little affected (Watanabe and Nakamura, 1996). However, an increase
in lung weight was noted in rats, mice and hamsters exposed chronically to 4,000 µg/m3
diesel exhaust particles (Heinrich et al., 1986).
Studies about effects of diesel exhaust on pulmonary function led to divergent conclusions. If
subacute exposures (1:14 dilution of diesel exhaust dilution for 28 days) show little effects
(Pepelko, 1982; Pepelko et al., 1980), chronic exposures revealed a decreased pulmonary
function in cats exposed to 6,000-12,000 µg/m3 for 62 weeks (Moorman et al., 1985) but an
increased pulmonary function in rats exposed to 1,500 µg/m3 for 612 days (Gross, 1981).
These divergent results could be explained by the experimental protocol and the species used.
As in humans, a pulmonary inflammation, characterized by an influx of macrophages,
neutrophils and sometimes eosinophils in alveolar spaces of exposed animals was noted
(Heinrich et al., 1986; Henderson et al., 1988b). In parallel, structural alterations of lung
tissue were noted in several species (rat, cat, guinea pig, hamster) exposed chronically to
diesel exhaust (200-6,000 µg/m3 particles). With time, lungs turn gray then black (Barnhart et
al., 1982; White and Garg, 1981). The first histological changes consist of some intra-alveolar
particle-laden macrophages. A few diesel particles are also present in the alveolar epithelial
cells. Particles were then present in the peribronchiolar lymphoid tissue as well as the
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mediastinal lymph nodes. After a few weeks of exposure, some of the alveolar macrophages
begin to be grouped in clusters within alveoli and some septal thickening, due to proliferation
of type II pneumocytes, was seen surrounding such agglomerates (Barnhart et al., 1981;
Barnhart et al., 1982; Plopper et al., 1983; White and Garg, 1981). Finally, fibrosis was noted
(Barnhart et al., 1982; Henderson et al., 1988b; Hyde et al., 1985). This evolution occurs
more or less rapidly according to exposure concentrations. For high exposures (6,000 µg/m3),
the first changes occur after 3-4 days (White and Garg, 1981). For lower concentrations
(750 µg/m3), changes occur after 15 days of exposure (Barnhart et al., 1981). Rats tend to
exhibit greater inflammation, epithelial hyperplasia and fibrosis than similarly exposed mice
(Henderson et al., 1988a; Henderson et al., 1988b), hamsters (Mauderly, 1994) and monkeys
(Nikula et al., 1997b).
The effects of diesel exhaust exposure on non-specific host defenses have been studied in
mice by assessing susceptibility to respiratory tract infections, with inconsistent results. It was
reported that acute and subacute exposures of mice to diesel exhaust (6,000-7,000 µg/m3)
caused an increase in mortality due to bacterial infection but not to viral infection (Campbell
et al., 1981). Hahon et al. found no increase in mortality or other measures of influenza viral
infection after one month of exposure to diesel exhaust (2,000 µg/m3); however, after longer
exposure (3 and 6 months), pulmonary consolidation and virus growth were greater in dieselexposed animals (Hahon et al., 1985). The most consistent finding relative to the potential of
diesel exhaust exposure to depress non-specific host defenses has been the decreased alveolar
clearance of indicator particles (Chan et al., 1984; Griffis et al., 1983; Heinrich et al., 1986;
Mauderly et al., 1989). There appears to be a threshold above which particle retention and
inflammation occurs. It has been calculated mostly on the basis of evidence from animal
studies that the threshold is about 500 µg/g lung tissue (Pritchard, 1989). However, it is
difficult to assess how this correlates to levels in the inhaled air, and there may be important
interactions between the actual concentration and the duration of exposure (Sydbom et al.,
2001).
Epidemiological studies have indicated that subjects with pre-existing lung disease may be
more susceptible to episodic high levels of airborne pollutants than normal subjects (Sydbom
et al., 2001). This has been studied in a rat model in which pulmonary emphysema was
induced in rats by intratracheal instillation of the proteolytic enzyme elastase, and manifested
as enlarged alveoli, alveolar ducts and ruptured alveolar septa (Mauderly et al., 1990). The
emphysematous rats and a group of control rats were then exposed for 24 months to diesel
exhaust (3,500 µg/m3) or air as control. The results showed that rats with experimentally
induced emphysema were not more susceptible to inhalation of diesel exhaust than control
rats. In fact, fewer soot particles accumulated in the emphysematous lungs.
Morphological changes have also been examined in a comparative study of Cynomolgus
monkeys and rats. Both species were exposed for 24 months to 2,000 µg/m3 diesel exhaust
particles or ambient air (Nikula et al., 1997b). It was found that monkeys retained relatively
more particulate matter than rats. Rats retained more material in the lumen of alveolar ducts
and alveoli whereas monkeys retained more in the interstitium. Rats but not monkeys showed
significant inflammation and fibrosis. The results indicate that particle retention patterns and
tissue reactions in rats exposed to diesel exhaust particles may not be predictive of the
reaction in primates (Nikula et al., 1997a). Primates may retain more particles but may also
beless sensitive to the harmful effects.
Laboratory studies using cells
Recent in vitro studies have emphasized the role of diesel exhaust particles in the
development of an inflammatory response of bronchial epithelial cells. It was shown that
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exposure of human bronchial epithelial cells to diesel exhaust particles (at a concentration of
50 µg/mL) significantly increased the cell electrical resistance and decreased the ciliary beat
frequency (Bayram et al., 1998).
Diesel exhaust particles (at concentrations of 50 and 100 µg/mL and 20 µg/cm2) are also able
to induce the release of inflammatory cytokines such as IL-8, IL-1 and GM-CSF (Bayram et
al., 1998; Boland et al., 1999a; Boland et al., 1999b; Boland et al., 2000). A more recent
study using an air-cell interface deposition technique, which allows direct contact between
particles and lung cells, has shown that diesel engine exhaust particles (DEP) were able to
induce the production of IL-8 in TNF- "primed" alveolar epithelial cells (A549) from 50 min
exposure with no delay (Cheng et al., 2003). Interestingly, the findings of Cheng et al.
indicate that, for a comparable concentration (about 1-2 million particles/cm3 for gasoline and
1.5-3.5 million particles/cm3 for diesel) gasoline particles (GEP) could induce the production
of higher levels of IL-8 than diesel particles (30 ng/ml versus 23 ng/ml), but with a 2-hour
delay of response. It should be noted that the number of particles larger than 20 nm was much
higher for GEP than for DEP, which could imply a greater concentration in mass for GEP
than for DEP.
Doornaert et al. propose that diesel exhaust particle exposure of human bronchial epithelial
cells would be able to alter cell-matrix interactions and cell cohesion through concomitant
decrease of 3 β1 integrin subunits and CD44 adhesion molecule (at 20 and 100 µg/mL) and
weakening of actin cytoskeleton (at 5, 20 and 100 µg/mL). These alterations are expressed by
reduced wound-closure capacity as well as enhancement of cell de-adhesion ability
(Doornaert et al., 2003a; Doornaert et al., 2003b). .
Taken together, all these results support the concept that diesel exhaust particle exposure
tends to break the link between cells and extracellular matrix suggesting increased potential of
cell detachment from underlying basement membrane in vivo.
I.2. Diesel and cancer
Several organizations have reviewed epidemiological and experimental studies related to
diesel engine exhaust and lung cancer, and they have classified (or proposed classifying)
exhaust gas mixtures (or the particles components of the mixtures) as "potential", "likely",
"probable", or "definite" carcinogens for humans (HEI, 1995; IARC, 1989; NIOSH, 1988; US
EPA, 2002; WHO, 1996). The different assessments were driven by a series of studies.
