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Nanomaterials Versus Ambient Ultrafine Particles: An Opportunity to Exchange Toxicology Knowledge

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BACKGROUND: A rich body of literature exists that has demonstrated adverse human health effects following exposure to ambient air particulate matter (PM), and there is strong support for an important role of ultrafine (nanosized) particles. At present, relatively few human health or epidemiology data exist for engineered nanomaterials (NMs) despite clear parallels in their physicochemical properties and biological actions in in vitro models. OBJECTIVES: NMs are available with a range of physicochemical characteristics, which allows a more systematic toxicological analysis. Therefore, the study of ultrafine particles (UFP, <100 nm in diameter) provides an opportunity to identify plausible health effects for NMs, and the study of NMs provides an opportunity to facilitate the understanding of the mechanism of toxicity of UFP. METHODS: A workshop of experts systematically analyzed the available information and identified 19 key lessons that can facilitate knowledge exchange between these discipline areas. DISCUSSION: Key lessons range from the availability of specific techniques and standard protocols for physicochemical characterization and toxicology assessment to understanding and defining dose and the molecular mechanisms of toxicity. This review identifies a number of key areas in which additional research prioritization would facilitate both research fields simultaneously. CONCLUSION: There is now an opportunity to apply knowledge from NM toxicology and use it to better inform PM health risk research and vice versa. https://doi.
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Nanomaterials Versus Ambient Ultrafine Particles: An Opportunity to Exchange
Toxicology Knowledge
Vicki Stone,
1
Mark R. Miller,
2
Martin J.D. Clift,
3,4
Alison Elder,
5
Nicholas L. Mills,
2
Peter Møller,
6
Roel P.F. Schins,
7
Ulla Vogel,
8,9
Wolfgang G. Kreyling,
10
Keld Alstrup Jensen,
8
Thomas A.J. Kuhlbusch,
11,12
Per E. Schwarze,
13
Peter Hoet,
14
Antonio Pietroiusti,
15
Andrea De Vizcaya-Ruiz,
16
Armelle Baeza-Squiban,
17
João Paulo Teixeira,
18,19
C. Lang Tran,
20
and
Flemming R. Cassee
21,22
1
Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot-Watt University, Edinburgh, Scotland, UK
2
Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, Scotland, UK
3
Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland
4
Swansea University Medical School, Swansea, Wales, UK
5
University of Rochester Medical Center, Rochester, New York
6
Department of Public Health, University of Copenhagen, Copenhagen, Denmark
7
IUF Leibniz-Institut für Umweltmedizinische Forschung, Düsseldorf, Germany
8
National Research Centre for the Working Environment, Copenhagen, Denmark
9
Department of Micro- and Nanotechnology, Technical University of Denmark, Lyngby, Denmark
10
Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Institute of Epidemiology, Munich, Germany
11
Air Quality & Sustainable Nanotechnology Unit, Institut für Energie- und Umwelttechnik e. V. (IUTA), Duisburg, Germany
12
Federal Institute of Occupational Safety and Health, Duisburg, Germany
13
Norwegian Institute of Public Health, Oslo, Norway
14
Center for Environment and Health, Katholieke Universiteit Leuven, Leuven, Belgium
15
Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
16
Departmento de Toxicología, Center for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV-IPN), México City, México
17
Paris Diderot University, Paris, France
18
National Institute of Health, Porto, Portugal
19
Instituto de Saúde Pública da Universidade do PortoEpidemiology (ISPUP-EPI) Unit, Universidade do Porto, Porto, Portugal
20
Institute of Occupational Medicine, Edinburgh, Scotland, UK
21
National Institute for Public Health and the Environment, Bilthoven, Netherlands
22
Institute of Risk Assessment Sciences, Utrecht University, Utrecht, Netherlands
BACKGROUND:A rich body of literature exists that has demonstrated adverse human health eects following exposure to ambient air particulate mat-
ter (PM), and there is strong support for an important role of ultrane (nanosized) particles. At present, relatively few human health or epidemiology
data exist for engineered nanomaterials (NMs) despite clear parallels in their physicochemical properties and biological actions in in vitro models.
OBJECTIVES:NMs are available with a range of physicochemical characteristics, which allows a more systematic toxicological analysis. Therefore, the
study of ultrane particles (UFP, <100 nm in diameter) provides an opportunity to identify plausible health eects for NMs, and the study of NMs pro-
vides an opportunity to facilitate the understanding of the mechanism of toxicity of UFP.
METHODS:A workshop of experts systematically analyzed the available information and identied 19 key lessons that can facilitate knowledge
exchange between these discipline areas.
DISCUSSION:Key lessons range from the availability of specic techniques and standard protocols for physicochemical characterization and toxicol-
ogy assessment to understanding and dening dose and the molecular mechanisms of toxicity. This review identies a number of key areas in which
additional research prioritization would facilitate both research elds simultaneously.
CONCLUSION:There is now an opportunity to apply knowledge from NM toxicology and use it to better inform PM health risk research and vice
versa. https://doi.org/10.1289/EHP424
Introduction
The idea of being able to manipulate materials and particles at the
molecular level sounds like a lm plot; however, over the last 25 y,
it has become rmly a part of science fact and a scienticeld in
its own right: nanotechnology. Although nanotechnology is a rap-
idly growing area of research with real-world applications in virtu-
ally every area of human activity (health care, food and nutrition,
water purication, manufacturing, and engineering, to name a few),
the introduction of a wide range of novel materials to the environ-
ment or to humans either by design or inadvertently raises the possi-
bility of harmful and/or unforeseen adverse eects. In response to
this burgeoning eld, governments and regulatory bodies have
attempted to balance nanotechnology promotion (e.g., the National
Nanotechnology Initiative in the United States and the Interagency
Working Group on Nanotechnology) with risk assessment and regu-
lation (e.g., the EU NanoSafety Cluster and associated projects such
as NANoREG). Nanotoxicology, the study of the toxicity of nano-
scale materials, has advanced in line with nanotechnology in terms
of the amount of literature being published. Indeed, unlike what has
been the case for harmful substances in the past, nanotoxicology is
running more in parallel with developments in nanotechnology.
Address correspondence to V. Stone, School of Life Sciences, Heriot-Watt
University, Edinburgh, UK. Telephone: +44 131 451 3460. Email: v.stone@
hw.ac.uk
V.S. currently receives grant funding from Byk Altana and from The
European Ceramic Fibre Industry Association (ECFIA).
In the past, V.S. has received funding from Unilever and GlaxoSmithKline.
In the past, A.B.S. has received funding for her laboratory from GDF-Suez
and BASF.
K.A.J. has research funding from private companies NanoCover A/S and I/S
Vestforbrænding.
N.L.M. has previously consulted for Abbott Diagnostics, Roche, Singulex,
and Beckman Coulter.
A.E. has received funding from Semiconductor Manufacturing Technology
(SEMATECH).
The other authors declare they have no actual or potential competing nancial
interests.
Received 17 December 2015; Revised 12 August 2016; Accepted 30
August 2016; Published 10 October 2017.
Note to readers with disabilities: EHP strives to ensure that all journal
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Environmental Health Perspectives 106002-1
A Section 508conformant HTML version of this article
is available at https://doi.org/10.1289/EHP424.
Review
The original concerns about nanotoxicology were born out of
research into particulate matter (PM) in air pollution (Figure 1;
Beelen et al. 2014;Benbrahim-Talla et al. 2012;Bouwmeester
et al. 2011;Brook et al. 2004;Donaldson et al. 2004;Homann
et al. 2007;IARC 2014;Künzli et al. 2005;Lelieveld et al. 2015;
Li et al. 2002,2003;Lim et al. 2012;Lucking et al. 2008;Lynch
et al. 2007;Lynch and Dawson 2008;Oberdörster 2010;
Oberdörster et al. 1990;Pedersen et al. 2013;Peters et al. 2001;
Pope et al. 1995;SCENIHR 2007;Stone et al. 2000a,2000b;
Unfried et al. 2007;WHO 2011,2014). This review examines key
ndings from air pollution and nanotoxicology health eects
research and the comparisons that can be drawn between these dis-
ciplines of particle toxicology. In May 2015, the COST MODENA
(European Cooperation in Science and TechnologyModelling
Nanomaterials Toxicity) project hosted a workshop to exchange
and merge knowledge in PM and nanoparticle toxicology. This
review outlines the systematic comparison of these overlapping
research elds and identies lessons for advanced understanding as
well as priority research gaps that must be addressed.
What Can Be Learned from PM Research
That Has Not yet Been Applied Effectively to
NM Research?
