Hindawi Publishing Corporation
Journal of Toxicology
Volume 2012, Article ID 782462, 23 pages
TheAdverseEffectsof AirPollution ontheNervous System
1Department of Neuroscience, Health Science Institute, Dokuz Eylul University, Inciralti, 35340 Izmir, Turkey
2Department of Molecular Biology and Genetics, Bogazici University, 34342 Istanbul, Turkey
Correspondence should be addressed to Kursad Genc, email@example.com
Received 30 May 2011; Accepted 15 November 2011
Academic Editor: Cinta Porte
Copyright © 2012 Sermin Genc et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Exposure to ambient air pollution is a serious and common public health concern associated with growing morbidity and
mortality worldwide. In the last decades, the adverse effects of air pollution on the pulmonary and cardiovascular systems have
been well established in a series of major epidemiological and observational studies. In the recent past, air pollution has also
been associated with diseases of the central nervous system (CNS), including stroke, Alzheimer’s disease, Parkinson’s disease, and
neurodevelopmental disorders. It has been demonstrated that various components of air pollution, such as nanosized particles,
can easily translocate to the CNS where they can activate innate immune responses. Furthermore, systemic inflammation arising
from the pulmonary or cardiovascular system can affect CNS health. Despite intense studies on the health effects of ambient air
pollution, the underlying molecular mechanisms of susceptibility and disease remain largely elusive. However, emerging evidence
suggests that air pollution-induced neuroinflammation, oxidative stress, microglial activation, cerebrovascular dysfunction, and
alterations in the blood-brain barrier contribute to CNS pathology. A better understanding of the mediators and mechanisms will
enable the development of new strategies to protect individuals at risk and to reduce detrimental effects of air pollution on the
nervous system and mental health.
Air pollution collectively describes the presence of a diverse
and complex mixture of chemicals, particulate matter (PM),
or of biological material in the ambient air which can cause
harm or discomfort to humans or other living organisms.
The sources of air pollution can either be natural (e.g., vol-
canic eruptions) or manmade (e.g., industrial activities), and
air pollution emerges as a serious health problem especially
in rapidly growing countries. Millions of people worldwide
are chronically exposed to airborne pollutants in concen-
trations that are well above legal safety standards . There-
fore, morbidity and mortality attributable to air pollution
continue to be a growing public health concern worldwide.
Air pollution ranks eighth among the leading risk factors for
mortality and accounts for 2.5% of all deaths in developed
countries . The World Health Organization (WHO) es-
timates that air pollution is responsible for over 3 million
premature deaths each year . Epidemiological and obser-
vationalstudiesidentified a stronglink betweentheexposure
to contaminants in the ambient air and adverse health out-
air pollutants has been associated with marked increases in
cardiovascular disease morbidity and deaths resulting from
myocardial ischemia, arrhythmia, heart failure, and respira-
tory diseases such as lung cancer and asthma [3, 4].
About a decade ago, the central nervous system (CNS)
has also been proposed to be a target organ for the detrimen-
tal effects of airborne pollutants . Indeed, emerging evi-
dence from recent epidemiological, observational, clinical,
and experimental studies suggest that certain neurological
diseases, such as Alzheimer’s disease (AD), Parkinson’s dis-
ease (PD), and stroke, may be strongly associated with ambi-
ent air pollution.
Mechanistically, air pollution may affect the nervous
system through a variety of cellular, molecular, and inflam-
matory pathways that either directly damage brain structures
or lead to a predisposition to neurological diseases. Although
ischemic stroke (chronic exposure to ambient air pollution),
multiple sclerosis (MS, exposure to second-hand smoking),
the only neurological disorders for which a strong link to
ambient air pollution has been established, it is not unlikely
2 Journal of Toxicology
It has been suggested from epidemiological and obser-
vational studies that exposure to airborne pollutants can
contribute to neurodegenerative disease processes already
from early childhood on, especially if the individuals are
chronically exposed to the contaminants [1, 9–11]. Air pol-
lutants affect the CNS either directly by transport of nano-
sized particles into the CNS or secondarily through systemic
inflammations. Either of the effects can be caused by the
physical characteristics of the particle itself or by toxic com-
pounds that adsorb on the particles [12, 13]. Although the
pollution are not fully understood, several lines of current
evidence point out that neuroinflammation, oxidative stress,
glial activation, and cerebrovascular damage might be the
primary pathways [1, 14].
In this paper, we provide an overview of the different
classes of air pollutants and their potential ways to entry by
which they could get into contact with the CNS. We sum-
marize findings of epidemiological, observational, clinical,
and experimental studies which describe a link between air
pollution and neurological diseases or neurodevelopmental
disturbances. Finally, we summarize the current understand-
ing of the adverse effects of air pollutants on the nervous
system and mental health on a cellular and molecular level.
2.Components of AirPollution
Air pollution represents a diverse mixture of substances in-
cluding PM, gases (e.g., ground-level ozone, carbon monox-
ide, sulfur oxides, and nitrogen oxides), organic compounds
(e.g., polycyclic aromatic hydrocarbons and bacterial endo-
toxins), and toxic metals (e.g., vanadium, lead, nickel, cop-
per, and manganese) that can be found in outdoor and
organic compounds, appear to be the most widespread and
harmful components. Of those, PM is especially relevant for
nervous system damage and can be found as a mixture of
solid particles and liquid droplets, that are suspended in the
air . Most individual components of atmospheric PM are
not especially dangerous and some major constituents, such
as sodium chloride, are harmless .
PM is characterized by its size and aerodynamic property
which is directly related to its biological effects. For instance,
only particles less than 10μm in diameter can be inhaled
deep into the lungs, whereas larger particles usually get
trapped in the upper airways. Generally,coarseparticles with
an aerodynamic diameter of 2.5 to 10μm (PM10), fine
Road and agricultural dust, tire wear emissions, products
of wood combustion, construction and demolition works,
and mining operations are the primary sources of PM10.
processing facilities, tailpipe and brake emissions, residential
fuel combustion, power plants, and wild fires . They
are formed from gas and condensation of high-temperature
vapors that are formed during combustion and industrial
activities. PM2.5can be composed of both organic and inor-
ganic compounds, including sulfates, nitrates, carbon, am-
monium, hydrogen ions, lipopolysaccharide (LPS), metals,
and water . Diesel exhaust particles (DEPs), however, are
the major components found among ambient fine particles.
UFPs are mostly combustion-derived NPs, which can
be produced by internal combustion engines, power plants,
incinerators, and other sources of thermodegradation. They
can carry soluble organic compounds, polycyclic aromatic
hydrocarbons, and oxidized transition metals on their sur-
face . UFPs have distinct features that render them more
dangerous than other PMs. For instance, they have been
responses . Although the effects of UFPs have been
evidence that the size of the particles is negatively correlated
with their adverse health effects .
Indeed, ambient UFP concentrations are found to be
directly correlated with mortality . Current national air
However, when compared to fine particles at similar mass
concentrations in the air, UFPs are much more numerous
and have a larger combined surface area, enhanced oxidant
capacity, greater inflammatory potential, and higher pul-
monary deposition efficiency [16, 17, 21, 22]. A major risk of
UFPs arises from the fact that they are not filtered out during
their passage through the nose and bronchioles but are able
to penetrate deep into the lung where they eventually enter
the blood circulation and can get distributed throughout the
3.Entryof AirPollutantsintothe Central
Sustained exposure to significant levels of airborne UFPs,
PM, and LPS may result in the direct translocation of
these pollutants to the CNS where they can result in neu-
ropathology through a variety of pathways and mechanisms
(Figure 1). Alternatively, air pollutants might not enter the
CNS directly, but could exert adverse effect on the CNS
by triggering the release of soluble inflammatory mediators
from primary entry organs or secondary deposition sites.
the susceptibility for neuroinflammation and neurodegener-
ation in the CNS.
Once taken up by the body, fine PM or NPs could rapidly
enter the circulatory system with the potential to directly
affectthe vascularsystem.For instance,NPs couldbe inhaled
and cross the alveolar-capillary barrier in the lungs. The
ability of NPs to cross this barrier is influenced by a number
of factors that include the size of the particles, their charge,
their chemical composition as well as their propensity to
form aggregates. Even though the translocation of inhaled
or instilled NPs across the alveolar-capillary barrier has been
clearly demonstrated in animal studies for a range of NPs
[23, 24], it has been more difficult to directly demonstrate
this mechanism in humans .
Journal of Toxicology3
Neuronal transport (?)
TNFα, IL-1β, IL-6
Figure 1: The impact of air pollution on the brain through multiple pathways.
Regardless of the route of entry, NPs that reach the
creating local oxidative stress or by causing proinflammatory
effects similar to those seen in lung tissue. Inflammatory
mediators that are produced in the respiratory tract as a
consequence of chronic pollutant-induced epithelial and
endothelial injury can lead to systemic inflammation .
The systemic inflammation is accompanied by the produc-
tion of proinflammatory cytokines such as tumor necrosis
factor alpha (TNFα), interleukin-6 (IL-6), and interleukin-
1beta (IL-1β), for which blood vessels in the brain exhibit
constitutive and induced expression of receptors [1, 26].