Epidemiology
Over 40 studies currently provide estimates of the risk of lung cancer associated with
occupational exposure to diesel exhaust (reviewed in US EPA, 2002) . Most of them have
consistently observed elevated lung cancer rates among exposed workers that cannot be
readily attributed to known sources of bias or confounding. The most frequently studied
occupational groups have been railroad workers (Garshick et al., 1988) and truck drivers
(Steenland et al., 1998). The studies of truck drivers show a 20-50% excess incidence and/or
mortality from lung cancer, which persists when cigarette smoking is accounted for in data
analysis. Studies of railroad workers have also consistently observed excess relative risks on
the order of 30-50% after analytic control for cigarette smoking (Cohen and Nikula, 1999).
Unfortunately, however, no current study provides quantitative estimates of the past exposure
of study subjects to any constituent of diesel exhaust. A special panel of the Health Effects
Institute recommended that existing epidemiologic studies should not be used for quantitative
risk assessment because of the large uncertainty in assessing retrospective diesel exposures
(HEI, 1999).
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Valberg and Watson recently compared information on the reported lung-cancer risk with
estimated diesel exhaust concentrations for several occupational groups (Valberg and Watson,
2000). According to the authors, although none of the epidemiological studies had concurrent
measurements of diesel-exhaust concentrations, such data are available from more
contemporary studies. Three groups were defined: truck drivers, dock workers, railroad
workers (5-100 µg/m3), bus garage workers, railroad shop workers and hostlers (5070 µg/m3), underground miners (500-2000 µg/m3). They found a lack of concordance
between reported lung-cancer risk and estimated exposure (i.e. more heavily exposed
population have a lower lung cancer risk), arguing against a causal role for diesel exhaust in
the epidemiological associations. Anyway, retrospective exposure assessment using data from
other populations is a very hazardous exercise and the conclusions obtained should be taken
cautiously.
The 70-year cancer mortality risks have been estimated from several epidemiological studies
and vary over 30-fold, from about 1.10-4 to 3.10-3 per µg/m3 of diesel particulate matter
(Lloyd and Cackette, 2001). According to the authors, many of these variations are
attributable to different estimates of exposure. These values were questioned in a critical
discussion (Chow, 2001). Due to the large uncertainties in using current studies to precisely
estimate risk, it was suggested that a risk range rather than a single value provides a better
basis for setting emission reduction targets (Chow, 2001).
A special report of the Diesel Epidemiology Working Group of the Health Effects Institute
has pointed out explicit research needs to improve risk estimates from diesel exhaust exposure
(HEI, 2002). It does not recommend initiating a new cohort study at that time until
improvement of exposure assessment, including the possibility of defining a signature that is
characteristic of diesel emissions. Assessment of possible biomarkers for lung cancer related
to diesel emission exposure should be conducted, based on anticipated advances in
understanding the molecular and cellular biology of lung cancer at medium term (3-10 years).
HEI might also consider establishing population for prospective observation so that exposure
assessment with sufficient validity could be implemented.
Experimental studies in animals
The carcinogenicity of inhaled diesel exhaust has been studied extensively in rodent
bioassays.
No effects were seen in hamsters or mice (reviewed by Mauderly, 1992).
In all the reliable studies in rats, diesel exhaust has been found to be carcinogenic with longterm exposure at particle concentrations of 2 mg/m3 or more, which is equivalent to
approximately 1 mg/m3 in terms of continuous exposure (Kagawa, 2002). Studies including
groups of rats exposed to whole or filtered exhaust have shown that filtered exhaust is not a
pulmonary carcinogen in rats. These studies demonstrated the importance of particulate
matter in causing lung tumors in rats, but they did not determine whether the particleassociated organic compounds or the particles themselves were responsible for the
carcinogenesis (Cohen and Nikula, 1999). Other studies have shown that carcinogenic
capacity of particles decreased when numerous organic substances, including mutagens and
carcinogens, were removed from them (Kagawa, 2002). Nevertheless, when rats were allowed
to inhale carbon black to which hardly any organic substances had adsorbed or other inert
particles such as titanium dioxide, it produced similar types of lung tumors. From these
results, it has been pointed out that lung cancer in rats might be non-specific and due to
particle overload in lung from prolonged exposure to high concentrations of particles
(Mauderly, 1996; McClellan, 1996).
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Review of cellular responses in rat lungs (Watson and Valberg, 1996) shows that the
mechanistic series of steps related to tumorigenesis in rats are not likely to be relevant to
humans. Moreover, Valberg and Crouch combined tumor data from eight chronic inhalation
studies in rats (Valberg and Crouch, 1999). Statistical analysis identified a threshold for
tumors at diesel exhaust particulate concentrations between 200-600 µg/m3 average
continuous lifetime exposure. Exposure-response analysis of all rats exposed at less than
600 µg/m3 average continuous lifetime exposure showed no tumorigenic effect. Thus, metaanalysis of studies exposing rats to diesel exhaust gives no evidence that it exerts a
tumorigenic effect at low exposure.
Mutagenic potency of diesel exhaust at environmental levels
Extensive studies with microbial bioassays have demonstrated mutagenic activity in both
particulate and gaseous fractions of diesel exhaust. Structural chromosome aberrations and
sister chromatid exchanges in mammalian cells have been also induced by particles and
extracts (US EPA, 2002).
For some, ambient levels of diesel exhaust (approx. 1.5 µg/m3) do not carry enough
mutagenic potency to pose a carcinogenic risk (Bunn et al., 2002). One can estimate the
"mutagenic dose" of diesel exhaust organic compounds and compare it quantitatively with
another combustion aerosol, cigarette smoke. A comparative potency analysis shows that
(assuming the mutagenic activity of diesel-engine exhaust to be 100% bioavailable)
continuous exposure to diesel exhaust at 1.5 µg/m3 would be equivalent to smoking a total of
eight cigarettes over a 70-year lifetime, starting at age 20 (Valberg and Crouch, 1999). This is
approximately one cigarette every 6 years. If diesel organic compounds were only 10%
bioavailable, the mutagenic dose of diesel exhaust at ambient levels would be equivalent to
approximately one cigarette par lifetime (Bunn et al., 2002).
II.3. Effects on allergic immune responses
There has been a rapid increase in the global incidence of allergic diseases such as asthma and
rhinitis in the last two decades, which cannot be attributed to genetic changes, and is assumed
to be related to changes in environmental factors (Sydbom et al., 2001).
It is about 10-15 years that attention has focused on the potential for diesel exhaust or diesel
exhaust particles to enhance allergic immune responses in the respiratory tract. One of the
first report was that of Takafugi et al., who found in 1987 that Immunoglobulin-E (IgE)
responses of mice to intranasally instilled ovalbumin antigen were increased by adding diesel
exhaust particles to the instillate (Takafuji et al., 1987).
Some epidemiologic evidence exists, associating exposure to high levels of diesel exhaust
with respiratory allergies. Three railroad workers, who traveled in locomotive units directly
behind the lead diesel-powered locomotive engine, developed symptoms consistent with
asthma, including hyperreactive airways, airflow limitation and reversibility with
bronchodilators (Wade and Newman, 1993). None of these workers had any known
preexisting respiratory conditions. It is recognized that the irritant effect of some components
of the diesel exhaust (acid aerosols, volatile organic compounds…) alone could potentially
trigger asthmatic symptoms at sufficiently high exposure levels (Pandya et al., 2002).