The Ultrafine Hypothesis and Nanomaterials
At the end of the previous century, several epidemiological stud-
ies identied health eects induced by airborne PM at levels that,
at that time, were considered safe (e.g., Brunekreef and Holgate
2002;Dockery et al. 1993). Particles <10 lm in aerodynamic di-
ameter (PM10) can be inhaled by humans and deposit in the respi-
ratory tract (ICRP 1994) (Appendix I), with smaller particles
having higher fractional deposition in the alveoli. Consequently,
ambient PM is frequently regulated as PM10 and PM2:5(<2:5lm
in aerodynamic diameter), the latter of which reects the ne
fraction of PM10. The composition of PM is complex and vari-
able (Appendix I). Although they do not contribute substantially
to the (regulated) mass, ultrane particles (UFPs) have also been
identied as one of the components that are responsible for the
adverse health eects observed at typical outdoor levels.
Evidence also exists for the involvement of other components in
the toxicity of PM, such as metals (Frampton et al. 1999;Jiménez
et al. 2000;Pope 1991) and biological components (Schins et al.
2004). The relative importance of each component is likely to dif-
fer with composition, reecting dierences in location and time.
In the 1990s, the UFP fraction was hypothesized to be respon-
sible for driving the acute respiratory and cardiovascular eects
of PM (Oberdörster et al. 1995;Seaton et al. 1995). The UFP
hypothesiswas derived from toxicological evidence from rodent
models that smaller titanium dioxide (TiO2) particles (20 nm)
were more likely than larger TiO2particles (250 nm) to cross the
lung barrier and induce inammation (Ferin et al. 1992;
Oberdörster et al. 1994). Soon after, this hypothesis was sup-
ported by epidemiological evidence (Peters et al. 1997). Owing
to the lack of readily available PM samples, health eect studies
in the following decade used surrogate particles (e.g., carbon
black, diesel engine soot, TiO2, and polystyrene beads) to investi-
gate the mechanisms of toxicity of UFPs, the results of which
were then extrapolated to PM (e.g. Li et al. 1996;Stone et al.
1998).
In contrast to ambient PM, which is derived from natural and
combustion processes, nanomaterials (NMs) are prepared deliber-
ately at the nanoscale because they exhibit properties that provide
technological advantages compared with the bulk form of the
same material (The Royal Society and The Royal Academy of
Engineering 2004) (Appendix I). For example, elemental
(graphitic) carbon has semiconductor properties at the nanoscale
(e.g., carbon nanotubes). These advantages expand the number of
possible products and applications, oering great opportunities
and economic gains. Although UFPs and NMs are often derived
from very dierent sources and processes, their physicochemical
characteristics can overlap (Appendix I), suggesting that their
properties, their behaviors, and importantly, their toxicities might
also overlap. In the early 2000s, a number of high-prole national
and international reports highlighted the importance of nanotech-
nology, but they also recognized the potential risks (e.g.,
SCENIHR 2005;The Royal Society and The Royal Academy of
Engineering 2004). These reports led to an increased interest
in UFP toxicology accompanied by a change in terminology
from the mid-2000s.
Ambient PM and UFP Health Effects
Cardiopulmonary: Epidemiologic Evidence
Epidemiology studies clearly demonstrate links between PM10
and PM2:5with both short-term and long-term health eects, par-
ticularly on the respiratory and cardiovascular systems (Dockery
et al. 1993). However, PM includes a range of particle sizes, and
very few studies have included UFP per se as a variable. Using
particle number concentration as a surrogate for UFP, exposure
has been associated with hospital admissions for acute asthma
and increased systolic blood pressure in children (Andersen et al.
2008;Pieters et al. 2015) as well as with rehospitalization in
patients with prior myocardial infarction (von Klot et al. 2005).
For hospitalization with ischemic stroke, a stronger association
was reported with particle number than with PM10 mass concen-
tration (Andersen et al. 2010). Conversely, greater associations
for particle number than mass metrics have been less convincing
for acute myocardial infarction (Lanki et al. 2006).
Particle number concentrations have also been associated
with surrogate markers of cardiovascular health. For example,
elevated levels of brinogen, prothrombin factors 1 and 2, and
von Willebrand factor are associated with exposure to UFP
(Hildebrandt et al. 2009). Independent associations have been
observed for UFP and PM2:5with heart rate and heart rate vari-
ability in patients with diabetes mellitus and glucose intolerance
(Peters et al. 2015;Sun et al. 2015). In patients undergoing car-
diac rehabilitation, modulation of the parasympathetic innerva-
tion of the heart, increased blood pressure, and markers of
systemic inammation were all associated with exposure to UFP
(Rich et al. 2012). Epidemiological studies involving biomarkers
related to oxidative stress and inammation revealed that primary
combustion markers from quasi-UFP (PM<0:25) were positively
associated with systemic changes in interleukin 6 (IL-6) and tu-
mor necrosis factor alpha (TNFa), platelet activation, and eryth-
rocyte antioxidant enzyme activity in an elderly population
(Delno et al. 2009). Similarly, elevated plasma brinogen and
white blood cells have been associated with UFP exposure (Gong
et al. 2014).
Cardiopulmonary: Preclinical and Clinical Evidence
Several preclinical and clinical studies have addressed the short-
term inhalation and respiratory eects of UFPs. For example,
eld studies have observed associations for UFPs and carbon
with reductions in lung function among asthmatics (McCreanor
et al. 2007), and healthy adolescents and adolescents with asthma
in New York exhibited an increase in indicators of inammation
(Patel et al. 2013). The majority of preclinical and clinical studies
on UFPs have been conducted with diesel exhaust and diesel
exhaust particles (DEPs), an especially rich source of UFPs
Environmental Health Perspectives 106002-2
Figure 1. Time line showing the increased interest in particulate matter (PM) and nanomaterials (NMs) over the last three decades, highlighting key studies
and research trends in both areas. Number of references per year (noncumulative) based on Pubmed (https://www.ncbi.nlm.nih.gov/pubmed/) search without
further limits applied.
Environmental Health Perspectives 106002-3
(Figure 2). These studies have shown airway inammation in
healthy individuals, including elevated levels of inammatory
cells and mediators (Ghio et al. 2012;Xu et al. 2013;Yamamoto
et al. 2013).
Inhaled UFPs modify numerous aspects of cardiac function,
reducing heart rate variability (Cassee et al. 2011;Pieters et al.
2012), a predictor of cardiovascular risk, and increasing the inci-
dence, duration, and severity of arrhythmia (Delno et al. 2005;
Robertson et al. 2014). Furthermore, UFPs in urban air
(Weichenthal 2012) or diesel engine emissions (Mills et al. 2007)
exacerbate myocardial ischemia (Cascio et al. 2007;Robertson
et al. 2014). Blood vessels nely regulate blood ow through
changes in the tone of vascular smooth muscle, and UFPs gener-
ally alter the balance in favor of constriction (Møller et al. 2011).
The resulting increased blood pressure (Bartoli et al. 2009) and
the reduced ability of the arteries to relax are usually detrimental.
Vascular dysfunction can be caused by a loss of mediators such
as nitric oxide released by the vascular endothelium (Courtois
et al. 2008;Miller et al. 2009;Møller et al. 2011), by increased
sensitivity to vasoconstrictor factors (Langrish et al. 2009), and
by alterations in baroreceptor/neuroregulatory feedback (Rhoden
et al. 2005;Robertson et al. 2012). Blood components are also
dysregulated, with UFPs tending to increase blood coagulability
(Kilinç et al. 2011;Nemmar et al. 2004), encourage platelet acti-
vation (Cascio et al. 2007;Lucking et al. 2011), and reduce blood
clot clearance (Mills et al. 2005). The cellular and biochemical
mechanisms underlying these eects are wide-ranging, with oxi-
dative stress and inammation being key drivers (Miller et al.
2012)(Figure 3). In combination, these actions promote cardio-
vascular disease. Indeed, long-term exposure to UFPs in animal
models (Araujo et al. 2008;Miller et al. 2013) has been shown to
worsen atherosclerotic vessel disease.
Other Target Organs
Although research has predominantly focused upon the inhalation
of UFPs and their impact upon cardiovascular function, a number
of additional, secondary target organs have been investigated (see
Figure 3). Such research has been based upon the hypothesis of
alveolar translocation of UFPs to the bloodstream allowing for
nonspecic interaction with other essential organs such as the
brain and kidneys. UFP exposure and mucociliary clearance from
Figure 2. Schematic providing an example of the complex composition of
ultrane particles (UFPs) [e.g., urban particulate matter (PM) or particles in
vehicle exhaust], which in urban air often have a carbon core coated with a
diverse range of chemical species including reactive transition metals and or-
ganic hydrocarbons. Detail is not to scale.