The cytokines could thus activate cerebral endothelial cells,
disrupt the blood-brain barrier (BBB) integrity, or trigger
signaling cascades that lead to the activation of mitogen-
activated protein (MAP) kinase, and nuclear factor kappa B
(NFκB) transcription factor-mediated pathways. Disruption
of the BBB could then be followed by trafficking of mast
cells and inflammatory cells expressing CD163, CD68, and
HLA-DR to the damaged site . In addition, circulating
cytokines that are released by inflamed peripheral organs or
endothelial cells could stimulate peripheral innate immune
cells, activate peripheral neuronal afferents, or enter the
condition synergistically [27, 28]. Accordingly, brain tissue
samples from individuals residing in highly polluted areas
show an increase in the number of infiltrating monocytes
or activated microglia, increased expression of IL-1β, BBB
damage, endothelial cell activation, and brain lesions in the
prefrontal lobe [10, 11].
directly by activating the brain’s innate immune system. The
effect of LPS on neuroinflammation is well studied in a
bacterial endotoxin/LPS-based experimental model of PD
that constitutes an important tool to delineate the mecha-
nisms of neuroinflammation-mediated loss of dopaminergic
neurons . This system could also be exploited in combi-
nation with exposure to other environmental toxins and air
pollutants. Brain uptake of circulating LPSs is usually low,
and most effects of peripherally administered LPS are likely
to be mediated through LPS receptors located outside the
BBB . Thus, LPSs might stimulate afferent nerves, act at
circumventricular organs, or alter the permeability of the
BBB. Circumventricular organs are specialized brain struc-
tures located around the third and fourth ventricle. They are
highly vascularised and lack a BBB; therefore, they allow for
a direct uptake of chemicals circulating in the blood stream
by neuronal cells .
The very small UFPs on the other hand easily penetrate
cell membranes because of their large surface-to-volume
ratio, which also enables them to traverse the classical bar-
riers in the lung and the brain. Their ability to cross cell
membranes easily explains why PM can be found inside neu-
rons or erythrocytes [1, 32]. It has also been proposed that
the close contact between endothelial cells and erythrocytes
could represent a route for the exchange of PM between
4 Journal of Toxicology
activated endothelial cells and UFP-loaded erythrocytes [1,
Another important and more direct route for UFPs to
enter the nervous system is through the olfactory mucosa,
which is a neuronal epithelium that is in direct contact with
the environmental air [35–37]. Thus, fine and UFPs may
reach the brain through olfactory receptor neurons or the
trigeminal nerve. Olfactory receptor neurons are bipolar
epithelium is covered by a layer of sustentacular cells, but
olfactory sensory neurons extend their dendrites into the
mucous layer covering the olfactory epithelium where they
directly interact with odorants inhaled with the air. Nasally
inhaled pollutants that reach the olfactory mucosa could
enter the cilia of olfactory receptor neurons by pinocytosis,
simple diffusion, or receptor-mediated endocytosis. Once
incorporated into sensory neurons, they could be trans-
ported by slow axonal transport along the axons to the olfac-
tory bulb . From there, pollutants could be transported
the olfactory bulb to multiple brain regions, including the
olfactory cortex, the anterior olfactory nucleus, the piriform
cortex, the amygdale, and the hypothalamus.
Accordingly, UFPs have been observed in human olfac-
tory bulb periglomerular neurons and trigeminal ganglia
capillaries . Similarly, a decreasing gradient of metal
(vanadium and nickel) deposition and accompanying tissue
damage from the nose to the brain has been reported in
the canine nervous system, confirming the importance of
the nasal route for the entry of air pollutants into the CNS
. Controlled exposures of rats to UFPs and metals also
demonstrated their accumulation in the olfactory bulb [40–
42]. Taken together, these findings suggest that NPs can be
taken up directly by the olfactory mucosa and enter the CNS
. Uptake through the nose might even be enhanced
by additional pollutant-induced systemic inflammation by
deteriorating the olfactory mucosal barrier, which would
result in increased neuropathology.
Additional direct neuronal entry routes for NPs have
been described that involve the retrograde and anterograde
transport in axons and dendrites such as the transport of
inhaled NPs to the CNS via sensory nerve fibers that inner-
vate the airway epithelia . Ground-level ozone exposure
activates the CNS through the vagal nerves without the
involvement of the thoracic spinal nerves . PM-related
LPS is likely to play an important role in these pathways, as
shown by vagal upregulation of CD14 .
Even though the translocation rate of NPs from their site
in the brain as a secondary target organ in significant
amounts . Thus, it is also important to obtain data on
the retention characteristics of NPs in both primary and
secondary target organs, including associated elimination
and clearance pathways . With regard to the CNS, no
data on NP elimination are available yet. It is conceivable,
however, that CSF circulation provides an excretory pathway
for NPs that enter via neuronal uptake. Usually, the CSF
serves as a fluid cushion for the brain, but also distributes
substances to all brain regions and acts as an elimination
route for metabolic waste products . NPs could be elimi-
nated from the CSF through the same mechanisms: uptake
of CSF by the blood circulatory system through arachnoid
vili or via the nasal lymphatic system. The exact details of NP
clearance from the brain, however, await future investigation
Results about the direct effects of air pollutants and airborne
particles on neuronal cells have been reported from experi-
mental studies in vitro, using cell culture systems and in vivo,
as from epidemiological and controlled clinical studies in
4.1. Experimental Studies
4.1.1. In Vitro Studies. A variety of in vitro studies assessed
uring changes in cell viability, alterations of apoptosis, the
dysfunction of mitochondria, the production of reactive
oxygen species (ROS), or the production of pro-inflamma-
tory cytokines as sensitive identifiers . Varying degrees
of proinflammatory- and oxidative stress-related cellular
responses and decreased cell viability were reported upon
bient air particles in different cell culture systems . Of
ial cell lines or primary cultures of those cells that were ex-
posed to concentrated ambient air particles (CAPs), diesel
exhaust particles (DEPs), toxic gases, such as ozone, bacterial
endotoxins, such as LPS, or toxic elements, such as man-
cell types were shown to be targets of the toxic effects of
air pollutants [46–48]. However, the underlying mechanisms
could be rather complex, and some insight into the inter-
action of different cell types was derived from coculture
of DEPs on dopaminergic neurons could be either direct
or indirect via the release of inflammatory mediators and
ROS from activated microglial cells [46, 49]. Interestingly,
pretreatment of neuron-glia cocultures with LPS increased
the vulnerability of the cells to the toxic effects of DEP, while
DEPs alone were not harmful .
An important aspect of in vitro toxicity studies is the
establishment of dose-response relationships. For instance,
low concentrations (20–40μg/mL of gas per mL of complete
medium) of oxygen-ozone were not toxic to astroglial cells,
while higher concentrations (60μg/mL) severely decreases
cell viability . Transcriptomic and proteomic profiling of
cultured cells upon exposure to CAPs may provide insights
into alterations of gene and protein expression. One such
study demonstrated the upregulation of inflammatory and
innate immunity pathway components in mouse immor-
talized BV2 cells when exposed to CAPs . Likewise,
Journal of Toxicology5
Table 1: The effects of air pollutants on neuronal and glial cells in vitro.
Species Cell type Assays Key findingsReferences
Rat VM Neuron-glia TH immunostaining
Increased microglial ROS
No DEP neurotoxicity
Increased NO production
Increased TNFα release
Increased ROS production
Increased TNFα release
Increased TNFα, IL6 release
DEPs + LPS
Upregulated inflammatory genes
OzoneRat AstrocyteIncreased lipid peroxidation
Decreased cell viability
Abbreviations: concentrated ambient particles (CAPs), 2?,7?-dichlorfluorescein-diacetate (DCFH-DA), dopaminergic (DA), diesel exhaust particles (DEPs),
enzyme-linked immunosorbent assay (ELISA), interleukin-6 (IL-6), lactate dehydrogenase (LDH), malondialdehyde (MDA) nitric oxide (NO), reactive
oxygen species (ROS), tyrosine hydroxylase (TH), tumor necrosis factor alpha (TNFα), ventral mesencephalic (VM).
the expression profiles of microRNAs, which emerged as
crucial mediators of posttranscriptional gene regulation,
might change during exposure to air pollutants . Indeed,
hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a common
environmental contaminant and explosive nitroamine that
is widely used in military ammunition, has been shown to
change brain microRNA expression in exposed mice .
(ENPs) and nanomaterials (NMs) might also contribute to
air pollution as new nanotechnologies are constantly devel-
oped, and NMs are used increasingly in daily life through the
advent of new products. In addition, ENPs are extensively
tested for their usefulness in medical diagnostic and thera-
peutic applications. Although no human ailments have been
directly attributed to NMs so far, preliminary experimental
studies indicate that NMs could initiate adverse biological
responses and that NPs could have toxicological properties
. Thus, ENPs constitute a novel neurotoxic risk and
several in vitro studies could demonstrate adverse effects of
ENPs on CNS cells (not included in Table 1). For instance,
titanium dioxide, aluminum oxide, and nanosized silica
particles were shown to decrease cell viability and to increase
apoptosis in neuronal and endothelial cell cultures [54–58].
These substances also increased the amount of ROS, which
resulted in concomitant activation of microglia [54–59].