There is also (indirect) epidemiological evidence that chronic exposure to diesel exhaust at
lower environmental levels may also be associated with increased levels of respiratory
symptoms. For instance, several studies found that children grown in more polluted regions of
a country are more likely to develop respiratory diseases and allergies compared with children
grown in "cleaner" regions (Heinrich et al., 1999; Van Niekerk et al., 1979). Within
communities, children living on busy streets have a higher likelihood of developing chronic
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respiratory symptoms than those living on streets with lower traffic volume (Brunekreef et al.,
1997; Oosterlee et al., 1996). When exposed to similar levels of Japanese cedar pollen (a
standard allergen), people who live in highly trafficked areas have enhanced allergic reactions
compared with people who live in rural areas (Ishizaki et al., 1987). However, despite reports
suggesting an association with diesel exhaust, it is difficult to evaluate the contribution of
diesel, because there are no specific markers of diesel particle exposure. Nevertheless, there
have also been reports of human volunteer and animal experiments suggesting that diesel
particles are related to respiratory allergies.
Direct immunologic effects of diesel exhaust particles
Diesel exhaust particles have been shown to potentiate IgE production in human respiratory
mucosal membranes. IgE is produced by activated B cells in response to a specific allergen.
Once produced, IgE attaches to mast cells and, when cross-linked by allergen, induces mast
cells to release histamine and leukotrienes. The chemicals released from mast cells cause
constriction of bronchial smooth muscle, mucus secretion and serum leakage into the airways
and result in acute asthma symptoms (Sydbom et al., 2001). In a study of healthy volunteers,
it was shown that exposure to diesel exhaust particles (0.3 mg) significantly increases IgE
levels in nasal fluids by greatly increasing the number of IgE-secreting cells and by altering
the expression of IgE mRNA isoforms (Diaz-Sanchez et al., 1994; Diaz-Sanchez et al., 1996).
In comparison, there was no effect on IgG, IgA or IgM antibody production. This suggests
that diesel exhaust particle exposure in vivo induces both a quantitative increase in IgE
production and a shift in the type of IgE that is produced. Although most studies support the
finding that diesel particles increase IgE synthesis, one study in mice failed to find an increase
in IgE synthesis from diesel particle alone (Ohta et al., 1999).
Diesel exhaust may also stimulate the proliferation of eosinophils. The granules of mature
eosinophils contain chemokines, leukotrienes and toxic proteins. Degranulation of eosinophils
in mucosal tissues results in bronchial inflammation and contribute to asthmatic symptoms.
Just as mast cells are regarded as the central cell for the acute asthmatic response, eosinophils
are often regarded as the critical cell type in chronic asthma (Pandya et al., 2002). Healthy
human volunteers exposed to diesel exhaust had increased eosinophils and other inflammatory
molecules on bronchial biopsies 6 hr after exposure (Salvi et al., 1999a). However, a similar
study did not detect increased eosinophils in induced sputa 4 hr after exposure to diesel
exhaust particles (Nightingale et al., 2000). Induced sputa are less sensitive than bronchial
biopsies at detecting subtle inflammatory changes in the lower airways (Pandya et al., 2002).
Eosinophils incubated with diesel exhaust particles had enhanced adherence to human nasal
epithelial cells and enhanced levels of degranulation (Terada et al., 1997). In animal assays,
the diesel particle-induced eosinophilia is enhanced in the presence of allergens such as
ovalbumin and is accompanied by enhanced airway hyperresponsiveness to acetylcholine
challenge (Ichinose et al., 1998; Takano et al., 1998).
Exposure to diesel particles may augment levels of many different cytokines (soluble protein
immune mediators such as interleukins) and chemokines (attractant proteins that induce
migration of different cell types). These molecules are key chemical messengers in the
inflammatory processes of asthma. For example, healthy humans exposed nasally to 0.15 mg
of diesel particles suspended in 200 µL saline solution expressed Th2-type cytokines (i.e.
cytokines involved in allergic reactions: IL-4, IL-5, IL-6, IL-10) in their nasal mucosa cells
18-24 hr after exposure (Diaz-Sanchez et al., 1996).
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Adjuvant immunologic effects of diesel exhaust particles
Although diesel exhaust particle exposure alone can elicit adverse biologic effects in the
airway, the effect of diesel particles has been repeatedly shown to be even greater in
conjunction with allergen i.e. diesel particles can act as an adjuvant to allergen. In a study of
13 non-smoking volunteers, it was shown that exposure to particles plus a ragweed allergen
results in increased expression of all the Th2-type cytokines in nasal fluid (Diaz-Sanchez et
al., 1997). Human nasal instillation studies involving exposure to 0.3 mg diesel particles
along with a ragweed antigen challenge showed that ragweed-specific IgE levels peaked far
higher in the presence of diesel particles, with a maximum 4 days after exposure. The levels
of ragweed-specific IgG4 (an isoform of IgG that is linked to IgE expression) also increased
in these studies, although other forms of IgG were not affected (Diaz-Sanchez, 1997; DiazSanchez et al., 1997).
An innovative study of 10 nonsmoking atopic human subjects tested the potential for diesel
exhaust particles to create a brand new immune response to an allergen. The investigators
exposed the atopic subjects on three occasions to the neoantigen keyhole limpet hemocyanin
(KLH), a compound to which humans are not normally sensitized. Twenty-four hours prior to
each exposure to the new antigen, the subjects were exposed nasally to a concentration of
particles roughly equivalent to 1-3 days of breathing Los Angeles air (0.3 mg). Subjects
exposed to KLH alone did not develop IgE antibodies to this compound, whereas subjects
exposed to diesel particles followed by KLH developed KLH-specific IgE and mounted a
Th2-type cytokine response with increased levels of IL-4 (Diaz-Sanchez et al., 1999). This
important study indicates that diesel exhaust may promote new allergic sensitization to
antigens in addition to aggravating existing allergic diseases.
Animal studies have provided definitive data that diesel exhaust particles affect IgE
production. Diesel exhaust particles increase the production of ovalbumin-specific IgE after
repeated intranasal or intratracheal instillation in ovalbumin-sensitized and challenged mice
(Takafuji et al., 1987). Diesel exhaust particles also enhance antigen-specific IgE responses
after repeated intraperitoneal injection of mice with diesel particles plus ovalbumin or
Japanese cedar pollen (Muranaka et al., 1986). Intranasal instillation of diesel particles and
ovalbumin cause in vitro proliferation in response to ovalbumin and increased IL-4
production compared with mice instilled with ovalbumin alone (Fujimaki et al., 1994;
Fujimaki et al., 1995). Inhalation of diesel exhaust has been shown to enhance the production
of antigen-specific IgE antibody in mice through alteration of the cytokine network (Fujimaki
et al., 1997). Guinea pigs exposed to diesel exhaust particles for 5 weeks with ovalbumin
sensitization once per week developed 7-fold greater anti-OVA IgG antibody than guinea pigs
exposed only to filtered air, indicating that the response is not specific to mice (Kobayashi,
2000). Similar results have been seen in rats, where intranasal or intratracheal co-exposure to
particles and pollen grains resulted in a much greater serum level of specific IgE and IgG1
antibodies than exposure to either alone (Steerenberg et al., 1999).
At the present time, the relative contributions of the particle core versus various adsorbed
chemicals to the adjuvant activity of diesel particles are unresolved (see § III.2). Also
unresolved is the relative Th2 adjuvant potency or potential contribution of diesel particles
compared with other inhaled particles in enhancing allergic respiratory disease. Lastly, and
most importantly, it is not clear that inhaled diesel exhaust at environmental or occupational
concentrations would have significant Th2 adjuvant effects.