Figure 3. Schematic illustrating some of the key mechanisms through which inhaled ultrane particles (UFPs) may inuence secondary organs and systemic
tissues, with emphasis on the means through which inhaled particles may cause cardiovascular events. Note that there are three main pathways linking the pul-
monary and cardiovascular systems (grey arrows, left to right): autonomic regulation, passage of inammatory mediators, and particle translocation. The
arrows between these three pathways highlight the degree of interaction between mechanistic pathways and the challenges involved in broad categorization of
the wide-ranging biological actions of inhaled UFPs. Added to these pathways is the potential for desorbed components to exert eects.
Environmental Health Perspectives 106002-4
the lungs into the gut might also be linked with adverse eects on
lipid metabolism and intestinal villus shortening (Li et al. 2015),
conveying evidence of eects with potential clinical relevance
for gut or liver diseases.
Starting approximately 15 y ago, the eects of PM in the
central nervous system (CNS) gained recognition with reports
that exposure to polluted Mexico City air resulted in oxidative
stress, inammation, neuropathology, and cognitive and behav-
ioral changes in humans and in animals (Calderón-Garcidueñas
et al. 1999,2011). Other studies using a myriad of PM collec-
tion techniques upheld the early ndings related to PM-
induced brain-centric inammatory processes, including those
in regions related to learning and memory (Campbell et al.
2005;Fonken et al. 2011). Such health outcomes could be
explained by ndings that inhaled particles can travel to the
brain via the blood following alveolar deposition, via nose
brain transport following olfactory mucosa deposition
(Balasubramanian et al. 2013;Elder et al. 2006), or via the
spillover of systemic inammation to the CNS; a combination
of these processes is also possible. Although acute CNS inam-
matory processes cannot be directly measured in living
humans, it is interesting to note that neurodegenerative dis-
eases are on the rise and that there is a well-establishedalbeit
mechanistically murkylink between inammation and neuro-
degeneration (Akiyama et al. 2000;Amor et al. 2010). Recent
research has focused on one area where animal and human out-
comes have good concordance, namely, behavior and cogni-
tion. For example, Fonken et al. (2011)showedthatmice
exposed to PM2:5(which includes UFPs) had decits in spatial
learning and memory. Using mice exposed to concentrated
UFPs as neonates, Allen et al. (2014a,2014b) showed that
males had behavioral outcomes that were associated with per-
sistent enlargement of the ventricles and innate immune cell
activation. In population-based studies, several investigators
have now reported associations between trac aerosol expo-
sures and reduced cognitive function in the elderly (Ranft et al.
2009) and in children (Freire et al. 2010;Suglia et al. 2008).
Two U.S.-based casecontrol studies have also reported
increased odds ratios for autism in association with early-life
exposure to trac-related pollution, specically PM2:5
(Becerra et al. 2013;Volk et al. 2013). With the exception that
NM research has demonstrated plausibility for PM transloca-
tion to the brain, very little has been investigated in terms of
the nervous system impacts of NM.
Priority research gap. PM research provides a basis for devel-
oping a strategy to identify potential neurological eects of NMs
in which physicochemical characteristics could be responsible.
Epidemiological studies have also related PM2:5and PM10 air
pollution to reproductive toxicity and to adverse eects on the prog-
eny. A recent systematic review (Stieb et al. 2012)reportedanasso-
ciation between exposure to PM2:5and PM10 and low birth weight,
preterm birth, and small-for-gestational-age birth. Additionally, van
Rossem et al. (2015) found that maternal exposure to PM2:5and
black carbon were associated with increased blood pressure in new-
born children. The eect seems to be mediated by altered placental
vascular structure induced by PM2:5(Veras et al. 2008). Preclinical
studies indicated that adverse health eects of UFP exposures can-
not be excluded, although the potential for hazard has not been well
characterized (Hougaard et al. 2015).
The evidence outlined above demonstrates the impact of am-
bient PM on a range of targets, but in particular the adverse
eects on respiratory, cardiovascular, neurological, and reproduc-
tive systems. For cardiovascular studies, this evidence extends to
UFPs, but direct evidence for the role of UFPs in the induction of
the other disease targets is, in general, still lacking.
Investigation of NM Impacts on Human Health
A few studies are now emerging that demonstrate the eects of
nanomaterials on human health, particularly in an occupational
setting. Lee et al. (2015) investigated workers manufacturing
multiwalled carbon nanotubes and found that although there was
no impact on hematology and blood biochemistry, they did see
an increase in a range of markers of lipid peroxidation in exhaled
breath condensates of the workers, including malondialdehyde,
4-hydroxy-2-hexenal and n-hexanal. Multiwalled carbon nano-
tubes have also been reported to have impacts on a range of end
points in workers exposed for 6 mo. These end points include
the targeting of genes associated with cell cycle regulation, pro-
gression, and control, as well as genes involved in apoptosis and
proliferation (Shvedova et al. 2016). The same study also identi-
ed targeting of pathways involved in pulmonary and cardiovas-
cular eects, as well as pathways involved in carcinogenic
outcomes in humans.
Another study followed workers in 14 nanomaterial manufac-
turing or application (or both) factories in Taiwan for 6 mo (Liao
et al. 2014). The nanomaterials that were produced or handled
included silver, iron oxide, gold, titanium dioxide, carbon nano-
tubes, and silicon dioxide. The group working with nanomaterials
exhibited higher levels of antioxidant enzymes and cardiovascu-
lar markers than the workers who handled other materials. In
addition, the study also reported that markers of small airway
damage (Clara cell protein 16) and lung function were signi-
cantly associated with handling nanomaterials.
Liou et al. (2015) reviewed 15 studies that investigated the
eects of engineered nanomaterials on workers. Of these 15 stud-
ies, 11 were cross-sectional, four were longitudinal, and one was
a descriptive pilot study. All of the 11 cross-sectional studies
showed a positive relationship between various biomarkers and
exposure to engineered nanomaterials. Three of the four longitu-
dinal studies demonstrated a negative relationship, with the
fourth showing a positive relationship after 1 y follow-up. In gen-
eral, the exposure levels identied were not very high compared
with those used in human inhalation chamber studies; however,
there were some exceptions with higher exposures. The studies
were generally found to be limited by small numbers of partici-
pants, a lack of consistent exposure information, the detection of
generally low exposures, and short intervals between exposure
and eect.
Taken together, these initial human health studies suggest that
occupational exposure to nanomaterials may have detrimental
impacts on human health. Further work is required over the long
term to ascertain the nature and extent of these eects as well as
their relevance to dierent types of materials.
Lesson 1. A rich body of literature exists that has demon-
strated adverse human health eects following exposure to PM,
with a proportion of that literature providing support for UFP
involvement. In contrast, although initial studies have suggested
an association between exposure to nanomaterials and human
health, relatively few clinical or epidemiology data are available
at the present time.
Figure 4 outlines a range of health eects and biological indi-
cators of disease reported in the literature. This information can
be used to better inform and justify NM study end points.
Mechanisms of UFP-Induced Health Effects
Cardiovascular Effects
Three hypothetical pathways to explain the cardiovascular eects
of PM predominate: inammation,”“autonomic regulation,
and particle translocation(Figure 3). The classical hypothesis
Environmental Health Perspectives 106002-5
is that particles inhaled into the lung are taken up by alveolar
macrophages, triggering an inammatory response within the
lung. A sucient particle dose, reactivity, or lack of clearance
leads to amplication of the response with a resultant spill-over
of inammatory mediators into the blood causing systemic
inammation (Seaton et al. 1995), which is strongly associated
with cardiovascular disease. Alternatively, inhaled particles (or
the inammatory response resulting from inhalation of the par-
ticles) stimulate alveolar sensory receptors (Ghelet al. 2010;
Hazari et al. 2011;Robertson et al. 2014), providing a signal to
the CNS. This response manifests through alterations in auto-
nomic nervous system activity, which directly regulates cardiac
function and, indirectly, other aspects of the cardiovascular sys-
tem (Pope et al. 1999;Rhoden et al. 2005). The identication of
the UFP fraction of PM paved the way for a third hypothesis:
The minute size of UFPs allows them to translocate across the
thin alveolar-capillary wall (by an as-yet undetermined mecha-
nism) and enter the circulation themselves to directly aect cardi-
ovascular function (Nemmar et al. 2001;Oberdörster et al. 2002).