An important point in in vitro nanoneurotoxicity studies is
as particles of different size might exert different effects or
similar effects to different degrees. In addition, a controlled
investigation of the physicochemical properties of the NPs
over time and their interactions with culture media should
also be considered [60, 61]. Although NPs in environmental
air samples might be much more heterogeneous, epidemi-
ological and toxicological studies with airborne ultrafine
particles can be viewed as the basis for the expanding field
of nanotoxicology .
In vitro studies bear several distinct advantages for
studying neurotoxic effects of air pollutants because the
technology is cheap, the cultured cells grow rapidly, and the
assays provide reproducible results. However, many times
immortalized cell lines are used, which might not correctly
reflect the more complex responses of native CNS cells or
of neurons in their natural complex environment. Unfortu-
nately, long-term and large-scale cultures of primary CNS
cells are still challenging and thus might not be useful for
high-throughput screening of toxicological effects. The
emerging field of induced pluripotent stem cells, which can
and keratinocytes, may provide a solution to this problem
and induced pluripotent stem cells could soon emerge as
a novel experimental paradigm for human neurotoxicity
studies [62, 63].
Despite their advantages, in vitro studies have also im-
portant limitations, some of which are methodological. The
research groups might be hampered because of the use of
particles with different chemical compositions or different
culture cells. The duration of exposure and concentrations
might differ across laboratories. More importantly, however,
responses of cultured cells might not faithfully reflect the re-
sponses of the entire body system or target organ. In general,
ultraphysiological doses of air pollutants are used in cell
cultures studies and the long-term study of the effect of
chronic exposure to low doses of potentially toxic material is
not feasible. Organotypic cell cultures and tissue explant cul-
tures might be more useful in this regard since the integrity
of tissue of interest is fully or partially preserved. Because
systemic effects and biodistribution of air pollutants cannot
6 Journal of Toxicology
be investigated in in vitro assays, in vivo studies provide
additional and important information on the adverse effects
of air pollutants.
4.1.2. In Vivo Studies. The confirmation of in vitro results
through realistic in vivo studies is mandatory to validate hy-
potheses generated from in vitro studies . In vivo studies
are invaluable tools for the examination of bio-distribution,
the biokinetic properties, and the pathophysiological effects
of air pollution on the whole body system. They also provide
an opportunity to study neurobehavioral effects of air pol-
lution in intact living animals. Novel noninvasive imaging
techniques can be used to visualize neuroinflammation, mi-
croglia activation, brain redox-status, and BBB integrity in
live animals [64, 65]. Importantly, in vivo studies allow the
use of experimental conditions, routes of administration,
and exposure regimes that are not available in cell culture
systems. For instance, they enable a comparison of the effects
of acute, subchronic, and chronic exposure of the whole
animal. Likewise, pollutants can be administered through
and intratracheal instillation, or intraperitoneal injection
(Table 2). Like cell culture studies, whole animal studies are
amenable to investigate alterations in gene and protein ex-
to air pollutants. Finally, prevention strategies and therapeu-
tic approaches can be tested in a preclinical setting.
To investigate the effect of certain gene products on the
susceptibility to damage by air pollutants, genetically modi-
fied animals can be used. For instance, one study used Apol-
ipoprotein E (ApoE) knockout (ApoE−/−) mice and could
show that ApoE deficiency enhances air pollutant-induced
neurotoxicity . Exposure to UFPs activates NFκB and
AP-1 transcription factors via JNK-activation in ApoE−/−
mice in a dose- and duration-dependent manner . In a
more recent study, these findings were confirmed, providing
evidence that air pollution can produce neuropathological
damage in individuals that are susceptible to oxidative stress
Tin-Tin-Win-Shwe et al. used wild-type male BALB/c
mice and instilled carbon black (CB) intranasally . Six
hours after instillation, the mice were intraperitoneally
injected with the bacteria cell wall component lipoteichoic
acid (LTA) and the authors could show that LTAtreatment
potentiates CB-induced neurological effects. CB modulates
the levels of extracellular amino acid neurotransmitter and
proinflammatory cytokine IL-1β mRNA expression syner-
gistically with LTA in the mouse olfactory bulb. In a recent
study by Zanchi et al., rats were exposed to residual oil fly
intranasal instillation and were treated with the antioxidant
N-acetylcysteine (NAC) intraperitoneally for 30 days .
ROFA instillation alone induced an increase in lipid perox-
idation levels in the striatum and the cerebellum, whereas
NAC treatment had preventive effects.
Ozone is by far the most important air pollutant in
terms of its concentration, its persistence, and its ubiquitous
occurrence. A list of preclinical studies that investigated
the neurotoxic effects of ozone inhalation using different
experimental paradigms is given in Table 2. For instance,
Pereyra-Mu˜ noz et al. showed that chronic (4h daily for 15 or
30 days) and low-dose (0.25ppm) exposure induces oxida-
tive damage to neurons in the striatum and substantia nigra
. Angoa-P´ erez et al. exposed ovariectomized rats to air
loaded with ozone for 7, 15, 30, or 60 days (0.25ppm, 4h per
day) . A second experimental group of ovariectomized
rats were treated with 17[beta]-estradiol 2h after ozone
suggest that chronic ozone inhalation produces oxidative
stress and loss of dopaminergic neurons in the substantia
nigra and that the effects can be reduced by treatment with
17[beta]-estradiol [71, 76]. Neural mechanisms underlying
adaptive responses to acute ozone exposure were also studied
in adult rats that were subjected to 0.5ppm ozone exposure
for 3h and were then allowed to recover for 3h before ex-
amination. In this paradigm, acute ozone exposure had an
effect primarily on glial cells in the CNS . The pro-
tein expression levels of vascular endothelial growth factor
(VEGF) were upregulated in central respiratory areas, such
as the nucleus tractus solitarius (NTS) and the ventrolateral
medulla (VLM). Persistent VEGF upregulation following
ozone exposure may contribute to brain repair and consecu-
tive functional adaptations. Rats that inhaled 0.5 or 2ppm ±
10% of ozone for 1.5–120h suffered from lung inflammation
that induced the activation of NTS neurons through the
vagus nerve. It also promoted neuronal activation in other,
stress-responsive regions of the CNS as could be demon-
strated by up-regulated levels of the immediate early-gene
product c-Fos .
As exemplified above, in vivo studies offer a unique pos-
sibility to test the potential of neuroprotective agents such
as hormones and antioxidants against air pollutants [71, 73,
76, 78]. Selective inhibitors of the cyclooxygenase-2 (COX-2)
enzyme have been tested in young healthy dogs which were
residents of highly air polluted urban regions. Inhibition
of COX-2 showed beneficial effects probably by reducing
frontal lobe IL-1β expression . Interestingly, treatment
with dark chocolate has also been found to be neuroprotec-
tive against long-term air pollution in mice .
Despite the clear advantages of in vivo studies that were
summarized here (studying pathophysiological mechanisms
or neurobehavioral responses and testing preclinical preven-
tive and treatment strategies), a long list of confounding
parameters experimentally may obscure the results. Method-
ological details such as sex, age, strain, dose, and the partic-
ular assay that was used to measure the outcome should be
considered carefully when comparing results across different
studies. In particular body size, age, gender, species, and
strain are known to have dosimetric effects in air pollution
research . Although there is growing epidemiologic evi-
dence that associations between air pollution and respiratory
health differ between females and males, comparative studies
or studies on female rodents in general are limited [72, 82].
Likewise, only a single study evaluated the influence
of age on air pollution-induced CNS injury . In this
study, ozone inhalation resulted in high-lipid peroxidation
in the frontal cortex of old rats, which is in contrast to
findings in young rats, where oxidative stress injury occurred
Journal of Toxicology7
Table 2: The effects of air pollutants on the central nervous system in vivo.
SpeciesRoute of administration AssaysKey findings and outcomeReferences
Mouse (ApoE−/−) Inhalation TH immunostaining
NFκB and AP-1 activation
CAPsMouse (ApoE−/−) Inhalation
Increased TNFα and IL-1 α
Increased HO-1 and COX2
mRNA and protein expression
Increased glutamate levels
Inreased NMDA receptor
subunits (NR1, NR2A, and
NR2B), and CaMKIV mRNA
NFκB and AP-1 activation
Increased TNFα and IL-1 α
levels and mRNA expression
Decreased locomotor activity
Inhalation (w/wo i.p.
Inhalation Open-field test
Increased TNFα, IL-1β, IL-6,
and MIP-1α levels
Increased TNFα, MIP-1α
Increased lipid peroxidation
Increased TNFα, IL-1β, and
chemokine mRNA expression
Increased glutamate glycine
Decreased motor activity
Increased lipid peroxidation
Loss of DA neurons (SN)
ROFARat In instillationTBARs
in instillation (w/wo ip
Rat Inhalation (temporal)
Motor activity test
Lipid peroxidation assay
Inhalation TH immunostaining Loss of DA neurons (SN)
Rat InhalationBehavioral tests
Increased freezing behavior
Increased lipid peroxidation
Changes in neurotransmitter
Impaired olfactory perception
and social recognition
Increased lipid peroxidation
Increased lipid peroxidation
Increased VEGF, IL-6 and
Lipid peroxidation assay
Lipid peroxidation assay
Lipid peroxidation assay
Rat InhalationIHC 
8 Journal of Toxicology
Table 2: Continued.