One interesting study examined the effects of oral ingestion of diesel particles in mice
because it is known that airborne particulate reaches not only the lung but also the mucosa of
the gastrointestinal tract. Particles in the gut mucosa also appear to act as an adjuvant,
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enhancing response to allergen and production of allergen-specific IgG1 (Yoshino and Sagai,
1999).
II.4. Effects on cardiovascular system
Epidemiological studies have associated increased mortality in cardiovascular diseases with
episodes of heavy air pollution characterized in particular by elevated concentrations of
ambient particles (Dockery, 2001; Peters et al., 2001; Peters and Pope, 2002). It is suggested
that the ultrafine particles would induce airway inflammation in susceptible individuals,
release of mediators and an increase in blood coagulability (Donaldson et al., 2001).
Experimental studies of the influence of diesel exhaust on various cardiovascular responses
however, remain very few. Healthy human volunteers were exposed to air and diluted diesel
exhaust for 1 hour with intermittent exercise (Salvi et al., 1999b). The exposures were
standardized by keeping the PM10 concentration at 300 µg/m3 which was associated with
1.6 ppm of NO2, 4.5 ppm NO, 7.5 ppm CO, 4.3 ppm total hydrocarbons, 0.26 mg/m3
formaldehyde and 4.3.106 suspended particles/cm3. Significant increases in neutrophils and
platelets were observed in peripheral blood following diesel exhaust exposure, showing that
acute exposure to diesel has effects on inflammatory cells in the blood.
Pretreatment of human serum with diesel exhaust particle extracts (500-2500 µg/mL) gave a
dose dependant reduction in complement hemolytic activity of up to 20% and activation of
the complement pathway (Kanemitsu et al., 1998).
In animals, a recent study examined the effects of diesel exhaust particles instilled into the
trachea of hamsters on experimental thrombosis (Nemmar et al., 2003). Doses of 5 to 500 µg
particles (SRM 1650 from the US NIST) enhanced experimental arterial and venous platelet
rich-thrombus formation in vivo. Blood samples taken from hamsters 30 to 60 minutes after
instillation of 50 µg of diesel particles induced a rapid activation of circulating blood
platelets. According to the authors, the kinetics of platelet activation was consistent with the
reported clinical occurrence of thrombotiv complications after exposure to pollutants.
A direct toxic action of diesel exhaust particles was studied in a model of isolated atria from
guinea pigs (Sakakibara et al., 1994). Diesel particles in lower doses (10-500 µg/mL) induced
a transient but dose-dependent increase in contractile force. Particles in doses > 500 µg/mL
only, decreased contractile force and induced cardiac arrest. It was concluded that cardiac
toxicity contributes to the lung edema that is known to be one prominent cause of death in
diesel exhaust particles exposed animals. It appears unlikely, however, that inhalation of
diesel exhaust particles by humans could lead to the concentrations employed in these
particular experiments.
Recent studies have examined the effects of other combustion particles on rat cardiovascular
responses. Changes in hematological and hemodynamic parameters were observed in rats
with ozone-induced lung inflammation and exposed to 0.5, 1.5 or 5 mg EHC-93 particles by
intratracheal instillation (Ulrich et al., 2002).
Spontaneously hypertensive (SH) or normotensive (WKY) rats were exposed either
intratracheally (0, 1 or 5 mg/kg in saline) or nose-only (15 mg/m3 for 1 to 4 weeks) to
combustion source residual fly ash (ROFA) with low metal content (Kodavanti et al., 2002).
ROFA administered intratracheally was temporally associated with increase in plasma
fibrinogen in both strains but only the SH rats responded to the acute 1-week ROFA
inhalation. Longer term ROFA caused progressive lung injury (SH>WKY) but did not sustain
the increase in fibrinogen. There was a small but consistent decrease in blood lymphocytes
and an increase in blood neutrophils in SH rats exposed to ROFA acutely. The authors
conclude that acute particulate matter exposure can provoke an acute systemic thrombogenic
response associated with pulmonary injury in cardiovascular compromised rats.
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Another study demonstrated cardiac lesions with inflammation and degeneration in rats
exposed for 16 weeks to 10 mg/m3 of oil combustion-derived particles containing bioavailable
zinc (Kodavanti et al., 2003).
Nevertheless, the possible mechanisms involved in the alleged role of particulate matter and
diesel exhaust in particular, on various cardiovascular events remain unknown.
II.5. Effects on reproduction and development
Recent reports have indicated a decrease in semen quality of men in some countries, in
Europe in particular (Jorgensen et al., 2001). Several environmental chemicals may affect the
male reproductive system.
Recent studies have demonstrated that organic extracts of diesel exhaust particles possessed
estrogenic or anti-estrogenic activities (Mori et al., 2002; Taneda et al., 2000; Taneda et al.,
2002). Extracts of diesel exhaust may be thus capable of affecting human health by disrupting
normal endocrine function through interaction with hormone (estrogen in that case) receptors.
The effects of diesel particulate material extracts on human spermatozoa were studied using
an in-vitro system (Fredricsson et al., 1993). Diesel extract interfered with sperm motility in a
dose-response fashion. The initial effects were moderate and mainly restricted to percent
motile sperm but upon exposure to 18 hours the effects became more pronounced and affected
all the movement variables (velocity, linearity, movement amplitude).
In animals, experiments were conducted to determine whether diesel engine exhaust affects
reproductive endocrine function in male growing rats (Watanabe and Oonuki, 1999). The rats
were assigned to three groups: a group exposed to total diesel engine exhaust containing
5,630 µg/m3 particulate matter, 4.10 ppm NO2 and 8.10 ppm NO; a group exposed to filtered
exhaust without particulate matter; and a group exposed to clean air. Dosing experiments
were performed for 3 months beginning at birth (6h/day, 5days/week). A significant decrease
in pituitary gland hormones (FSH, LH) and an increase in serum levels of sexual hormones
(testosterone and estradiol) were observed. Although testis weight did not show any
significant difference among the groups, sperm production and maturation were affected.
Because these effects were not inhibited by filtration, the gaseous phase of the exhaust
appears to be more responsible than particulate matter for disrupting the endocrine system.
In a more recent study, 13 month-old rats were exposed to clean air or whole diesel exhaust at
particle concentrations of 300, 1,000 or 3,000 µg/m3 for 8 months (Tsukue et al., 2001). The
particles had a mass median aerodynamic diameter of 0.4 µm. Diesel exhaust did not
markedly affect testicular and body weights. However, diesel exhaust at 300 µg/m3
significantly decreased prostate and coagulating gland weight, accompanied by a reduction in
thymus and adrenal gland weight. In contrast, there was a significant rise in the weights of
prostate, seminal vesicles and coagulating glands in the 3,000 µg/m3 group. In rats exposed to
0.3 or 1 mg/m3, serum luteinizing hormone (LH) and testosterone increased significantly,
while a rise in testicular testosterone was noted with 3 mg/m3 particles. In conclusion, diesel
exhaust appeared to exert greater effects on accessory glands than on testes in rats.
Another study in mice suggests that diesel exhaust (300 µg/m3 particulate matter) may also
affect the testicular function by direct action on the testis (Yoshida et al., 1999).
Diesel exhaust seems to affect also fetal development. Pregnant rats were exposed to total
diesel engine exhaust containing 5,630 µg/m3 particles (90% measured less than 0.5 µm), 4.10
ppm NO2 and 8.10 ppm NO or to filtered exhaust or to clean air (Watanabe and Kurita, 2001).