There is a wealth of evidence for and against each of these
hypotheses, but in truth, all three are likely to occur; the contribu-
tion of each is dependent on the physicochemical properties of
the UFPs, the cardiovascular end point under investigation, and
the susceptibility of the person/model being explored (Miller
2014). Furthermore, it is highly likely that many of the subtleties
of these pathways have yet to be identied. The intricacies of
these processes may encompass nonclassical inammatory/oxida-
tive biological mediators such as acute-phase proteins (Saber
et al. 2014) or oxidized phospholipids (Kampfrath et al. 2011),
the release and accumulation of chemical particle surfaces and
constituents within biological compartments (Murphy et al. 2008;
Totlandsdal et al. 2015), particle/plasmaprotein interactions
(Deng et al. 2011;Monopoli et al. 2012), and the role of proteins/
inammatory cells in carrying/accumulating particles to suscepti-
ble areas of the body (Schäer et al. 2014). Reports are rapidly
emerging from preclinical models that demonstrate cardiovascu-
lar eects for NMs similar to those shown for UFPs, such as
altered autonomic function (Harder et al. 2005), impaired vasodi-
latation (Leblanc et al. 2010;Møller et al. 2011), blood hypercoa-
gulability (Kim et al. 2012;Radomski et al. 2005), and
aggravated atherosclerosis (Li et al. 2007;Mikkelsen et al. 2011;
Niwa et al. 2007). Identication of the biological mechanisms for
these parallel observations will have important consequences for
both elds of research.
Lesson 2. The information obtained from PM epidemiology
and mechanistic research has provided an evidence base on
which to develop hypotheses to stimulate research into the poten-
tial modes of action for NMs.
Genotoxicity/Carcinogenicity
Markers of genotoxicity, such as elevated levels of oxidized DNA
nucleobases, bulky DNA adducts, and clastogenic end points in
leukocytes have been documented in biomonitoring studies of
humans. Positive associations between UFP and oxidatively dam-
aged DNA in mononuclear blood cells have been observed
(Bräuner et al. 2007;Vinzents et al. 2005). In contrast, there is a
paucity of studies on UFP-generated oxidative DNA oxidation
products in cultured mammalian cells and experimental animal
models (Møller et al. 2014), as well as a lack of studies on neo-
plastic lesions in the respiratory tract (a priority research gap).
Studies including exposure to trac-related outdoor air pollution
or to DEPs (Stinn et al. 2005;Valberg and Crouch 1999) have
identied increased lung adenomas in rodent models.
The carcinogenic mechanism is believed to involve genotoxic-
ity by both oxidative reactions and formation of bulky DNA
adducts from polycyclic aromatic hydrocarbons (PAHs), which
may give rise to mutation and structural chromosome damage.
Early genotoxic events such as DNA adducts and small nucleobase
oxidative lesions can be generated by primary (direct) mechanisms
in relevant target cells, whereas oxidative-mediated DNA damage
may also occur as a consequence of secondary inammation-
driven events (Schins and Knaapen 2007). Importantly, the last
mechanism has been discussed as a major contributor to the muta-
genic and carcinogenic properties of DEPs, as well as to those of
poorly soluble nanoparticles such as carbon black and titanium
dioxide in long-term high-dose inhalation studies in rats (Knaapen
et al. 2004). However, the relevance of this mechanism towards
the carcinogenicity of PM and the specic contribution of the UFP
component herein remains to be elucidated (a priority research
gap). Recent reviews comparing the genotoxicity of DEPs and
NMs have indicated similar mechanistic causes of DNA damage
(Magdolenova et al. 2014), although the dose metric of mass con-
centrationcauses diculties in comparison across studies
(Møller et al. 2015). Interestingly, it has recently been reported
that single- and multiwalled carbon nanotubes could interfere with
the mitotic spindle apparatus (Sargent et al. 2012;Siegrist et al.
2014).
Lesson 3. NM research provides an opportunity to better
understand the (mechanistic) role of UFPs in the genotoxicity,
mutagenicity, and carcinogenicity of PM.
Lessons Learned from NM Toxicology That Could Be
Applied to PM
Regulatory standards for ambient PM are promulgated from a
rich body of literature that has demonstrated adverse human
health eects following exposure. Conversely, the toxicological
research on NMs is motivated by a desire to dene material
Figure 4. A range of health eects and biological indicators of disease that
can be used to identify relevant end points for study design.
Environmental Health Perspectives 106002-6
properties that are linked to adverse health eects, thus support-
ing eective risk management. To achieve engineering for safety
goals, it is necessary to understand the toxicity of the NMs them-
selves while recognizing that toxicological assessment is only
one part of an overall risk assessment process. However, it is
impossible to determine safer exposure levels and safer materials
design without rst understanding dose-specic toxicological
eects. Studies that address these questions have provided a
wealth of knowledge that might now be useful to better under-
stand the toxicology of PM.
Characterization of Test Materials
In the past, characterization of UFPs has focused on mass and
number concentrations, chemical composition, and size distribu-
tion. Studies initially used existing methods obtained from PM
and materials science research to characterize NMs, but over
time, the methodology has been rened. This renement has
allowed NM toxicological researchers to generate a list of desired
characterization information (https://www.iso.org/obp/ui/#iso:
std:iso:ts:17200:ed-1:v1:en). A similar list of requested charac-
terization end points was not developed in PM research owing to
the lack of understanding of how dierent physical and chemical
characteristics could interact to inuence PM toxicity. However,
a comparable list for PM health eect studies could be benecial
for understanding mechanisms as well as for enhancing compara-
bility across elds. In the future, these techniques will provide an
opportunity for improved monitoring of PM.
Lesson 4. A range of particle characteristics have been shown
to inuence their toxicity. These characteristics should also be
considered in UFP research, where appropriate characteristics
that can be used for the prediction of toxicity have not yet been
identied. Furthermore, the variance of these characteristics in
space and time should also be determined comparably to the
methods used for NMs.
Before the development of NM toxicity testing, standard
operating procedures (SOPs) for particle characterization were
rarely in place. Development of SOPs is currently ongoing in the
nanosafety communities as well as via standardization projects
conducted by the European Committee for Standardization
(CEN) and the International Organization for Standardization
(ISO) (European Commission. 2010).
Especially relevant particle parameter protocols for standardi-
zation are those of dispersion, size, agglomeration, and aggrega-
tion in dierent environmental and biological media. Techniques
that can (semi-)automatically obtain multiple size parameters
using transmission electron microscopy will help to satisfy the
challenges of regulatory NM denitions such as those proposed
by the European Commission (2011). Advances in dynamic light
scattering and coupling to inductively coupled plasma mass spec-
trometry (ICPMS) technologies such as eld-ow fractionation
multiangle light scattering ICPMS and single-particle ICPMS have
been driven by requirements for particle sizing and behavior in liq-
uid dispersions. Great improvements have also been made in under-
standing the applicability of dierent measurement devices for
airborne particles. These improvements include knowledge of the
care needed in using and interpreting data obtained from charge-
based instruments, particularly instruments using unipolar charging
such as surface area monitors and the Fast Mobility Particle Sizer
(Asbach et al. 2009;Levin et al. 2016). These instruments can pro-
vide erroneous results when signicant amounts of agglomerates
and aggregates that are approximately 200 nm or more in size are
present in the aerosol (Todea et al. 2015).
Advances have also been made in chemical analysis of NMs,
where ICPMS is often the preferred method, using either the
single-particle mode or particle extraction protocols (Lee et al.
2014). Nondestructive methods such as Instrumental Nuclear
Activation Analysis (INAA) and X-ray uorescence (XRF) may
be preferable for bulk chemical characterization to avoid chal-
lenges in developing material-specic extraction techniques.
New developments also include procedures to identify and
quantify specic surface coatings/functionalization of NMs using
combinations of dierential thermal gravimetric/dierential ther-
mal analysisgas chromatography and chemical-specic methods
such as high-performance liquid chromatographymass spec-
trometry/optical emission spectroscopy or gas chromatography
mass spectrometry. Combinations of these methods are particu-
larly important for second- and third-generation NM analysis,
and methods have recently been developed as part of the EU FP7
NANoREG project (European Commission 2013). Further
knowledge transfer of techniques between materials science,
environmental, and nanosafety researchers continues and is
highly likely to be applicable to PM research.
Analysis of surface charge via zeta potential measurements
is straightforward in simple systems (e.g., a pure NM in pH-
controlled water with moderate ionic strength) but becomes chal-
lenging in multicomponent complex systems such as PM in air
pollution. From a toxicological perspective, because zeta poten-
tial varies signicantly with pH and with the composition of the
test medium, a full assessment should consider all likely media
and biological compartments of interest.
Particle reactivity is currently not well dened, perhaps
understandably given that it is unlikely to be a single parameter.