Pollutants Species Route of administrationAssays Key findings and outcome
Increased c-Fos expression in
different brain regions
RatInhalation IHC 
Abbreviations: apolipoprotein E (ApoE), calcium/calmodulin-dependent protein kinase type IV(CaMKIV), concentrated ambient particles (CAPs),
cyclooxygenase-2 (COX-2), dopaminergic(DA), diesel exhaust particles (DEPs), electrophoretic mobility shift assay (EMSA), enzyme-linked immunosorbent
assay (ELISA), heme oxygenase (HO), high-performance liquid chromatography (HPLC), allograft inflammatory factor 1(IBA1), immunohistochemistry
(IHC), interleukin-1 alpha (IL-1α), interleukin-1beta (IL-1β), interleukin-6 (IL-6), c-Jun N-terminal kinases (JNK), macrophage inflammatory proteins
(MIPs), nanosized carbon black (NSCB), lipoteichoic acid (LTA), nuclear factor kappa B (NFκB), nucleus tractus solitarius (NTS), residual oil fly ash (ROFA),
reverse transcription polymerase chain reaction (RT-PCR), substantia nigra (SN), thiobarbituric acid-reactive substances (TBARS), tyrosine hydroxylase
(TH), tumor necrosis factor alpha (TNFα), and vascular endothelial growth factor (VEGF).
in the hippocampus. Region specific inflammation and al-
terations in gene expression were also seen after DEPs expo-
sure, suggesting a selective vulnerability of specific neuronal
subpopulations similarly to the selective loss of specific
neurons that is typical for certain neurodegenerative diseases
[69, 83]. Although strain difference is an important variable
in a variety of lung injury studies, it is a widely neglected
parameter in air pollution-induced CNS injury research [84,
Variations in the geographic location of sample collec-
tion, and seasonal climate variations during the collection of
ambient air samples are neglected oftenly as well. However,
these parameters have a crucial impact on the results and
should be clearly described in all studies. Use of filtered
ambient air samples may, on one hand, simulate real-world
exposure conditions, on the other hand, the samples also
contain unidentified or unmeasurable components. Thus,
the inherent heterogeneities of in vivo experimental para-
enables a more reliable comparison between studies from
different laboratories. The lack of such a standardized system
also hampers the translation of data from preclinical studies
and the nasal cavity, the breathing pattern (nasal breathing
is obligatory for rodents), and brain anatomy differ greatly
across species and impede generalization of the results. For
instance, while the olfactory mucosa lines more than 50%
of the surface of the nasal cavities in rodents, the human
olfactory tissue is restricted to a mere 3–5%. The use of
nonhuman primates would provide results more relevant to
humans, but poses great ethical concerns.
4.2. Epidemiological, Postmortem, and Clinical Studies
4.2.1. Stroke. While cardiorespiratory effects of air pollution
have been extensively investigated , only preliminary
findings are available on the effects of airborne pollutants on
the CNS. Stroke is one of the most prevalent CNS disorders
which can be caused by air pollution. A relationship between
air pollution and stroke was first reported after the Great
London fog , but similar results were obtained from dif-
ferent geographic regions that include Canada, Japan, Italy,
ever, a one-to-one comparison of these studies is difficult
because each study measured different pollutants, inves-
tigated populations with different genetic background, or
people exposed to different environmental conditions, in
addition to evaluating different stroke-related parameters.
ies demonstrated a positive correlation between stroke mor-
tality rates, hospital admission, and outdoor pollution [87–
90, 92–95], although contradictory results were reported as
well [91, 96]. Interestingly, a Canadian study showed that
only a specific subgroup of patients, those suffering from
diabetes mellitus, was at high risk for ischemic stroke .
Age and gender may also differentially affect the risk of air
It also appears that the air pollution-related ischemic stroke
risk is higher than the risk for hemorrhagic stroke [8, 86].
Hemorrhagic and ischemic strokes have distinct pathogene-
sis and also differ in terms of other risk factors.
Mechanistically, the correlation between air pollution
and stroke might be due to the observation that fine PM
and UFPs exert procoagulant effects in vivo [97, 98]. Yet,
the stroke risk increases with both, short-term and long-
effects of long-term exposure on stroke risk are less promi-
nent . In addition to these epidemiological findings, a
limited number of in vivo studies also support a close cor-
relation between air pollution and stroke. For instance, SO2
inhalation caused cerebral changes similar to the alterations
aggravated histological changes in ischemic brain regions
Air pollution will continue to become a major health
problem, especially in developing countries and rapidly
growing economies. Unfortunately, booming economic de-
velopment increases air pollution and related disease includ-
ing stroke. Thus, there is a great demand to organize popula-
preventive measures against the unfavorable effect of air
pollution on severe cerebrovascular diseases, such as ischem-
4.2.2. Neurodegenerative Diseases. Concomitant with a gen-
eral increase in life expectancies worldwide, the incidence
and prevalence of common neurodegenerative diseases grow
as well, thereby increasing the financial and social burden
on individuals and society. Alzheimer’s disease (AD), the
most prevalent neurodegenerative disease, is characterized
by extracellular deposition of amyloid-beta (Aβ) peptide
Journal of Toxicology9
fibrils known as amyloid plaques and intracellular protein
aggregates called neurofibrillary tangles (NFTs) . AD
is the most common cause of dementia in aged people,
affecting 27 million people globally. Parkinson’s disease
(PD), the second common neurodegenerative disorder, is
caused by the degeneration of dopaminergic neurons in the
substantia nigra and a progressive loss of dopaminergic neu-
rotransmission in the caudate and putamen of the neos-
triatum . This severe movement disorder affects 1-2%
of the population above the age of 50. Most AD and PD cases
are sporadic, and age is the leading risk factor. The etiologies
of the diseases, however, are multifactorial, and the risk
factors include environmental factors and genetic predis-
position. Environmental exposures to metals, air pollution,
and pesticides as well as nutritional factors are common risk
factors for neurodegenerative diseases . Although dif-
ferent neurodegenerative diseases have distinct pathologies
and clinical presentations, they often share common mech-
anisms such as protein aggregation, oxidative stress injury,
neuroinflammation, microglial activation, apoptosis, and
mitochondrial dysfunction, which ultimately result in the
loss of specific neurons [101, 102]. Accumulating evidence
suggest that exposure to air pollution can trigger these com-
pollution and neuropathology came from studies that were
carried out on animal populations that are naturally exposed
to polluted urban environments in Mexico City . Using
light and electron microscopy, Calder´ on-Garcidue˜ nas et al.
reported significant inflammatory and neurodegenerative
changes in the olfactory mucosa, the olfactory bulb as well
as in subcortical and cortical structures in otherwise healthy
mongrel canines, whereas similar changes were not evident
in control groups inhabiting less-polluted rural areas .
Breakdown of nasal and olfactory barriers, alterations in the
BBB, and degeneration of cortical neurons were observed
even in animals that were younger than 1 year of age. With
growing age, and therefore extended exposure, the dogs
exhibited reactive astrogliosis, white matter glial cell apop-
tosis, ApoE immunoreactivity in vascular cells, and nonneu-
ritic plaques and NFTs. These findings suggest an accelerated
AD-like neuropathology in chronically exposed animals.
Feral dogs naturally exposed to urban air pollution also
DNA . Cerebral inflammatory responses were associated
with the neurohistological findings as demonstrated by
nuclear translocation of the neuronal NFκB p65 subunit,
increased inducible nitric oxide synthase (iNOS) immunore-
activity in endothelial, glial and neuronal cells, and increased
endothelial and glial COX-2 immunoreactivity [39, 104].
Animals from polluted areas exhibited deposits of diffuse
amyloid plaques a decade earlier than control animals from
less-polluted regions [39, 104]. Although most animals do
not develop the full human pathology of AD, aged dogs
are known to suffer from cognitive dysfunctions that re-
semble key aspects of AD . The decline in executive
functions and the impairment of learning and memory
represent a spectrum that comprises normal aging, mild
cognitive impairment, and early/mild AD in humans .
However, dense core neuritic plaques and NFTs could not be
observed consistently in the dogs. Because of the numerous
atmospheric contaminants found in the highly polluted air
of Mexico City, postmortem studies on resident feral dogs
could only link the neuropathology to the complex mixture
of ozone, PM, LPS, and unmeasurable air pollutants .
Thus, whether airborne UFPs are causatively involved in
the observed CNS change remain to be determined [5, 16].
However, the oil-combustion PM-associated metals nickel
and vanadium, as well as UFPs were detected in the dogs
brains, indicating that brain uptake of metals and UFPs may
occur in natural exposure settings [11, 39].
Similar findings were recently observed in postmortem
examinations of human samples and in laboratory animals
of Mexico City exhibit significantly higher COX-2 expression
in the olfactory bulb, the hippocampus, and the frontal
cortex, and greater neuronal astrocytic accumulation of Aβ42
when compared to age-, gender-, and education-matched
subjects from cities with low pollution levels [9, 13]. Based
from relatives and coworkers by 2 physicians, each subject
was considered cognitively and neurologically fit when alive
. The neuropathology, however, could be observed in
subjects as early as in the second decade, suggesting that
neuropathologies induced by chronic exposure to high levels
of air pollution share similarities with the pathology of AD
. Although NFTs or Aβ neuritic plaques could not be
observed because of the relative young age of the subjects,
neuroinflamation and intraneuronal Aβ42 accumulation in
target brain areas may be compatible with a premature
accelerated process preceding AD neurodegeneration. Most
interestingly, a recent postmortem study on children and
young adults who died suddenly has shown that lifelong
exposure to air pollution is associated with neuroinflam-
mation, altered innate immune responses, disruption of the
BBB, endothelial activation, and accumulation of disease
proteins (Aβ42and α-synuclein) in the CNS . Moreover,
Aβ42-immunoreactivity was higher in brain tissue derived
from carriers of the ApoE ε4 allele than those of ApoE ε3
carriers suggesting that a specific genotype constitutes a
higher risk for developing AD in a polluted environment.