The exposure period was from day 7 until day 20 of pregnancy. The main observation was the
masculinization of the fetus in total or filtered exhaust groups. This distorted fetal
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development may be the consequence of an accumulation of sexual hormones due to altered
metabolism.
To clarify the toxic effects of diesel exhaust on delivery in mice and growth of young, female
mice were exposed to 300, 1,000 or 3,000 µg/m3 diesel exhaust particles (mass median
aerodynamic diameter of 0.4 µm) or filtered air for 4 months (12 h/day, 7 days/week) (Tsukue
et al., 2002). After exposure, some females from each group were examined by necropsy and
the reminders were mated with unexposed males. Estrous female had significantly lower
uterine weight than the control estrous females. In the mated females, some of the pregnancies
resulted in abnormal deliveries (abortion and unable delivery) in diesel exhaust-exposed mice
but this was not significant. The rate of good nest construction by delivered females exposed
to 3 mg/m3 was significantly lower. Body weight of young of dams exposed to 1 and 3 mg/m 3
was significantly lower and some malformations were noted (for example a shorter anogenital
distance, a decrease in some organ weights or early opening of vaginal orifices). These results
show that toxic substances in diesel exhaust might cause abnormal delivery in mice and that
exposed females affected the growth and sexual maturation of their offspring.
A recent study has also shown that inhalation of diesel exhaust during fetal and neonatal
periods (i.e. during immune system differentiation) caused enhanced serum IgE to cedar
pollen, which could end up to greater sensitization (Watanabe and Ohsawa, 2002).
An earlier study investigated the effect of exposure to diesel exhaust on lung development
(Mauderly et al., 1987). Rats were exposed to diesel exhaust (3,500 µg/m3) or to air as a
control. One group was exposed first in utero (by exposing the mother from conception and
throughout gestation) and then from birth up to 6 months of age. Another group, representing
an adult model, was exposed between the ages of 6 and 12 months. It was found that particles
altered the airway fluid constituents and tissue collagen in both groups. In the adult group,
there was an increase in pulmonary neutrophils, a delayed clearance of particles and an
increase in lung weight. However, none of these changes were seen in rats exposed during
development. In adult rats, there was also a focal aggregation of soot-laden alveolar
macrophages but only scattered individual macrophages were found in the young rats. The
authors concluded that there was no evidence for developing rats being more susceptible to
the toxic effects of diesel exhaust.
III.
Volatile nanoparticles
Nanoparticles, as defined in the framework of Particulates, are volatile particles in the size
range below about 50 nm, not adsorbed on solid particles. The main formation mechanism in
this size range is the nucleation of condensable species present in the exhaust gas (Samaras,
2003). Humidity and hydrocarbon concentrations have a strong effect on nanoparticle
formation (Mathis et al., 2001). Using a nanodifferential mobility analyzer to size-select
nanoparticles from diesel exhaust, coupled with mass spectrometry, it has been shown that
branched alkanes and alkyl-substituted cycloalkanes from unburned fuel and/or lubricating oil
contributed most of the diesel nanoparticle mass (Tobias et al., 2001). Sulfuric acid was also
detected at estimated concentrations of a few percent of the total nanoparticle mass (Tobias et
al., 2001). According to the authors, the mechanism of nanoparticle formation should involve
nucleation of sulfuric acid (maybe ammonium sulfate) and water, followed by particle growth
by condensation of organic species (Tobias et al., 2001). In a recent work, it has been shown
that the volatile component of diesel nanoparticles is comprised of at least 95% unburned
lubricating oil (Sakurai et al., 2003).
To our knowledge, no information regarding the potential toxicity of such nanoparticles is
currently available.
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Some studies have examined health effects of sulfuric acid aerosols in both humans (Avol et
al., 1990; Frampton et al., 1995; Linn et al., 1995; Linn et al., 1997; Tunnicliffe et al., 2001)
and animals (Kilgour et al., 2002; Kimmel et al., 1997; Kleinman et al., 1999; Last and
Pinkerton, 1997; Uleckiene and Griciute, 1997). The main differences, however, are the lack
of condensed organic species and the droplet size, which is generally above the ultrafine range
(400 to 800 nm). In a well-conducted study, the importance of the aerosol droplet size on lung
toxicity was investigated by exposing rats for 2 days to 0.5 mg/m3 fine (aerosol mass median
diameter = 300 nm) or ultrafine (60 nm) sulfuric acid alone or in combination with ozone
(Kimmel et al., 1997). There were no differences between the ultrafine or fine acid exposure
groups. A synergistic interaction between ozone and ultrafine (but not fine) sulfuric acid
particles was found for lung tissue injury (Kimmel et al., 1997). Even if those observations
are not transposable to diesel engine nanoparticles, this study demonstrates that ultrafine
aerosols present a toxicity which is potentially more important than that of fine aerosols.
There is therefore a clear need for studies about the inhalation toxicity of diesel nanoparticles.
The main difficulty is obviously to generate such nanoparticles in the laboratory, in order to
expose animals in an environmentally representative way. This leads to the question of what
is an "environmentally representative way of exposure". Some work has been done to
characterize the physics and chemistry of diesel nanoparticles (Sakurai et al., 2003; Shi and
Harrison, 1999; Tobias et al., 2001). The formation and characteristics of these particles is
strongly dependent on the experimental setup, the diesel engine and fuel oil used and the
sampling method. There will be as many sorts of particles as there will be experiments to
generate them. It is therefore important to make measurement of those particles in different
diesel exhausts, in order to have a good idea of their permanent and variable features and also
to determine the modifying parameters (i.e. humidity, oil composition…). The first results
obtained on the physics and chemistry of diesel nanoparticles should be confirmed and
specified, especially in term of chemical composition.
Yet, it is not necessary to have a complete knowledge of diesel nanoparticle formation and
characteristics to design toxicological studies. What is important is to provide a precise
description of the equipment design and operating parameters and to give the most data on
aerosol characteristics, at least the particle mass and number concentrations, the size
distribution and (non) exhaustive chemical composition.
In studying the toxicity of diesel nanoparticles, two way of research should be undertaken.
1/ It is important to consider the aerosol alone, to determine which property(ies) of the
nanoparticles condition the observed toxicity. This implies the ability:
(a) to generate representative ultrafine organic aerosols that could be used in animal
inhalation exposure experiments – on this aspect, the particle generator of Veranth et al.
(2003) seems to be promising,
(b) to make one specific aerosol feature vary with the others remaining constant.
2/ This reductionist approach should be completed by the study of the relative toxicity of
diesel nanoparticles with respect to the whole exhaust. Two research strategies can be used
(McDonnell, 1993):
(a) the "top down" approach involves study of the mixture as a whole, with further studies of
fractions of the mixture to identify the causative agents and interactions among them –
this implies, in our case, to be able to remove selectively gas-phase and/or solid
copollutants from the whole exhaust,
(b) the "bottom up" approach involves study of the individual compounds as a first step,
followed by examination of the joint effects of mixtures of these individual compounds.
This could be done by coupling the condensation organic particle generator of Veranth et
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al. with a nuclei generator to create mixtures of organic compounds and carbon or metal
oxides (Veranth et al., 2003).
To conclude, there is a clear need for data on toxicity of diesel volatile nanoparticles. Their
physical-chemical characteristics (i.e. size, mass and number concentrations, chemical
composition) need to be confirmed but the overall knowledge of the physical-chemical
characteristics of nanoparticles is now sufficient to design toxicological studies of these
materials. The main difficulty is to control the diesel nanoparticle aerosol in a way that could
be used in animal inhalation exposure experiments.