Simplemethods include measurement of reactive oxygen spe-
cies, pH and redox potential, and band gap. In recent years, band
gap has been shown to be related to the toxicity of metal oxide
NMs (Zhang et al. 2012). In vitro dissolution can also be an im-
portant indicator of reactivity for some NMs insofar as it is indic-
ative of biodurability/biopersistance, methods which are under
development (CEN/ISO). Recent work has shown that great care
must be taken in the design and harmonization of such experi-
ments to achieve reproducible results (Tantra et al. 2016). These
developments are relevant for both NM and PM research,
although the weight has been strongly tilted towards NM research
in the last decade.
Lesson 5. A range of new and improved techniques for
assessing the physicochemical and nanoscale characteristics of
NMs have been developed. These techniques should be applied
appropriately to inhalation exposure assessment in population
studies to better determine the relationship between particle char-
acteristics and health eects.
It is worth noting that the procedures used for sample prepara-
tion and the mode of exposure used in toxicology studies deter-
mine the requirements for characterization of exposure and fate.
For example, quantication and characterization of aerosolized
particles might include aerosol monitors and lter samples,
whereas particles used as dispersions for exposure would require
analysis via hydrodynamic size distribution, agglomeration state,
sedimentation, and reactivity in the dispersion medium. NMs are
often dispersed in protein-rich media, which can aect both the
biokinetics and the toxicity, whereas ambient UFPs can be dis-
persed to some extent without these additives (Moore et al. 2015).
The improvements in characterization have revealed the
need for correct storage of NM test items over time. For NMs
analyzed by the Organisation for Economic Co-operation and
Development (OECD) working party of manufactured nanomate-
rials (WPMN), this has resulted in storage under argon, in single-
use vials, in the dark. Previously, both nanosafety and PM
researchers have stored dry powders, lter-bound particles, and
wet suspensions under many dierent conditions. For many am-
bient PM samples, storage under argon at <0C could potentially
Environmental Health Perspectives 106002-7
prevent oxidation/loss of toxicologically relevant (semi)volatile
substances.
Exposure Characterization
Since the emergence of nanotoxicology as a discipline, it has
become increasingly recognized that particles can be modied
upon interactions with cells and tissues, for example, owing to
the inuence of the surrounding media (e.g., proteins). Such bio-
molecule interaction is likely to impact the fate of the particles by
modifying the surface properties and the behavior of the particle
(e.g., agglomeration, solubility, bioavailability, biodurability) as
well as the adsorbed protein properties (Brown et al. 2010a;
Deng et al. 2011). These modications could, in turn, alter how
particles are taken up into cells, how they trigger signaling path-
ways, and in what physicochemical format they are translocated
between cells and to distal organs.
Characterization of NMs at various stages throughout the life
cycle of the material is far from simple. For PM, this is further
complicated by the complex mixtures present in ambient air.
Furthermore, it is important to note that consumer exposure to
NMs may be dierent than occupational exposure depending on
the state of the material (e.g., native NMs, NMs embedded in a
product, NM degradation and disposal). In toxicology studies,
many NMs have been studied as dispersions (usually of agglom-
erates) in biological or culture media. Several protocols for NM
dispersion have been developed using dierent dispersion princi-
ples and media (Hartmann et al. 2015); rst attempts have also
been made to establish harmonized dispersion for regulatory test-
ing (e.g., HA Jensen, H Crutzen, A Dijkzeul, unpublished work,
2014). In contrast, harmonized dispersion protocols have largely
been lacking in PM research.
Lesson 6. Harmonized dispersion protocols can be transferred
to PM research to increase harmonization and comparability
between test methods and results.
There is, however, a major obstacle for PM research, in that
much smaller amounts of PM (obtained via collection) are nor-
mally available than for NM research, where direct synthesis is
often possible. For ambient PM, it is necessary to collect and
extract PM from the collector (e.g., scraping, sonicating, or
chemically extracting from a lter), which may alter the state of
the PM before its use in toxicology studies. Transformation and
loss of toxicologically important semivolatile compounds during
sampling is also an issue that must be taken into account when
testing collected PM.
Lesson 7. The need to extract PM from lters can be avoided
by moving the laboratory to the eld, for example, using in vivo
or airliquid interface (ALI) systems. In addition, systems such
as particle concentrators (Gupta et al. 2004;Kim et al. 2001)
have been developed; these systems help to ensure sucient
doses over the period of the experiment. However, such toxico-
logical studies in the eld can be expensive and additionally
complicated.
Inhalation Exposure and Deposited Dose
The experimental data for total lung deposition of particles are
highly consolidated (ICRP 1994); however, the regional deposi-
tion of NMs is weakly supported by direct experimental data to
validate the models (a priority research gap).
The deposition of NMs depends on three sets of parameters:
particle dynamics, lung geometry, and gas ow dynamics. Owing
to their size, the primary region for the deposition of NMs is the
alveolar region of the human lung (a priority research gap), which
means that the rst biological matrix encountered is lung surfac-
tant (Gasser et al. 2010). Interaction with NMs may alter the
structure and function of the surfactant proteins (Beck-
Broichsitter et al. 2014;Valle et al. 2015) and subsequently inu-
ence the specic mammalian cell interactions (Schleh et al.
2013).
Lesson 8. NM and parallel UFP inhalation studies can provide
important information on pulmonary deposition and surfactant
interactions and would facilitate the investigation of comparabil-
ity between both types of particles.
For PM and NM toxicology studies, consideration of relevant
particle doses is required. A daily inhalation mass dose for PM
that relates to maximal air-quality standards has been suggested
[WHO Global Air Quality Guidelines (WHO 2016): 10 lg=m3
for PM2:5]. A daily inhaled volume for a moderately active adult
human (75 kg) is typically 20 m3=d. If the mean deposited frac-
tion is 0.3 (Price et al. 2001), the suggested daily mass dose
would be 60 lg=d. However, PM mass is often dominated by
coarse particles, so only a fraction of the 60 lg=d would actually
represent UFPs.
Therefore, dose might be better expressed as particle num-
ber for such small particles because of their low mass. There is
great variability in ambient UFP numbers, ranging from 500
10,000 particles=cm3in rural areas to 750025,000 particles=
cm3in urban background (Putaud et al. 2010), and with a
European mean concentration of 31,500 particles=cm3at hot
spots (busy streets). To estimate in vivo exposure conditions
based on particle number concentrations, a healthy adult breath-
ing during moderate exercise in ambient air with an assumed
moderate concentration of 30,000 particles=cm3(of which 80
90% of the particle count is assumed to be UFPs) will inhale
6×10
11 particles=cm3. Assuming a mean deposition probability
forUFPsof0.5,thiscorrespondstoadoseof3×10
11 particles
deposited per day or 1:2×10
10 particles deposited per hour
(Geiser and Kreyling 2010).
To obtain a more relevant assessment of health eects in
vitro, the UFP and NM dose per cell number or area should
reect real inhalation. The initial use of high UFP or NM doses
may be justied by the need to be able to detect eects of expo-
sure, but such high doses need to be accompanied or followed up
with studies using realistic doses in relation to current informa-
tion concerning occupational and ambient exposures to UFPs or
to specic NM types.
The respiratory zone of the lung represents by far the largest
compartment for NM deposition. Estimates of lung physiological
data (Stone et al. 1992), together with the above-mentioned
European mean ambient exposure concentration of 31,500
particles=cm3, suggest that on average 8 nanoscale particles de-
posit per day per cell of the alveolar epithelial surface (Geiser
and Kreyling 2010). According to limits imposed by thermody-
namic conditions, the highest possible NM aerosol number con-
centration is approximately 106=cm3, which translates to 700
particles/d/cell or 30 particles/h/cell of the alveolar epithelium.
Because mass is a more frequently used metric, these numbers
must be converted using the eective density of the particles.
This information is useful when verifying relevant in vitro doses.
Models have been published (including experimental verica-
tion) that can estimate particle deposition onto a monolayer of
cells in vitro (Cohen et al. 2013;Hinderliter et al. 2010;
Teeguarden et al. 2007). These studies identify NMs, and the
eective density of NMs, as important factors in modeling cellu-
lar dose. In essence, only submicrometer and larger NM agglom-
erates will deposit within one hour, whereas NMs <100 nm may
remain suspended for >24 h.
Lesson 9. The models of in vitro NM deposition should be
more widely used for estimation of NM dose in cultured cells to
rene models so that they better reect anticipated airborne
Environmental Health Perspectives 106002-8
exposures. Their application to PM would be more dicult
owing to the size and density diversity of such a mixed particu-
late sample. However, such dosimetry models (DeLoid et al
2015) can in principle deal with size distribution data as well as
with mean size data in cases where such distribution data are
measured. Thus, the lack of a narrow size distribution for PM
should not preclude the more eective use of such modeling
advances to improve dosimetry in both the NM and PM elds.