The ApoE ε4 allele is known to contribute to a genetic
predisposition for late-onset AD, although the mechanisms
by which ApoE ε4 influences onset and progression of the
disease are not well understood [101, 108].
The accumulation of α-synuclein in the brain of young
people that were exposed to air pollution lifelong is notewor-
thy . α-synuclein is a major component of Lewy bodies,
a pathological hallmark of PD . Dopaminergic neurons
were found to be selectively vulnerable to DEPs both in
vitro and in vivo [46, 49]. However, a recent epidemiological
study from Canada did not support a direct link between the
markers of traffic-generated air pollution and PD, although
an association between ambient manganese pollution and
the risk of physician-diagnosed PD was reported .
A further interesting similarity between air pollution-
induced neuropathologies and neurodegenerative disorders
10 Journal of Toxicology
dysfunction, especially in ApoE ε4 carriers, can be seen from
childhood in individuals that grew up in highly polluted
environments. Yet, olfactory dysfunction is also among the
first clinical signs of AD and PD . In sporadic PD,
olfactory impairment precedes motor symptoms by years
and is independent of the loss of dopaminergic neurons. In
AD, however, olfactory dysfunction and disease progression
Recent epidemiological studies combined with psycho-
logical tests support an association between chronic expo-
sure to traffic-related air pollution and decreased cognitive
function in both genders [113, 114]. Altogether, these find-
ings warrant further and more extensive epidemiological,
forensic, and toxicological studies that aim to understand the
association between chronic exposure and the risk of neu-
rodegenerative diseases development. Such efforts may lead
to the development of preventative strategies for these devas-
tating diseases in certain risk groups.
4.3. Implications for Neurodevelopment and Mental Health.
Normal brain development is a complicated process that
involves controlled cell proliferation, neuronal migration
fromtheir place of birth to their finallocation, and the estab-
lishment of specific connections between neurons and target
tissues . All of these processes are tightly controlled,
but are also influenced by environmental conditions. Air
pollutants can affect the brain at any age, but the developing
brain is particularly vulnerable because of its high neuronal
proliferation and differentiation rates and its immature me-
tabolism and imperfect BBB . Disturbances of devel-
opmental processes in the brain can lead to permanent
abnormalities that translate into later life.
In developing embryos, the placenta serves as a barrier
against many environmentally hazardous substances, but it
might not be protective against all components of air pol-
lution. Among documented hazards that affect neurodevel-
opment are certain industrial chemicals, maternal smoking,
alcohol, certain drugs, noise, diet as well as maternal stress
. This section, however, only focuses on the effects of
air pollutants on neurodevelopment.
Ozone is one of the best studied substances in preclinical
examinations that assess the effects of exposure to an air
pollutant during the prenatal period. Prenatal ozone expo-
sure leads to permanent damage of the cerebellum  and
disruption of the cerebellar monoaminergic system .
In addition, prolonged prenatal ozone exposure altered the
levels neurotrophic factor in the brain. CD-1 rats showed
reduced nerve growth factor levels in the hippocampus and
increased brain-derived neurotrophic factor levels in the
striatum when exposed to ozone . Changes in neuronal
responses and neuronal injury were also evaluated by
immunohistochemistry in rats using c-Fos immunolabeling
as a marker for neuronal activity and tyrosine hydroxylase
exposure during the prenatal period induced long-lasting
changes in the nucleus tractus solitarius (NTS), important
respiratory control center .
In vivo studies have shown that prenatal exposure to
DEPs can also affect brain development [122, 123]. In utero
administration of a low dose of DEPs (1.0mgDEP/m3)
reduced locomotor activity and dopamine turnover in the
striatum  and affected monoamine metabolisms in a
variety of brain regions generally . Other air pollutants
can also adversely affect the brain during development. For
instance, when silica and titanium dioxide NPs were injected
intravenously to pregnant mice, they could be detected in
the fetal brain . This suggests that NPs can cross the
maternal-fetal barrier in the placenta and could cause neu-
rotoxicity in the offspring. Using an ex vivo human placental
perfusion model, Wick et al. found that nanosized material
can cross the placenta without affecting the viability of the
placental explant per se . However, the ability of ambi-
ent air pollutants to cross the placenta needs further evalua-
tion to understand the full spectrum of possible effects.
Epidemiological and clinical studies demonstrating a
negative impact of air pollution on neural development in
humans were performed in children living in Mexico City
can origin from New York City, who had valid prenatal
polycyclic aromatic hydrocarbons (PAHs) monitoring data,
were evaluated for mental and psychomotor development at
was found to be associated with a lower mental development
index at age 3. A second study from Boston, examined the
relation between CB and cognition . Long-term expo-
sure to CB particles was associated with a decrease in
status, birth weight, smoking, and blood lead level. These
studies, however, have certain limitations, such as limited
monitoring of pollutants levels and significant reduction in
the sample from the original cohort over time. Moreover, the
presence of confounding factors was not addressed in these
studies. A third study showed that early-life exposure to
emissions from indoor gas appliances is negatively correlated
with neuropsychological development through the first 4
years of life. Children that carried the glutathione-S-trans-
ferase gene Val-105 allele were particularly susceptible to the
effects . Electrophysiological examinations confirmed
disturbances in brain development as a result of exposure to
polluted air. Brainstem auditory-evoked potentials (BAEPs)
were compared across children from highly and lowly pol-
ments displayed significant delays in the central conduction
time of BAEPs, suggesting that exposure to air pollution may
be a risk for auditory and vestibular impairment.
Prenatal exposure to air pollutants may also constitute a
risk factor for neurodevelopmental disorders such as autism
and neuropsychiatric diseases such as schizophrenia. Schizo-
phrenia is a chronic disease of the brain that is characterized
by positive and negative psychiatric symptoms as well as
cognitive dysfunction. The incidence of schizophrenia in
the population is about 1% . Schizophrenia is caused
through a combination of genetic factors and environmental
insults, for instance prenatal infection . An increased
risk for schizophrenia is evident in people inhabiting
urban regions . The exact reasons remain unclear, but
Journal of Toxicology11
exposures to infectious agents or toxins from the urban
environment have been suggested as possible causes. An
important feature of airborne PM is that they may interact
with other pathogens to serve as transporters for viruses,
bacteria or molecules with infectious or antigenic properties,
for instance, bacteria cell wall components . Contrary
results, however, were reported in a study from Finland,
showing a correlation between living in rural regions and
that nontraffic source of air pollution, such as firewood,
could have been a possible risk factor .
Autism on the other hand is a neurodevelopmental
disorder that is characterized by impairments in social inter-
action, verbal and nonverbal communication, and repetitive
behavior . The prevalence for autism in the general
population has been reported to range from 0.2 to 0.6% with
an increasing trend over the recent years. Although, the exact
etiology of autism is still unclear, genetic, environmental,
and social factors may contribute to the development of the
disease . Maternal exposure to air pollution during the
and 657 healthy controls were examined in a San Francisco
study that evaluated the possible effects of air pollution
on autism development . An association was found
between the estimated concentrations of metals and solvents
in the ambient air around the birth residence and autism. An
association was also found between autism and residential
alerting results suggest that subtle health effects, such as
functional delays in brain maturation and impairment of
neurobehavioral competences, should be included in studies
of chronic effects of urban air pollution .
As derived from studies on the aged population, air
pollution also has adverse effects on mental health during
strated that neurobehavioral effects are associated with long-
term exposure to ambient PM and ozone in adults .
Further longitudinal studies are urgently needed to fully
explore the relationship between long-term exposure and
neurobehavioral changes and subsequent development of
neurocognitive impairment, such as cognitive decline and
dementia. Ten human volunteers were exposed to dilute
amounts of DEPs (300μg/m3), and brain activity was moni-
tored by quantitative electroencephalography (EEG) show-
ing a significant increase in the EEGs median power fre-
quency and fast wave activity . Additional studies need
to determine whether other types of air pollutants elicit
comparable effects on brain activity. The use of recent and
more sophisticated technology, such as functional MRI and
recording of event related potentials, in future studies will
contribute to a better understanding of the relationship be-
tween air pollution and mental health.
Air pollution can produce its adverse effects in the CNS
through a variety of cellular and molecular mechanisms
CNS pathology is probably a result of the synergistic inter-
action of multiple pathways and mechanisms . Although
the exact mechanisms that are responsible for air pollution-
induced neurotoxicity are poorly understood, postmortem
and experimental studies suggest that air pollution causes
oxidative stress, neuroinflammation, cerebrovascular dam-
degenerative disorders. Genetic and epigenetic mechanisms
might also be involved.