IV.
Which properties of particles are important for their toxicity?
The relationship between particulate matter pollution and health effects has been established
for years (see § II.). But the exact biological causes of the observed effects are unclear.
Particle mass, size, composition, number of particles, their available reactive surface area,
chemical composition may be all important physico-chemical properties but to what extent
and combination is still unclear (Reynolds and Richards, 2001).
Many works are done at that time to understand which properties of particles are responsible
for noxious effects. A lot of studies are performed with various particles other than diesel
exhaust ones. However, particles of diesel soot are ubiquitous in urban air and contribute to
particulate air pollution of aerodynamic diameter 10 µm (PM10). With diesel exhaust particles
being part of ambient particulate matter (PM), the question of what is seen in ambient PM
data is of interest.
IV.1. Size parameters
Among the physical and chemical characteristics of inhaled particles, which can have a
profound effect on the nature of the toxicity produced in both laboratory animals and humans,
the particle size (and the interrelated parameters: volume, surface and number) is a major
determinant.
The mass median diameter of diesel exhaust particles is approximately 0.2 µm with over 90%
being < 1 µm (Salvi and Holgate, 1999). A recent electron microscopy study showed that
distribution of the particle sizes by number was 10.1% ultrafine (< 0.1 µm), 89.5% fine (0.12.0 µm) and 0.4% coarse (> 2.5 µm) (Bérubé et al., 1999).
Information regarding the role that particle size have in PM toxicity has come from studies
employing laboratory-derived surrogate insoluble particles such as mineral oxides (TiO2),
cobalt and carbon black (CB) (Li et al., 1999; Oberdorster et al., 1994; Osier and Oberdorster,
1997; Zhang et al., 2000). These studies have shown that on an equivalent mass exposure
dose metric, ultrafine particles (14-21 nm) have a greater ability than fine particles (250320 nm) to induce acute and persistent lung injury.
This mainly appears to be due to the large surface area available on smaller-sized particles
(Salvi and Holgate, 1999). In fact, more health effects were observed for particles with high
surface area when compared to particles of similar composition but having less surface area
(Brown et al., 2001; Hohr et al., 2002; Lison et al., 1997). As particles get smaller, their
surface area for the same mass becomes greater, and hence their capacity to carry toxic
substances and free radicals increases (Salvi and Holgate, 1999). More free radicals were
detected in ultrafine particulate samples compared with coarser samples of the same substance
(Donaldson et al., 1998; Zhang et al., 1998). This is generally due to an increased surface area
but other factors can contribute to this. For example, the free radical activity of ultrafine TiO2
(size  20 nm) was found much more higher than those of commercial TiO2 (size  250 nm)
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but the authors could not explain this difference on the basis of relative surface areas
(Gilmour et al., 1997). According to a report by Concawe, it is not surprising since
commercial TiO2 is often surface-coated with other materials such as silicon and binders
while the ultrafine TiO2 used by virtually all investigators is Degussa P25, a specially
prepared ultrafine material with high surface oxidant-catalytic activity (Hext et al., 1999).
Anyway, these differences in themselves add further to the evidence that surface properties of
insoluble particles influence their biological activity and in particular, the differences in their
ability to cause lung inflammation can be explained on the basis of different amount of free
radical activity. The resulting cellular oxidative stress may lead to an impairment of
macrophage phagocytosis. Decreased phagocytosis could allow enhanced interactions
between ultrafine particles and the epithelium, which occur in any case because of the sheer
number of particles. This leads to pro-inflammatory cytokine production by macrophages
because of oxidative stress from the surface of the ultrafine particles and chemokine
production by the epithelium (figure 5) through similar pathways. Increased interstitial
transfer of particles may also arise because of interactions between particles and epithelial
cells (Donaldson et al., 2001). It should be noted that under the experimental conditions used
in most studies, the ultrafine particles are generally aggregated (Hext et al., 1999). The ability
to translocate into the epithelium with resultant inflammatory response is related to the extent
to which these will deaggregate into primary particles once deposited (Hext et al., 1999). This
may explain, in part at least, the differences between pulmonary translocation of ultrafine
carbon black and ultrafine TiO2 particles (Hext et al., 1999).
Figure 5. Diagrammatic representation of the hypothetical events after exposure to ultrafine particles (right)
compared with fine particles (left). The essential elements of the ultrafine response are many particles outside
and inside macrophages. Release of mediators from the macrophages and epithelial cells due to activation of
signaling pathways mediated by oxidative stress, may then lead to inflammation. The enhanced interaction of
particles with the epithelium leads to their transfer to the interstitium (Donaldson et al., 2001).
The potential toxicological importance of particle number has only recently been
recognized. The first published studies, dealing with particle number, show that it may be an
important driver of health effects. In a study from Germany (Peters et al., 1997), the number
and mass concentration of PM in the range of 0.01 to 2.5 µm was determined during the
winter season. Most of the particles (73%) were in the ultrafine fraction (< 0.1 µm), whereas
most of the mass (82%) was attributable to particles in the size range of 0.1 to 0.5 µm. In this
study, adverse respiratory effects, in a group of patients with asthma, were associated with the
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number of ultrafine particles. Similar findings were made in a study in Finland, in that the
daily mean number concentration of particles was dominated by the ultrafine particles. The
daily mean number concentration of particles, but not particle mass (PM10, PM2.5-10, PM2.5,
PM1), was associated with daily deviation in peak expiratory flow. The strongest effects were
seen for particles in the ultrafine range (Penttinen et al., 2001). However, no association was
found in patients with chronic airflow obstruction, between symptoms, peak flow rate and the
number of ultrafine particles (Osunsanya et al., 2001).
The effect of different physical particle characteristics was investigated in a series of
experiments in which the total dose-weight, particle size, total particle number, or total
surface area of particles were kept constant, by the use of well-characterized, spherical
polystyrene particles (PSP) (Granum et al., 2000). Mice were given two intraperitoneal
injections with ovalbumin plus different doses of PSP (it was not possible to use diesel
exhaust particles since they have a tendency to form aggregates of varying shapes and sizes).
The serum level of allergen-specific IgE increased with both an increasing number and
increasing surface area of PSP, whereas there seemed to be no covariation between the doseweight and the level of allergen-specific IgE. There were no clear associations between the
levels of IgE and the size of PSP, but according to the authors, this may be due to the
relatively small size range of PSP. These findings indicate that the total number and total
surface area of PSP, rather than the dose weight, are important parameters for the IgE
adjuvant activity from PSP, and possibly also for particles in general.
These studies illustrate the fact that there may be a large number of small particle that hardly
contribute to the total mass and still have important biological effects. Weight measurement
alone, therefore, leave out possibly important physical characteristics of PM, such as the
number concentration, size distribution, and the total surface area, even if the relative
contribution from the different "size particle properties" is difficult to assess since they are
closely related to each other (Granum and Lovik, 2002). When particles have a smooth
surface and a simple shape, their approximate geometric surface area can be calculated, but
the estimation of the true surface area becomes more complicated when the particles have a
complex shape. Measurements of the total surface area in an automated way are, therefore,
virtually impossible to perform, and other measures of particles must be sought (Ayres, 1998).
Since both the size distribution and number concentration of particles can be measured in an
automated way (e.g. by using a photon correlation spectrometer), this procedure may be an
appropriate alternative to the measurement of the total surface area (Granum and Lovik,
2002).