Uptake, Clearance, and Fate following Pulmonary Exposure
In rodent models, within 12 d of exposure, NM clearance is rela-
tively low compared with clearance of micrometer-sized par-
ticles, and it is associated with more rapid and extensive uptake
into epithelial cells (Kreyling et al. 2002;Semmler-Behnke et al.
2007;Semmler et al. 2004). However, the rodent model is not a
good reection of clearance in humans. For humans, there is evi-
dence that both NMs and micrometer-sized particle clearance
from the conducting airways is less extensive, leading to
long-term particle retention (Möller et al. 2008). Interestingly,
in dogs (Kreyling et al. 1999) and monkeys (Nikula et al.
1997), long-term retention increases substantially in conduct-
ing airways with decreasing particle size. Considering long-
term clearance predominantly from the alveolar region in
these species, macrophage-mediated clearance occurs at a rate
that is one order of magnitude lower than that in rodents
(Kreyling 2013).
Priority research gap. For the human distal regions of the
lungs, neither macrophage-mediated long-term clearance kinetics
data nor translocation data for NMs into the circulation are cur-
rently available.
Particle clearance from the lungs can also occur via transport
toward lymph nodes and translocation into the blood circulation,
leading potentially to accumulation in secondary organs and tis-
sues. These pathways will have a limited or lesser relevance for
the clearance of rapidly or moderately soluble particles, respec-
tively (Oberdörster et al. 2005).
Lesson 10. NM inhalation studies provide details of the
potential for UFP biopersistence and transport based upon their
solubility.
Pioneering studies in the 1990s rst demonstrated detectable
translocation of nanoscale TiO2particles into the lung intersti-
tium and that translocation is lower for larger particles (e.g.,
21 nm vs. 250 nm diameter) (Ferin et al. 1991;Oberdörster et al.
1994). More recently, a comprehensive list of inhalation studies
and instillation studies using various NMs has provided consen-
sus that, in rodents, relatively small fractions (approximately
1%) of inhaled NMs are translocated across the airblood bar-
rier, leading to accumulation in secondary organs, including the
liver and spleen (Balasubramanian et al. 2013). Notably, this
rat inhalation study used gold NM of dierent primary particle
sizes but agglomerated to give the same diameter in air of
45 nm. The authors demonstrated size-related translocation
with the smaller 7 nm primary gold particles translocating more
than the 20 nm primary gold particles, suggesting deagglomera-
tion of the 45 nm agglomerates in the lung.
Priority research gap. Based on the observation that in
humans, NM retention in the lung is likely to be longer than in
rodents, it is necessary to consider whether the relatively small
translocation proportions identied in rodents might be greater in
humans.
NM research has facilitated understanding of the biokinetics
and biodistribution of particles such that there is now clear evi-
dence that inhaled NMs can reach and accumulate in secondary
organs (Geiser and Kreyling 2010;Kreyling 2013). Quantitative
biokinetics analysis of NMs applied via the lungs of rodents
demonstrated small fractions of NM (iridium, carbon, gold,
TiO2) in all secondary organs studied, including the brain and
heart, and even in fetuses (Kreyling et al. 2002;Semmler-Behnke
et al. 2007;Semmler-Behnke et al. 2014). An inhalation study
using 20-nm iridium NM was extended to six months after a sin-
gle 1-h inhalation and yielded signicant retention in the liver,
spleen, kidneys, heart, and brain (Semmler-Behnke et al. 2007;
Semmler et al. 2004). Although the fractions of the total dose that
reach these tissues are very small in rodents, the studies have
highlighted the importance of epithelial barrier health (Heckel
et al. 2004) and that the particles protein corona aects biodistri-
bution (Kreyling et al. 2014). As a corollary to biodistribution
studies, in vitro studies with NMs have helped to dene the abil-
ity of particles to breach cellular membranes (Bachler et al.
2015), their interactions with subcellular structures, and there-
fore, the toxicological mechanisms related to particle uptake.
Lesson 11. NM translocation studies provide clear evidence
of the potential for UFPs to translocate from the lung surface into
blood and to distribute throughout the body, accumulating in a
range of secondary organs. The knowledge gained from NM bio-
kinetics and biodistribution studies provides an evidence base to
predict the fate and health eects of UFPs in the body.
In addition to translocation, rodent studies also suggest that
NMs can relocate from the interstitium and epithelium back onto
the epithelial surface via an unknown mechanism (e.g., via mac-
rophages) (Semmler-Behnke et al. 2007). The fraction removed
via the lymphatic or cardiovascular system is relatively small in
contrast. Studies using NMs (sometimes at doses higher than a
few hundred micrograms per lung) such as TiO2, carbon black,
gold, quantum dots, silver nanowires, and carbon nanotubes have
identied accumulation of NMs in the lung-associated lymph
nodes (Schinwald et al. 2012).
Priority research gap. The development of this research area
using dierent types of NMs and dierent investigative protocols
will be useful in determining the importance of this potential
route of uptake and hence potential translocation within the body.
Pathways of relocation of inhaled NMs in rodent lungs are sche-
matically sketched in Figure 5.
Lesson 12. The dierential clearance and uptake of NMs and
micron-sized particles could also apply to the varied size fractions
of PM, adding to the plausibility of a dierence in their toxicity.
Toxicological Mechanisms
There are a number of mechanisms by which UFPs and NMs
may have an impact on cells, and these mechanistic studies pro-
vide a great opportunity for comparison or alignment of our
understanding of NM and UFP toxicity. Mechanisms including
reactive oxygen species (ROS) and oxidative stress (Miller 2014;
Nel et al. 2006;Stone et al. 2007) feature widely in the literature
for both NMs and UFPs (see below). Endothelial cells and epithe-
lial cells may also generate nitric oxide in response to NMs and
UFPs via stimulation of NOX4 (e.g., in addition to the respiratory
burst generated by inammatory cells exposed to particles, it
appears that particles can also generate ROS directly, including
PM10 (Gilmour et al. 1996), DEPs (Miller et al. 2009), and many
dierent NMs including carbon black (Stone et al. 1998;Wilson
et al. 2002), polystyrene beads (Brown et al. 2001), and a range
of metal/metal oxide particles (Dick et al. 2003;Rushton et al.
2010). Dierent material compositions vary in their potential to
induce ROS production, ranging from copper (Rushton et al.
2010), which has an inherent ability to generate ROS, to amor-
phous nanosilica, which exhibits no intrinsic capacity to generate
oxidants (Napierska et al. 2012). Some NMs do not exhibit intrin-
sic oxidant-generating capacity but will generate ROS upon
Environmental Health Perspectives 106002-9
interaction with cellular targets, causing changes in the intracellu-
lar redox status (Hussain et al. 2009).
However, studies with NMs suggest that the mechanisms of
toxicity may be more diverse than via oxidants, including
direct physical NMcell interaction, receptor-mediated, or other
unknown mechanisms (the last of which is a priority research
gap) (Thomassen et al. 2011). Increased epidermal growth fac-
tor receptor expression and phosphorylation have also been
observed for DEPs (Pourazar et al. 2008).
Lesson 13. The ability of PM, UFPs, and NMs to generate
ROS and to induce oxidative stress, either intrinsically or via cel-
lular sources, has been well documented and is frequently associ-
ated with mechanisms of toxicity. In addition, both NM and DEP
studies have demonstrated receptor activation, and NM research
has also identied other potential mechanisms such as direct
physical cell interaction and unknown mechanisms that require
further investigation.
Lesson 14. For NMs and UFPs that generate ROS, the amount
of ROS production is likely to be associated with their physical and
chemical properties. This is important for UFPs because dierent
toxicities could result as compositions vary with time and location.
The induction of oxidative stress by NMs and by PM has been
linked to proinammatory intracellular signaling responses and
cytokine production (Baulig et al. 2009;Brown et al. 2004a,b)as
well as to cytoprotective intracellular molecules such as heat shock
protein 70 (HSP70) (Xin et al. 2015) and nuclear factor (erythroid-
derived 2)-like 2 transcription factor (Nrf2) (Brown et al. 2010b).
Inammatory cells are crucial to clearance; however, excessive
inammation can lead to exacerbation of preexisting diseases
(e.g., asthma, cardiovascular disease) (Donaldson et al. 2000)orto
increases in the incidence of autoimmune, allergic, and other
immune-related diseases (Hussain et al. 2012). Evidence exists
that environmental PM and DEPs can interact with allergens to act
as an adjuvant, leading to allergic sensitization (Alessandrini et al.