5.1. The Interaction of Air Pollutants with Cells and Cellular
Organelles. Possible mechanisms by which air pollutants can
interact with biological tissue depend on the size, the struc-
ture, and the composition of the components in the polluted
air, determining their spectrum of molecular activity and
entry routes. PMs can be taken up by mammalian cells in
different ways, including phagocytosis, pinocytosis, passive
diffusion, receptor-mediated endocytosis, direct penetration
of the cell membrane, or transcytosis. Which route is taken
largely depends on the physicochemical properties of the
toxic components. PM that cannot enter cells directly could
still interact with surface proteins and change cellular signal-
ing and behavior.
and the ways by which it can be taken up by cells. While
the uptake of fine particles (0.1–2.5μm diameter) by macro-
the uptake of ultrafine particles (<0.1μm diameter) can
occur by other, nonspecific mechanisms. These mechanisms
may include electrostatic, van der Waals, and steric interac-
tions and are subsumed under the term adhesive interaction,
although the exact mechanisms remain to be determined
[13, 33]. As mentioned before, ultrafine PMs can cross red
blood cell membranes rapidly and easily; a process that
appears to be mediated by an unidentified non-phagocytic
mechanism . Particles smaller than 100nm could be
observed in intraluminal erythrocytes that were collected
from frontal lobe and trigeminal ganglia capillaries from
to-volume ratio and are not enclosed by membranous or-
ganelles, which allow them to directly interact with intracel-
lular proteins, organelles, or DNA. Such particles may reach
specific organelles, such as mitochondria, lysosomes, and
nuclei, where they could induce an oxidative burst within
their membranes by interfering with NADPH-oxidase activ-
ity. They may also induce the release of inflammatory
mediators and cytokines by the cell . A recent study has
shown that exposure to airborne UPMs is associated with
number of mitochondrial DNA (mtDNA) . Damaged
mitochondria may then contribute to increased oxidative-
stress through altered ROS production and subsequently
antioxidant defense mechanisms.
result in protein degradation and protein denaturation. Loss
of enzyme activity and formation of autoantigens are possi-
ble consequences . Environmental NPs can also signifi-
12 Journal of Toxicology
a possible link between air pollution and neurodegenerative
disorders [143, 144]. If these findings can be confirmed
under realistic in vivo conditions, it would have far-reaching
consequences with respect to the mechanisms underlying
neurodegenerative diseases . Other key molecular path-
ways that are affected in neurodegenerative diseases lead to
misfolding, aggregation, and accumulation of proteins in the
brain [145, 146]. PMs that have the capability to enter nerve
cells could contribute to these processes, so could oxidative
stress that is induced by the air pollutants.
Cellular responses to oxidative stress can lead to changes
in mitochondria and other organelles, notably the endoplas-
mic reticulum (ER), and eventually triggers the cell to enter a
cell death pathway [147, 148]. Mitochondria, as regulators
of cellular energy metabolism and apoptosis, are critical
organelles in switching between different cellular responses
leading to death or survival of the cell. Perturbed ER calcium
homeostasis may also contribute to neuronal dysfunction
and degeneration in neurodegenerative disorders . The
ER is critical for early protein biosynthesis steps of secreted
and membrane proteins, which occurs in the lumen of the
ER, where the ER machinery assists in their folding.
Loss of ER homeostasis triggers stress responses, which
diseases . Recent studies have shown that exposure to
airborne PM causes ER stress in lung tissue [151, 152].
Neurodegenerative disorders are often characterized by the
aggregation and accumulation of misfolded proteins .
Protein folding stress in the ER may lead to activation of
the unfolded protein response (UPR). Organic DEP chemi-
cals induce an UPR and proinflammatory effects in human
bronchial epithelial cell line . However, the possible
relationship between ER stress and exposure to air pollution
has not been studied in the context of CNS cells. The in-
signaling that regulates the intensity and duration of innate
immune responses should also be considered in neuroin-
flammation-induced by air pollution .
5.2. Neuronal and Glial Cell Death. Air pollution-induced
loss of neurons is a consistent finding in postmortem and
experimental studies, and neuronal cell death may be direct
or indirect via microglia activation. It is noteworthy that
mitochondria directly [42, 142]. This can lead to disruption
of the mitochondrial electron transport chain, which leads
to increased superoxide radical production. Furthermore,
ambient UFPs perturb the permeability of the mitochondrial
transition pore, resulting in the release of proapoptotic
factors and ultimately programmed cell death . It has
NP-mediated changes in glutamatergic neurotransmission,
which can result in neuronal damage and finally neurode-
In addition to neurons, other CNS cells may also be
target of air pollution. Indeed, astroglial cell death has been
reported upon exposure to high dose of ozone in vitro .
As suggested by MRI studies in dogs and children of Mexico
City, oligodendroglial cells may be affected by air pollution
[11, 80], and prefrontal white matter hyperintense lesions
were observed in these studies. However, any experimental
study specifically focusing on the effects of air pollution on
oligodendrocytes and myelin has not been reported so far.
Brain endothelial cells and pericytes are other candidate tar-
get cells. Exposure to DEPs resulted in endothelial activation
and dysfunction in rat brain capillaries, but cell viability was
not assessed in the study .
Besides apoptosis and necrosis, additional cell death
mechanisms may also contribute to air pollution-induced
CNS injury. Increased levels of autophagic vacuoles were
observed upon exposure of cells to NMs in vitro . Au-
tophagy is a cellular process for the disposal of damaged or-
ganelles or denatured proteins through a lysosomal degra-
dation pathway. The interaction of NMs with the autophagy
pathway may be disruptive to neurons, leading to severe
structural changes and ultimately cell death. Impaired auto-
phagy is also implicated in the pathogenesis of neurodegen-
erative disorders . However, the exact role of autophagy
in CNS injury induced by air pollutants remains to be
tive stress refers to an imbalance between the production of
ROS and the cells ability to detoxify reactive intermediates or
to repair cellular damage caused by ROS. They are highly
reactive molecules because of their unpaired electrons and
form as natural byproducts of a cell normal oxygen metab-
olism. They also fulfill important roles in cell signaling and
homeostasis. However, during times of environmental stress
such as air pollution, ROS levels can increase dramatically,
resulting in significant damage to cellular components, in-
cluding proteins, lipids, and DNA. Disturbances in the nor-
mal redox-state of tissues can cause toxic effects through
the production of peroxides and free radicals (e.g., chemical
species that contains one or more unpaired electrons). The
two most important oxygen-derived free radicals are super-
oxide and hydroxyl radicals. Free radicals are important for
a number of biological processes, such as the elimination
of bacteria by phagocytic cells. Excessive ROS accumulation,
however, poses a challenge for cell survival, and cells have
developed defense mechanisms against excessive amounts of
ROS that include antioxidant enzymes (superoxide dismu-
tase, catalase, and glutathione reductase, glutathione perox-
ides) and antioxidant molecules (glutathione, taurine, sele-
nium, vitamins E and C).
Under normal conditions, ROS are generated at low
concentrations and are easily neutralized by cellular antiox-
idant defenses such as glutathione (GSH) and antioxidant
enzymes . However, under conditions of excess ROS
production, antioxidant and detoxification enzymes (phase
these enzymes contain antioxidant response elements (ARE)
in their promoter regions, which contains a binding site
for the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)
transcription factor . At moderate levels of oxidative
stress, the Nrf2 protective response pathway is activated;
resulting in mitogen-activated protein kinase- (MAPK)
Journal of Toxicology13
and NFκB- (a redox-sensitive transcription factor) induced
proinflammatory responses . Increased intracellular
calcium levels also mediate the activation of these signaling
pathways. At high levels of oxidative stress, perturbation of
the mitochondrial permeability transition pore and the elec-
tron transfer chain cause apoptotic and necrotic cell death.
Nrf2 regulates the expression of numerous cytoprotective
genes that function to detoxify reactive species produced
during ambient air pollutant metabolic reactions, highlight-
ing the important role of Nrf2 in the defense against air
pollutant-induced toxicity . Dysfunction of Nrf2 may
also be a risk factor for neurodegenerative diseases such as
induced injury has not yet been studied in the context of
The brain is especially vulnerable to oxidative stress
injury because of its high metabolic activity, its low activity
of antioxidant enzymes (superoxide dismutase and catalase),
its low content of endogenous radical scavengers, such as
vitamin C, its high cellular content of lipids and proteins,
which can act as a potent catalyst for ROS production [103,
160]. Oxidative stress has been consistently linked to aging-
related neurodegenerative diseases leading to the generation
of lipid peroxides, carbonyl proteins, and oxidative DNA
damage in tissue samples from affected brains [103, 161].
Metals, pesticides, and air pollutants, all of which have been
associated with neurodegeneration share a common feature,
namely, their capacity to lead to increased production of
reactive oxygen and nitrogen species. Although each pollu-
tant has its own mechanism of toxicity, several air pollutants,
like ozone, sulfur dioxide, volatile organic compounds, and
and disturb physiological functions [17, 162–164]. Some of
these pollutants go through a series of metabolic reactions
catalyzed by phase II enzymes, in order to be detoxified and
excreted. These reactions involve chemical modifications,
like oxidation, to increase the solubility of the original
compound so that it can be excreted. During these metabolic
reactions, many reactive intermediates, particularly ROS, are
produced . Both postmortem and in vivo studies have
recently revealed a link between oxidative stress and air pol-
ical screening studies, more refined approaches, for example,
the use of nanosensors to detect ROS generation by NPs will
emerge with time [32, 142].