IV.2. Chemical composition
Diesel exhaust particles consist of a carbonaceous core similar to carbon black, onto which an
estimated number of 18 000 different high-molecular-weight organic compounds are adsorbed
(Salvi and Holgate, 1999). Diesel exhaust, in addition to particles, contains a complex mixture
of gases such as carbon monoxide, nitric oxides, sulfur dioxide, hydrocarbons, formaldehyde,
transition metals and carbon particles (Sydbom et al., 2001). An electron probe X-ray microanalysis demonstrated the presence of C, O, Na, Mg, K, Al, Si, P, S, Cl and Ca along with a
range of metals (Ti, Mn, Fe, Zn, Cr) that were heterogeneous in distribution (Bérubé et al.,
1999).
It is not entirely clear which diesel exhaust particle components produce toxicity. Some
studies suggest that the majority of the toxicity is attributable to the adsorbed organic
compounds (Boland et al., 1999b; Ohtoshi et al., 1998; Sagai et al., 1993; Yang et al., 1999),
whereas others conclude that the most toxic portion of a diesel particle is the carbonaceous
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core (Lovik et al., 1997; Samet et al., 2000). Most likely and as developed below, there must
be a combined effect between these two factors.
Effects of particles per se
Several studies, using different model particles such as polystyrene, carbon black, amorphous
silica, Teflon or titanium dioxide, indicate that particles per se can exert toxic effects
independent of adsorbed chemical substances found on environmental particles (reviewed in
Granum, 2002).
Effects of particle-associated compounds
Perhaps the best research outcome, which could be hopes for, would be the identification of a
given minor chemical component of particulate matter, which is solely responsible for adverse
effect on health. However, the UK Department of Health Committee on the Medical Effects
of Air Pollution concluded in 1995 that no known chemical substance is of sufficient toxicity
given the current levels of exposure to PM to explain the observed magnitude of health effects
(Harrison and Yin, 2000). Epidemiological studies, investigating the connection between total
mortality and PM10, are remarkably consistent irrespective of where they are carried out,
which also argue against chemistry having an especially important influence (Harrison and
Yin, 2000). On the other hand, it is difficult to imagine that chemical composition does not
play a role (Harrison and Yin, 2000) and experimental evidence of this is developed below.
Inorganic constituents
Inorganic constituents of airborne PM such as sulfate, nitrate, ammonium and metals
represent potential causal constituents for PM-associated adverse health effects (Dreher,
2000). Major components such as sulfate, nitrate, ammonium or chloride do not appear to
affect toxicity strongly (Harrison and Yin, 2000) but nevertheless, acute exposure of mice to
sulfate-coated carbon black was found to impair alveolar macrophage phagocytosis and
intrapulmonary bactericidal activity (Clarke et al., 2000; Jakab et al., 1996).
It has long been recognized that some trace metals such as lead, cadmium and mercury are
highly toxic in sizeable doses, but exposures through inhalation of urban airborne PM in the
developed world are likely to be wholly insufficient to cause toxic effects through classical
mechanisms of toxicity (Harrison and Yin, 2000).
However, some works have suggested that transition metals, and particularly iron, may have
adverse effects through non-classical mechanisms such as contributing to the production of
hydroxyl radicals through the Fenton reaction (Donaldson and MacNee, 2001; Gilmour et al.,
1996; Wilson et al., 2002). In particular, extensive research in combustion emission
particulates, residual oil fly ash (ROFA), which is rich in metals (including iron, vanadium
and nickel) with little organic component, has been carried out. These studies have provided
direct evidence that the soluble transition metal component, especially vanadium, promotes
lung injury in animals and humans (Ghio et al., 2002).
The potential toxicity of various metals found in urban particulate dusts has been investigated
in mice (Prieditis and Adamson, 2002). Solutions of metal salts (Zn, Cu, V, Ni, Fe, Pb) were
instilled to mouse lung at the same concentration as Zn EHC-93 dust content (i.e. 4.8 µg/mg
in 0.1 mL). It was shown that Zn, and to a lesser extent Cu induced lung injury and the
magnitude of response was similar to that seen after administering the dust at 1 mg/0.1 mL.
For that particular dust, the results indicate that Zn and Cu are most likely to cause lung injury
and inflammation as compared to metals such as Ni, Fe, Pb and V at the same concentrations.
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Numerous in vitro toxicology studies have also demonstrated the ability of deferoxamine, a
metal chelator, to inhibit a number of biological responses induced by ROFA and air PM,
implicating particle-associated metals as causal properties of these particles in a variety of in
vitro biological effects (reviewed in Dreher, 2000). This supports metals as a potential causal
property for PM-associated health effects, even if epidemiological data to support that
transition metals at realistic ambient concentrations are related to health endpoints are still
largely lacking (Brunekreef, 2000).
In particles that are carbonaceous (like diesel exhaust particles), concentrations of metals can
be extremely low. However, it has been shown that exposure to diesel exhaust particles with
low content of metals, resulted in an accumulation of biologically active iron in the rat lung,
with both oxidative stress and lung injury (Ghio et al., 2000).
It must be underlined that specific trace metal elements, such as platinum, rhodium or
palladium for example, may be found on diesel exhaust particles, due to engine wear,
lubricant and catalyst. Because of their belonging to the transition metal family, these
elements should hold toxicologist attention. Spare data exist on mostly platinum salts and
further works are requested to study the valence state and the solubility of these compounds
when adsorbed to particles and their potential role in the toxicity of PM. Due to its role in
adverse health effects mediated by ROFA, vanadium (and especially vanadium pentoxide
which is widely used as a catalyst for a variety of reactions) is also of interest. A recent study
has shown that this compound is a pulmonary carcinogen in rats and mice (Ress et al., 2003).
Organic constituents
Information on the potential role of organic constituents (especially PAH) as contributors to
PM-associated adverse health effects has come from studies examining the toxicology of
diesel exhaust particles.
PAH extracted from diesel exhaust particles and other PAH (e.g., pyrene, phenanthrene),
often adsorbed to diesel exhaust particles, have been found to have several effects on allergic
immune responses, including upregulation of IL-4 production (Bommel et al., 2000),
increased production of IgE (Suzuki et al., 1993; Takenaka et al., 1995; Tsien et al., 1997),
and induction of inflammatory responses (Fahy et al., 1999; Terada et al., 1997).
However, these results must be taken cautiously. Indeed, an important consideration in
estimating the potential dose of PAH delivered by inhaled diesel particles is the bioavaibility
of these compounds. When working on organic extracts, chemical constituents are forced to
be desorbed from the particle core and are therefore highly bioavailable. Moreover,
considering carcinogenic effects, the chemicals adsorbed onto the particles may not be as
mutagenic in vivo as they are in most in vitro assays because the material extracted using
physiological fluid such as saline or serum is generally less genotoxic than that extracted
using the non-physiological organic solvents employed in most in vitro assays (Cohen and
Nikula, 1999).
Many other factors affect the bioavailability of organic constituents from diesel exhaust
particles. The degree of particle agglomeration is one determinant of the release of organic
chemicals. There may be a greater degree of agglomeration with intratracheal instillation and
high exposure concentrations, used in animal bioassays, than of particles associated with
typical environmental exposures (Cohen and Nikula, 1999). This may explain the
contradictory results observed between instillation and inhalation studies (Osier and
Oberdorster, 1997). In the latter, ADN adducts were observed in rats exposed by inhalation
either to diesel exhaust particles (organic compounds 30%) or to carbon black (organic
compounds 0.04%), suggesting a major role of the carbonaceous core (Bond et al., 1990).