2009;Hussain et al. 2011;Li et al. 2008). Although such observa-
tions have also been made for some NMs (e.g., TiO2)(Larsen
et al. 2010), the mechanism of NMallergen interaction cannot be
related to the particle size alone; instead, other physical and chem-
ical factors such as surface reactivity and chemistry play a role
(Smulders et al. 2015).
Lesson 15. The proinammatory eects of UFPs and NMs
may exacerbate existing disease and increase the incidence of
Figure 5. Exposure to nanomaterials (NMs) via the lungs results in rapid transport into the epithelium and interstitial spaces and long-term retention as
a result of substantial endocytosis by epithelial cells (Type I and Type II) and limited initial phagocytosis by alveolar macrophages. Pathways exist for
the transport of inhaled NMs into the alveolar epithelium and interstitium of rodent lung and further across the endothelial vascular membrane of
blood circulation as well as into the lymphatic drainage system. Some evidence suggests that a predominant route of clearance from the lung tissue is
then via reentrainment back onto the alveolar epithelial surface (via an unknown mechanism) for long-term macrophage-mediated transport toward cili-
ated airways and the larynx. Nanosized NMs may cross the epithelium, whereas larger aggregates/agglomerates are likely to be phagocytosed by alveo-
lar macrophages.
Environmental Health Perspectives 106002-10
other immune-related diseases. These eects are likely to
be related to multiple physicochemical characteristics of the
particles.
The relationship between an NMs physicochemical charac-
teristics and the observed responses is a key research endeavor
in nanotoxicology. Quantitative structureactivity relationship
(QSAR) models have been developed to identify these key char-
acteristics (Puzin et al.). The roles of some dose metrics such as
particle surface area, solubility (and the ability to release ions),
and aspect ratio have already been conrmed (Brown et al. 2001;
Dun et al. 2002,2007;Johnston et al. 2013;Kermanizadeh
et al. 2016;Oberdörster et al. 1994;Poland et al. 2008;Prach
et al. 2013;Schinwald et al. 2012). QSAR research is an impor-
tant component of nanotoxicology (MODENA MPNS COST
2013). Because PM is a complex mixture, a comprehensive char-
acterization of PM samples is needed, and QSAR methods can be
used to determine which physicochemical characteristics of PM
drive the observed adverse responses. Thus, the QSAR modeling
approach currently being developed for nanotoxicology can also
be relevant to air pollution research.
Lesson 16. There is now an opportunity to review in detail
this rather large mechanistic body of research to look for syner-
gies and dierences between NM and UFP modes of action and
to relate them to physicochemical characteristics. Clarifying the
relationships between these mechanistic end points and the physi-
cochemical characteristics of the NMs or UFPs will be essential
in the further development of QSAR- and modeling-type
approaches.
In the last decade, NM surfacebiomolecule (proteins, lipids,
etc.) corona interactions have been characterized in dierent
media and body uids to investigate their actions and the fate of
NMs should they enter the circulation. These interactions, when
concerning intracellular proteins, can alter the eects of NMs as
has already been shown for xenobiotic-metabolizing enzymes
(Sanns et al. 2011). In addition, results suggest that the NM
properties can inuence the composition of the corona and that
its composition changes over time and with passage through dif-
ferent tissue and subcellular compartments (Wang et al. 2013).
Lesson 17. Our understanding of the composition of the mo-
lecular corona for NMs can be applied to UFPs because it is
likelytoinuence their uptake, fate, and eects within the
body.
The acute-phase response has been proposed as a mechanism
of particle-induced cardiovascular disease. The acute-phase
response is a general alarm response of the body to various
assaults including bacterial and viral infections, trauma, and so
on. The most widely studied acute-phase protein is C-reactive
protein (CRP), the serum levels of which are associated with risk
of cardiovascular disease in prospective epidemiological studies
(Ridker et al. 2000). Serum Amyloid A (SAA) may also play a
causal role in cardiovascular risk by promoting plaque progres-
sion and atherosclerosis.
Inhalation of TiO2nanoparticles has been shown to cause up-
regulation of the Serum Amyloid A3 (Saa3) gene in the lung
(Halappanavar et al. 2011). Similarly, inhalation and intratracheal
instillation of NMs and carbon nanotubes also increased expres-
sion of Saa3 (Saber et al. 2013,2014). Lung Saa3 mRNA levels
were shown to correlate with deposited surface area of carbon
black and TiO2NMs and with neutrophil inux into bronchioal-
veolar lavage uid (Saber et al. 2013,2014), consistent with
SAA being a neutrophil chemoattractant (Badolato et al. 1994).
Moreover, Saa3 mRNA levels in lung tissue were shown to cor-
relate with SAA3 levels in plasma following pulmonary exposure
to carbon nanotubes (Poulsen et al. 2015). DEPs have also been
shown to induce a pulmonary acute-phase response such as up-
regulation of C-reactive protein and serum amyloid A (Saber
et al. 2014).
Priority research gap. Both PM and NM research studies
need to consider a wider array of biological mediators, for exam-
ple, acute-phase proteins or new carriers of the oxidative signal.
Methodological Considerations
NM research also oers methodological renements for biologi-
cal assessment of ambient PM, including means to account for
particle-related interference (e.g., light absorbance, uorescence
quenching, protein binding), which may occur in some cellular
(Pulskamp et al. 2007;Wörle-Knirsch et al. 2006) and mutage-
nicity assays (Clift et al. 2013).
Lesson 18. Evidence for the ability of NMs to interfere in var-
ious assays means that study designs for NM and UFP research
require consideration of control procedures to limit the potential
to confound result interpretation.
Over the past decade, there has been a progressive approach
toward standardized protocols to provide a better understanding
of the biological impact of NMs [the International Alliance for
NanoEHS Harmonization, the EU FP7 (Risk Assessment of
Engineereed Nanoparticles) ENPRA and NanoTest projects].
These projects have helped in understanding the pitfalls and
advantages of the dierent biochemical test systems used within
nanotoxicology (e.g., Guadagnini et al. 2015).
Lesson 19. Standardized protocols for assessing biological
responses to NMs, once wholly available, could be applied to
both UFPs and PM.
Conclusions and Recommendations
A comparison of the UFP and NM literature has identied at least
19 immediate unifying lessons, as well as a number of areas
where further research is needed to better understand both elds
of research. In fact, in this review, we identied that UFP and
NM toxicology are not two distinct elds; rather, they overlap
extensively with the potential to extrapolate from one to the other
in many respects. Firstly, ambient PM research provided evi-
dence of potential health impacts for UFPs, and NM toxicology
has largely provided essential evidence of the mechanistic plausi-
bility of these health eects. PM research provides indications of,
at least in part, the potential disease eects to consider, and early
initial human health studies involving workers suggest this may
also be true for other materials; however, more work is required
to conrm this hypothesis. It seems safe to conclude that UFPs
and NMs share the same general biological mechanisms of
adverse eects, such as oxidative stress and inammation.
However, NM toxicology has also provided a signicantly better
understanding of the role of physicochemical characteristics of
particles regarding their toxicity, including factors in addition to
size and surface area, such as solubility, charge, composition,
coating, and agglomeration/aggregation. This information is im-
portant because it means that not all NMs are created equal in
terms of their toxic potential, and likewise, not all ambient PM or
UFPs have the same potential to induce health eects. Although
more work could be performed to compare the mechanism of tox-
icity of UFPs with that of NMs, a more eective use of resources
might be to translate the techniques for physicochemical charac-
terization into the PM eld to better enable identication of PM
sources that are responsible for health eects, allowing their
more eective management. Integration of both elds of research
will provide greater potential for justication, interpretation, and
application of the wealth of important knowledge that has been
gathered over the last few decades.
Environmental Health Perspectives 106002-11
Editor's Note: In the Advance Publication of this article,
João Paulo Teixeira was missing from the author list. The author
and his aliation have been added in this version of the article.
Acknowledgments
The authors thank B. Ross and D. Balharry for practical support
for the workshop. This work was supported by and performed
with the context of the European Union Modelling Nanomaterial
ToxicityEuropean Cooperation in Science and Technology (EU
MODENA COST) action (TD1204), which funded the workshop
in May 2015 (http://www.modena-cost.eu).
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Appendix I. Physicochemical characteristics of ambient ultrafine particles
(UFPs) and engineered nanomaterials (NMs).
Ambient ultrafine particles
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coarse (2:510 lm), ne (<2:5lm), and UF (<100 nm) particles.
Urban UFPs derive mainly from combustion processes (e.g., trac) and
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particle in the lung.
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particles.
Controlled exposures are impeded by temporal variability, which com-
plicates mechanistic studies.
UFPs are always surrounded by gaseous pollutants.