Exposure to combustion particles is consistently associ-
ated with oxidative damage to DNA and lipids in humans
detected from leukocytes, plasma, urine, and exhaled breath
[165, 166]. The evaluation of apurinic/apyrimidinic sites in
nasal and brain genomic DNA in healthy dogs naturally
exposed to urban pollution in Mexico City showed DNA
damage suggesting a link to air pollution . DNA damage
is also crucial in aging and in age-related disorders, such as
AD. The processes involved in particle-induced genotoxicity
complex and of diverse physicochemical characteristics .
Interestingly, a recent study evaluating the link between
gaseous air pollutants and brain cancer mortality did not
provide evidence for an increased risk of mortality due to air
5.4. Microglial Activation. Microglia, the macrophage-like
cells of CNS, are the principal players in the brain’s innate
immune response. They are the immunocompetent cells of
the brain that continuously survey their environment with
highly motile extensions . Microglial cells normally
provide tissue maintenance and immune surveillance to the
brain and exert a neuroprotective role by their ability to
phagocytose aggregated disease proteins and pathogens and
to secrete neurotrophic factors. Microglia cells rapidly
change their cell morphology in response to any disturbance
of nervous system homeostasis and are then referred to as
activated on the basis of morphological changes and expres-
main cellular event during neuroinflammation. The activa-
tion of microglia results in the production and release of a
myriad of inflammatory cascade mediators, including Nitric
oxide (NO), chemokines, proinflammatory cytokines, ROS,
and reactive nitrogen species (RNS) those are deleterious
to the CNS . Microglial activation and inflammation
are also associated with progressive neuronal apoptosis in
human neurodegenerative diseases [170–172]. However, it
is not clear whether activation of microglia and the inflam-
matory responses play a role in the cause of the disease or
whether cell activation is a response to the early changes
associated with the disease process.
Microglia are also activated in response to aggregated
disease proteins (Aβ and α-synuclein), bacterial endotoxins
(LPS), proinflammatory cytokines, MMP-3 released from
apoptotic neurons, and environmental neurotoxins [1, 173].
An important molecular component of microglial responses
to initiates an inflammatory cascade in response to various
CNS stimuli . LPS, as the prototypical endotoxin,
binds to a CD14/TLR4/MD2 receptor complex and enables
TLR4signaling. Human autopsy studies showed evidencefor
increased CD14 expression in response to chronic exposure
to high levels of air pollution, indicating an activation of
either infiltrating monocytes or the resident microglial cells
healthy dogs exposed to air pollution . As demonstrated
by morphological changes and increased superoxide pro-
duction in a neuron-glia cell culture system, DEPs can also
activate microglia in vitro . Furthermore, neuron-glia
cocultures treated with DEP showed selective dopaminergic
indicating that activated microglia cells mediate the neu-
ronal damage. Neuron-glia co-cultures derived from mice
lacking functional NADPH oxidase, the enzyme responsible
for extracellular superoxide production, were insensitive
to DEP-induced neurotoxicity, indicating that microglia-
derived ROS mediate DEP-induced dopaminergic neurotox-
icity [1, 46]. Interestingly, cytochalasin D, a phagocytosis
inhibitor, reduced DEP-induced superoxide production in
enriched-microglia cultures, implying that DEP is phagocy-
tized by microglia to trigger the production of superoxide
, whereas UFPs themselves can inhibit phagocytosis in
14 Journal of Toxicology
alveolar macrophages . This difference may result from
the differences in cell or particle type. A very recent in vivo
study could also demonstrate DEP-induced microglial acti-
Metals associated with air pollution are also able to
activate microglia. Manganese, a component of industrial-
derived air pollution, is able to activate rat microglia in vitro
. Microglial activation by manganese chloride also in-
duces dopaminergic neurotoxicity in vitro and application
of antioxidants, such as superoxide dismutase/catalase, glu-
tathione, NAC, or inhibitors of NO biosynthesis significantly
protected dopaminergic neurons against damage .
LPS on the other hand amplifies neurotoxicity induced by
activated microglia in response to manganese chloride .
activators differ, although both cell types are regarded as
cellular components of the brain’s innate immune system.
5.5. Neuroinflammation and Inflammasome Activation. Neu-
roinflammation is a complex and innate response of neural
tissue against harmful stimuli such as pathogens, damaged
cells, and other irritants within the CNS. A crucial compo-
nent of innate immunity in the CNs involves the produc-
tion of proinflammatory cytokines mediated by inflamma-
some signaling . The innate immune cells in the CNS,
microglia and astrocytes, express pattern-recognition recep-
tors (PRRs), for example, TLR4, which participate in the
assembly and activation of the inflammasome . The
inflammasome itself is a multiprotein complex that consists
of caspase 1, PYCARD, NALP (a NOD-like receptor serving
as a PRR), and sometimes caspase 5 or caspase 11 .
Nucleotide-binding domain, leucine-rich repeat, pyrin do-
main containing 3 (NLRP3) are a key component of the
inflammasome complex, which also includes ASC (apoptotic
speck-containing protein with a card) and procaspase-1
. The exact composition of the inflammasome depends
on the activator which initiates its assembly, that is, dsRNA
will trigger one inflammasome composition, whereas as-
bestos will induce the assembly of a different variant. The
inflammasome promotes the maturation of inflammatory
cytokines such as IL-1β and interleukin 18 (IL-18). It has
also been shown to induce cell pyroptosis, a process of
programmed cell death that is distinct from apoptosis .
The inflammasome orchestrates the activation of caspase
precursors, which in turn, cleave the precursor forms of the
cytokines as IL-1β, IL-18 and interleukin-33 (IL-33), which
triggers an inflammatory response, or the release of toxins
from glial and endothelial cells .
Inflammasome activation was recently shown to be in-
duced in acute brain injury as well, thus the NLRP1 in-
flammasome may constitute an important component of the
CNSs’ response to traumatic brain injury . An inflam-
masome complex also forms after experimental focal brain
analysis of inflammasome proteins in neurons, astrocytes,
microglia, and macrophages . The NLRP3 inflam-
masome also plays an important role in an experimental
model of MS, which is mediated by caspase-1 and IL-18
. Although it has recently been shown that the NALP3
inflammasome is involved in the innate immune response
to Aβ in microglia , the specific pathophysiologic role
of the inflammasome in neurodegenerative disorders still
remains to be clarified .
The organic substances adsorbed onto airborne Asian
sand dust activate the NALP3 inflammasome in macrophage
CB induces inflammasome activation and pyroptosis .
The identification of pyroptosis as a cellular response to
carbon NP exposure is novel and has important consequen-
ces for environmental and health-related issues. Another
study showed that TiO2and SiO2NPs activate the NLRP3
inflammasome in cultured keratinocytes, murine lung, and
dendritic cells [189, 190]. Whether air pollutants induce in-
flammasome activation in CNS and neuroglial cells remains
to be identified.
5.6. Reactive Astrogliosis. Astrocytes are characteristic star-
shaped glial cells that outnumber neurons in the brain about
fivefold. They perform many functions, including biochem-
ical support of cerebral endothelial cells that form the BBB,
provision of nutrients to the nervous tissue, maintenance of
extracellular ion balance, buffering of excess neurotransmit-
ters, secretion of neurotrophic factors, control of cerebral
blood flow, supporting neurogenesis as well as repair of
injured brain and spinal cord . Reactive astrogliosis
is a ubiquitous feature of CNS pathologies . At later
stages of CNS disorders, astrocytes become activated and
contribute to neuroinflammation and neurodegeneration.
Astroglia were reported to be activated in humans that
were chronically exposed to high levels of air pollution, as
evidenced by enhanced glial fibrillary acidic protein (GFAP)
expression [9, 10]. Animal studies investigating ozone ex-
posure showed that astroglial cells that are located close
to brain capillaries have enhanced expression of IL-6 and
TNFα  or are increased in number . However,
it is unclear whether the astroglia respond to components
of air pollution, to the inflammation, and oxidative stress
produced from other cell types or to cellular damage .
site of controlled blood-CNS exchange. This physical barrier
protects the CNS from potential toxins and pathogenic
agents. An intact BBB is important for the proper function-
ing of the CNS by actively controlling cellular and molecular
trafficking between the systemic circulation and the brain
parenchyma . Cerebral endothelial cells have luminal
tight junctions that form the physical barrier of the inter-
endothelial cleft. Endothelial cell are covered on the outside
by a basement membrane, which also surrounds pericytes.
Around these structures end-feet processes from nearby
astrocytes can be found which seal the BBB additionally
. The BBB integrity is impaired in many common CNS
disorders such as AD, PD, and stroke . Activation or
damage of the various cellular components of the BBB facil-
itates leukocyte infiltration leading to CNS injury. Systemic
inflammation induced by inhaled air pollutants can disturb
Journal of Toxicology 15
the integrity of the BBB through the effects of circulating
proinflammatory cytokines and LPS on cerebral endothelial
cells . Furthermore, an increase in ROS is a common
trigger for many downstream pathways that directly mediate
BBB compromise such as oxidative damage, tight junction
modification and matrix metalloproteinases (MMP) acti-
vation . Air-borne particulate matter has been iden-
tified both in human brain capillaries and in the brain
parenchyma, although the exact transport mechanisms are
unclear . Additionally, increased expression of intercel-
lular adhesion molecule (ICAM) and vascular cell adhesion
molecule (VCAM) was observed in cerebral vasculature
suggesting endothelial activation. As demonstrated by an ex
vivo study, DEPs induce oxidative stress, proinflammatory
signaling, and P-glycoprotein up-regulation in the rat brain
capillaries . These findings suggest that the BBB is an
important target for air pollutants. Therapeutic strategies
effects of air pollutant on the CNS.