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This was also observed by Gallagher (Gallagher et al., 1994). On the contrary, studies by
instillation showed that diesel exhaust particles with no adsorbed organic chemicals induced
less tumors than native diesel exhaust particles (Dasenbrock et al., 1996; Iwai et al., 1997).
Other studies addressing specifically the question of bioavailability of chemicals adsorbed on
diesel exhaust particle should be undertaken.
Effects of particle core per se versus adsorbed chemical substances
The effects of environmental particles are probably not due to the particle core only or to the
adsorbed chemical substances only, because both of these components appear to contribute to
adverse health effects (Granum and Lovik, 2002).
Exposure of rats to intratracheally administered diesel exhaust particles induced lung
inflammation and injury which were not substantially different from those elicited by carbon
black or silica (Yang et al., 1999). But only diesel exhaust particles suppressed alveolar
macrophage cytokine release in response to lipopolysaccharide (a bacterial endotoxin)
stimulation. The contrasting cellular response with respect to diesel exhaust particles and
carbon black exposures may be due to the presence of adsorbed organic compounds on diesel
exhaust particles, which may contribute to the increased susceptibility of hosts to pulmonary
infections after diesel exhaust particle exposure.
Particles may have also the ability to carry adsorbed material to regions of the lung were they
might not normally reach and possibly induce effects not associated generally with the core
particle (Hext et al., 1999). Carbon black particles (10 mg/m3) were co-generated with
10 ppm SO2 in order to assess the ability to form H2SO4 and hence act as a potential carrier
for this into the lungs (Hemenway et al., 1996). At relative humidity up to 60%, 4 µg SO42- /
mg carbon black was formed whereas at 85% humidity, this increased to 13.7 µg SO42- / mg
carbon black. The impairment of alveolar macrophage phagocytosis was only significant at
85% humidity. The authors concluded that fine carbon particles could be an effective vector
for delivery of toxic amounts of SO42- to the periphery of the lung under conditions of
elevated relative humidity. Carbon black particles were also used for co-exposure of mice
with acrolein vapour, which is normally absorbed in the upper respiratory tract regions (Jakab
and Hemenway, 1993). Exposure during 4 days (4 hours/day) to 10 mg/m3 carbon black and
2.5 ppm acrolein resulted in effects in the lower respiratory tract regions, characterized by
alteration of alveolar macrophage properties (phagocytosis and LPS-induced TNF-
production) in co-exposed animals only.
Attempts have been made to evaluate the relative importance of the particle core versus
specific adsorbed chemicals to the adjuvant activity of diesel exhaust particles. It was shown
that both diesel particles and carbon black injected in the footpad in conjunction with
ovalbumin can have an adjuvant activity of popliteal lymph node inflammation and systemic
ovalbumin-specific IgE in mice. This effect was however slightly lower for carbon black
(Lovik et al., 1997). These results suggest that the particle core contributes to the adjuvant
activity of diesel exhaust particles. A similar observation was made after injection of
ovalbumin plus diesel particle extracts or insoluble diesel particles (the remaining part of
particles after extraction) to mice (Heo et al., 2001). Although the diesel particle organic
extract induced a significantly increased production of allergen-specific IgE compared to mice
given ovalbumin alone, the adjuvant effect was less than that elicited by the insoluble diesel
particle core. In another study, Kanoh et al. demonstrated that both pyrene, fluoranthene,
anthracene, benzo(a)pyrene and diesel particles have adjuvant activity on allergen specific
IgE antibody production when mice are immunized by intraperitoneal injection of ovalbumin
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or Japanese pollen cedar allergen (Kanoh et al., 1996). These results suggest that chemical
compounds contained in diesel soot can also have adjuvant activity in mice.
These three studies indicate that a larger proportion of the adjuvant effect of diesel exhaust
particles is associated with the particle core, with an additional contribution from adsorbed
chemical.
V.
Conclusion
This review has focused on the existing knowledge, but also on the gaps, in the field of health
effects of diesel emissions. These latter are a complex mixture of gases, vapors, semivolatile
organic compounds and solid (soot) or volatile particles.
If the toxicology of soot particles and total exhaust is well documented, fewer data are
available concerning ultrafine diesel exhaust particles (commonly considered to be 100 nm or
less) and particularly nanoparticles (commonly considered to be 50 nm or less), which are
composed of condensed organic material and sulfur compounds having little or no elemental
carbon content (Mauderly, 2001). Attention has been drawn to ultrafines by data suggesting
that emissions of these particles may increase as mass emissions of soot are reduced (Bagley
et al., 1996) and by studies showing an inverse relationship between particle size and health
responses (Mauderly, 2001). Laboratory research on ultrafines has focused almost exclusively
on solid, poorly soluble particles. Toxicological data are needed on particles comprised
largely of organic compounds, perhaps condensed on sulfuric acid nuclei that are droplets
when inhaled. Even if additional studies are requested to confirm and generalize the first
results obtained on the physical-chemical characteristics of such particles, the knowledge is
now sufficient to design toxicological studies of these materials (Mauderly, 2001). The main
difficulty is to create an environmentally relevant diesel nanoparticle aerosol that could be
used in inhalation studies. It is also important to determine the contribution of diesel
nanoparticles in the toxicity of the whole exhaust.
Toxicological studies have provided supportive evidence that certain particle characteristics
such as size and organic and metal constituents elicit biological responses in humans and
animals, which has enhanced their plausibility of being the properties responsible for PMassociated health effects (Dreher, 2000).
Nevertheless, at this time, there are considerable gaps in our knowledge about the way in
which the surface of particles might react with biological media. We do not know which
molecules bind to which particle layers or, indeed, how quickly such molecules may change.
We understand only poorly how close to the surface of a particle a cell can come and whether
a particle surface could influence cell differentiation and activity. We need to understand
more about the chemical composition of the surface of particles and also try to define how
important the microstructure and topography of the particle surface may be in term of
permitting differing degrees of chemical reaction at the particle surface or indeed, the way in
which such a surface may govern cellular responses (Ayres, 1998).
At this stage of knowledge, it seems that several physical or chemical properties of particles
are responsible for their adverse effects on health. In order to improve our assessment of
health effects of particulates, we must therefore, consider complementary ways to the
gravimetric measure of particles, for measuring the exposure of an individual. The metrics
above could be used to complete the mass concentration measurement:
a/ the number concentration,
b/ the size distribution,
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c/ the surface area,
d/ the ratio solid particles (soot) / volatile nanoparticles, which can be completed by
measurement of particle volatility and hygroscopicity; these latter parameters may also be
useful in the determination of the toxicity mechanisms,
e/ the redox potential, i.e. the capacity of the exhaust to generate free radicals, which are
recognized to play a role in particle-mediated toxicity.
Different measures of particle may then reflect different aspects of health effect (Ayres,
1998).
In that perspective, the work done in Particulates provides significant advances in
measurement of automotive exhausts. The main innovation is the split of the flow to two
branches, a "dry" branch and a "wet" branch, which allow real-time characterization of
respectively solid (i.e. that do not evaporate at 250°C) and volatile particles (Samaras, 2003).
Particle properties recorded include total active surface equivalent, size segregated solid
particle number, total particle number, gaseous pollutants, number weighted size distribution,
particulate matter mass and particle mass distribution (Samaras, 2003). These characteristics
are completed by chemical analysis (metals and organic compounds). This measurement
system should now benefit toxicological studies, in order to get further information on the
role of particle properties in their toxicity.
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