5.8. Gene-Air Pollution Interaction and Epigenetic Mecha-
nisms. Individual differences that were observed upon expo-
sure to the same polluted ambient air suggest that genetic
susceptibility is likely to play a role in response to air pol-
lution . Gene-air pollution interaction was extensively
studied in pulmonary and cardiac disorders [198, 199].
There are, however, only a limited number of studies that
address gene-air pollution interaction in CNS injury. The
by exposure to air pollution . Air pollution-induced
olfactory dysfunction, also an early indicator for neuro-
degeneration, was higher in Apo ε4 carriers  and
more vulnerable to neuropathology induced by air pollution
Another susceptibility gene for the effects of air pollution
in the brain may be the glutathione-S-transferase gene
(GSTP1) because of its important role as radical scavenger
. Adverse effects of exposure to nitrogen dioxide on
cognitive function are more significant in children with any
GSTP1 Val-105 allele. Since oxidative stress, and inflamma-
tory processes are common denominators of air pollution-
induced neuropathology, oxidative stress and inflamma-
tory pathway genes including Glutathione S-transferase
Mu 1(GSTM1), GSTP1, NAD(P)H dehydrogenase quinone
1(NQO1), TNF, and TLR4 are further logical candidates for
the study of the association with the susceptibility to air
Air pollutants can change gene expression through a
broad array of gene regulatory mechanisms. Epigenetics is
a posttranscriptional control mechanism in gene regulation.
Changes in DNA methylation and histone acetylation leads
to imprinting, gene silencing, and suppression of gene ex-
pression without altering the sequence of the silenced genes
. Epigenetic alternations are often involved in the path-
ogenesis of neurological disorders [202, 203]. Air pollution
related neurological damage may occur via epigenetic effects
and could be demonstrated [204, 205]. Nano and microsized
SiO2exposure significantly decreased genomic DNA methy-
lation and levels of the related methyl transferase in normal
2 gene expression by increasing histone H4 acetylation and
histone deacetylase 1 (HDAC1) degradation in bronchial
epithelial cells . Similar results were also obtained from
in vivo studies . Exposure of inbred mice to particulate
air pollution caused hypermethylation in spermatogonial
stem cells. Human studies showed that either short- or
long-term exposure to air pollution in elderly can cause
hypomethylation in peripheral lymphocytes [207, 208]. In
addition, higher exposure to traffic-related air pollution is
in how much epigenetic changes contribute to neurological
symptoms caused by air pollution.
Air pollutants have been, and continue to be, major con-
tributing factors to chronic diseases and mortality, thereby
dramatically impacting public health. Air pollution is a glob-
al problem and has become one of the major issues of public
health as well as climate and environmental protection. The
effects of air pollutants are thus at a high level of interest for
scientific, governmental, and public communities. An in-
creasing number of people are exposed to a complex mixture
of inhalable NPs and toxic chemicals occupationally or as a
result of man made and natural disasters, such as war, fires,
and volcanic eruptions [210, 211]. Air pollution is increas-
ingly recognized as an important and modifiable determi-
nant of cardiovascular and respiratory diseases in urban
communities [3, 16]. Although adverse cardiopulmonary
outcomes have been the focus of many studies, air pollution-
related damage to the CNS has been widely neglected.
However, there is mounting evidence that air pollution also
contributes to CNS damage or increased progression of
that UFPs rapidly translocate from the lungs into the cells
and into the blood circulation. There is good evidence that
oxidative stress occurs in other organs, such as the heart
and the brain. The breadth, strength, and consistency of the
preclinical and clinical evidence provide a compelling argu-
ment that air pollution, especially traffic-derived pollution,
causes CNS damage and that there is a clear link between
air pollution and neurological diseases. Airborne particles
cause neuropathology, which seem to be mediated by direct
or indirect proinflammatory and oxidative responses. Both,
the physical characteristics of the particle itself and toxic
compounds adsorbed on the particle may be responsible for
the damage. The time of exposure has a key role in damage.
when this exposure is acute, but the same doses administered
chronically lead to an oxidative stress state that can produce
neurodegeneration. Astroglia, cerebral endothelial cells, and
microglia in particular respond to components of air pol-
lution with chronic activation, inflammation, and oxidative
16 Journal of Toxicology
stress . CNS effects can be chronic, can begin in early
childhood, and may accumulate with age .
Given the enormous complexity of the CNS and the
complex natureof air pollution, theresulting CNS pathology
can have many underlying causes and pathways and could
be due to synergistic interaction of multiple pathways and
mechanisms making it difficult to pinpoint a clear stimulus-
response relationship. While epidemiological data link in-
creased risk for stroke, MS, and PD to the exposure to
specific air pollutants, further experimental and mechanistic
studies aiming at the association between the components
of air pollution and the development of CNS diseases are
of pressing importance for mental health . The adverse
effects of the complex mixtures of polluted air components
are poorly understood. For instance, the contribution of
direct effects of airborne UFPs to CNS injury remains to be
worked out in detail, and data on the presence of UFPs in the
human CNS are still lacking to date. The biological studies
can be strengthened by the use of recent discovery tools
and platforms, such as proteomics and genomics, to develop
biomarkers for toxicity screening . The main problems
that are encountered in testing air pollutants toxicity in
humans are dosimetry, the lack of appropriate standardized
protocols, and good quantitative descriptions of real-world
exposure conditions [60, 142]. Novel detection methods
need to be developed for exposure assessment and dosimetry
Our current knowledge provides a basis for much more
extensive epidemiological, forensic, and toxicological studies
aimed at identifying the underlying mechanisms of neural
damage, and strengthening of the association between
chronic exposure to air pollutants, and the risk of developing
neurological diseases. However, epidemiologic and observa-
tional data are limited by imprecise measurements of pol-
lution exposure, the potential of environmental, and social
factors to confound the apparent associations. Since genetic
susceptibility is likely to play a role in response to air
pollution, gene-environment interaction studies can be a
tool to explore the mechanisms and the importance of mo-
lecular pathways for the association between air pollution
and CNS damage . Inconsistencies between studies
sometimes prevent us from drawing firm conclusions. The
limited sample size of most studies, difficulty in quantifying
exposure, providing a qualitative description of active com-
ponents from complex environmental air samples, method
of ascertainment, time of measurement, and collinearity be-
tween pollutants make difficult to use for the study of
gene by gene interactions . More studies and more
intensive collaborationsare needed to generate larger and
more diverse cohorts and standardized data that would allow
us to draw stronger conclusions . The roles of gene-air
pollution interactions and epigenetic mechanisms need to be
considered . Better understanding of the mediators and
mechanisms of CNS injury due to air pollution will help to
develop preventive and treatment strategies for the protec-
tion of individuals at risk. Improving air quality standards,
minimizing personal exposures, and the redesign of engine
and fuel technologies will also reduce air pollution and its
consequences for neurological morbidity and mortality.
Antioxidant response element
Apoptosis-associated speck-like protein
containing a CARD
Brainstem auditory evoked potentials
Central nervous system
Concentrated ambient air particles
DEPs: Diesel exhaust particles
ER: Endoplasmic reticulum
ENPs: Engineered nanoparticles
GFAP: Enhanced glial fibrillary acidic protein
GSTP1: Glutathione-S-transferase gene
GSTM1: Glutathione S-transferase Mu 1
HDAC1: Histone deacetylase 1
iNOS: Inducible nitric oxide synthase
IL-18: Interleukin 18
ICAM: Intercellular adhesion molecule
LTA: Lipoteichoic acid
MMP: Matrix metalloproteinases
MCAO: Middle cerebral artery occlusion
mtDNA: Mitochondrial DNA
MAP: Mitogen-activated protein
MAPK: Mitogen-activated protein kinase-
MS: Multiple sclerosis
NQO1: NAD(P)H dehydrogenase quinone 1
NP: Nano-sized particulate
NFTs: Neurofibrillary tangles
NO: Nitric oxide
NFκB: Nuclear factor kappa B
Nrf2: Nuclear factor (erythroid-derived 2)-like 2
NLRP3: Nucleotide-binding domain, leucine-rich
repeat, pyrin domain containing 3
NTS: Nucleus tractus solitarius
PRRs: Pattern Recognition Receptors
PD: Parkinson’s disease
PM: Particulate matter
PAHs: Polycyclic aromatic hydrocarbons
RNS: Reactive nitrogen species
ROS: Reactive oxygen species
ROFA: Residual oil fly ash
WHO: World Health Organization
TLR4: Toll-like receptor 4
TNFα: Tumor necrosis factor alpha
UPR: Unfolded protein response
Journal of Toxicology17
VEGF: Vascular endothelial growth factor
VCAM: Vascular cell adhesion molecule
VLM: Ventrolateral medulla.
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