Nanomaterials
A number of denitions exist that usually stipulate at least one dimen-
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in the nanoscale, making them nanoparticles.
NMs are often referred to as engineered or manufactured because they
are designed and generated for a specic purpose.
NMs are made in a wide variety of chemistries, consisting of single ele-
ments (e.g., carbon, metals), compounds (e.g., metal oxides, salts), or
complex composites (e.g., core-plus-shell structure).
NMs can vary signicantly in particle morphology and chemical com-
position but are well dened at production and close to production
levels.
Spatial and temporal variance in airborne concentration may vary
signicantly.
Controlled exposures are possible, enabling detailed
mechanistic studies.
NMs can be handled in a standardized manner, facilitating studies of
dened properties.
Environmental Health Perspectives 106002-12
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... Nanotechnology has moved from the focused research environment to wider application in the workplace, and engineered nanomaterials (ENMs) have been incorporated into novel products and technological solutions in the past decade or more. However, uncertainty remains about possible adverse human health effects from exposure to ENMs (Halappanavar et al. 2020;Oberdorster et al. 2005;Riediker et al. 2019;Stone et al. 2017). A unique hazard potency cannot be attributed to all ENMs since their toxicity can change considerably based on their physical and chemical characteristics. ...
... Poorly soluble ENMs can also translocate from the original site of deposition, can become systemically available and can accumulate in secondary organs (ISO 2019;Landsiedel et al. 2012). For example, effects of ENMs have been found in the brain (Heusinkveld et al. 2016), liver (Modrzynska et al. 2018) and cardiovascular system (Stone et al. 2017). Also, translocation of very small engineered particles is possible via the olfactory epithelium in the nose or via uptake into the circulation in rodent studies (Heusinkveld et al. 2016). ...
... Exposure to UFPs can result in adverse short-term associations with inflammatory and cardiovascular changes (Ohlwein et al. 2019). In addition, some exposure studies compared UFP dust with dust exposure in the workplace (Stone et al. 2017;Viitanen et al. 2017). Thus, UFP data could be used in a weight of evidence approach or sensitivity analysis. ...
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Unique physicochemical characteristics of engineered nanomaterials (ENMs) suggest the need for nanomaterial-specific occupational exposure limits (OELs). Setting these limits remains a challenge. Therefore, the aim of this study was to set out a framework to evaluate the feasibility of deriving advisory health-based occupational limit values for groups of ENMs, based on scientific knowledge. We have used an expert panel approach to address three questions: 1) What ENM-categories should be distinguished to derive advisory health-based occupational limit values (or health-based Nano Reference Values, HNRVs) for groups of ENMs? 2) What evidence would be needed to define values for these categories? And 3) How much effort would it take to achieve this? The panel experts distinguished six possible categories of HNRVs: A) WHO-fiber-like high aspect ratio ENMs (HARNs), B) Non-WHO-fiber-like HARNs and other non-spheroidal ENMs, C) readily soluble spheroidal ENMs, D) biopersistent spheroidal ENMs with unknown toxicity, E) biopersistent spheroidal ENMs with substance-specific toxicity and F) biopersistent spheroidal ENMs with relatively low substance-specific toxicity. For category A, the WHO-fiber-like HARNs, agreement was reached on criteria defining this category and the approach of using health-based risk estimates for asbestos to derive the HNRV. For category B, a quite heterogeneous category, more toxicity data are needed to set an HNRV. For category C, readily soluble spheroidal ENMs, using the OEL of their molecular or ionic counterpart would be a good starting point. For the biopersistent ENMs with unknown toxicity, HNRVs cannot be applied as case-by-case testing is required. For the other biopersistent ENMs in category E and F, we make several recommendations that can facilitate the derivation of these HNRVs. The proposed categories and recommendations as outlined by this expert panel can serve as a reference point for derivation of HNRVs when health-based OELs for ENMs are not yet available.
... Due to their small size, UFPs are able to deposit in the alveolar space of the lungs, bypassing the normal defenses against inhaled particles (mucociliary clearance), where they can cause the development of respiratory diseases and/or the exacerbation of existing diseases [15,16]. This region of the lungs is involved in gas exchange and, here, UFPs can translocate into the bloodstream where they can impact other organs including the brain and heart leading to the development of neurodegenerative and cardiovascular diseases [15,18,19]. Additionally, UFPs can damage lung epithelial cells by inducing reactive oxygen species (ROS) production which can cause DNA damage, among other negative effects [18]. ...
... This observation, consistent with prior literature [12], is particularly important because UFPs are known to deposit in the gas exchange area of the lungs where they can cause respiratory disease [15,16]. Additionally, due to their small size, UFPs are able to translocate into the bloodstream where they can travel to other organs causing damage [15,18,19]. Together, these data highlight the notion that current OELs for Cu and CO are not sufficient to assess occupational exposure of emissions to range instructors as symptoms still develop (Table 4) despite not reaching the OEL. ...
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Full-text available
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... Finally, several components of tobacco smoke may linger in the maternal and fetal organism and remain biologically active long after actual exposures (e.g. particulates and heavy metals) [57][58][59]. Hence smoking prior to the fetal masculinization programming window might still be able to cause damage long after actual exposure. ...
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Maternal smoking during pregnancy constitutes a potential, major risk factor for adult male reproductive function. In the hitherto largest longitudinal cohort, we examined biomarkers of reproductive function according to maternal smoking during the first trimester and investigated whether associations were mitigated by smoking cessation prior to the fetal masculinization programming window. Associations between exposure to maternal smoking and semen characteristics, testicular volume and reproductive hormones were assessed among 984 young men from the Fetal Programming of Semen Quality (FEPOS) cohort. Maternal smoking was assessed through interview data and measured plasma cotinine levels during pregnancy. We applied negative binomial, logistic and linear regression models to estimate differences in outcomes according to levels of maternal smoking. Sons of light smokers (≤ 10 cigarettes/day) had a 19% (95% CI − 29%, − 6%) lower sperm concentration and a 24% (95% CI − 35%, − 11%) lower total sperm count than sons of non-smokers. These estimates were 38% (95% CI − 52%, − 22%) and 33% (95% CI − 51%, − 8%), respectively, for sons of heavy smokers (> 10 cigarettes/day). The latter group also had a 25% (95% CI 1%, 54%) higher follitropin level. Similarly, sons exposed to maternal cotinine levels of > 10 ng/mL had lower sperm concentration and total sperm count. Smoking cessation prior to gestational week seven was not associated with a higher reproductive capacity. We observed substantial and consistent exposure–response associations, providing strong support for the hypothesis that maternal smoking impairs male reproductive function. This association persisted regardless of smoking cessation in early pregnancy.
... The best examples under this category are burning gas, candle, and biomass (John et al. 2017). The nanomaterials produced are ultrafine particles that contribute to air pollution (Stone et al. 2017). ...
... Dans le cadre des études de toxicologie de l'inhalation, et au regard des dosages de tungstène dans les différentes sous-structures, il est important de considérer certains mécanismes par lesquels les particules inhalées peuvent influencer des tissus et des organes secondaires à ceux directement concernés par l'inhalation, notamment au niveau systémique et cardio-vasculaire [244,245]. L'implication des différentes voies de passage pourrait ainsi avoir une influence sur la diversité des réponses observées à 24 heures. La proximité des structures avec le liquide céphalo-rachidien peut conditionner l'impact de la composante systémique, et les projections anatomiques des nerfs olfactif et trijumeau peuvent conditionner l'impact de la composante directe. ...
Thesis
L’exposition à des aérosols particulaires est la principale cause de contamination dans les installations nucléaires, et le cerveau peut en être une cible directe. Ce projet de thèse a étudié les effets biologiques induits par l’inhalation de particules de tungstène, présenté comme un contaminant émergent. Pourtant peu de connaissance sont disponibles concernant ses effets sur le cerveau. Du fait de son usage pour ses propriétés physico-chimiques remarquables, les situations d’exposition sont principalement professionnelles, notamment en lien avec les activités nucléaires (démantèlement, réacteurs de fusion nucléaire). Des travaux préalables de métrologie ont été réalisés pour maitriser la génération de l’aérosol à des concentrations de l’ordre de la Valeur Limite Moyenne d’Exposition Professionnelle, et caractériser sa granulométrie. L’extrême densité de ces particules leur confère des propriétés particulières, notamment une grande différence entre les diamètres aérodynamiques et de mobilité électrique qui dictent leur comportement dans les voies aériennes. Des campagnes d’exposition par inhalation ont été réalisées à deux concentrations différentes (5 et 80 mg.m-3) en mode aigu et répété sur 1 semaine chez le rat adulte. L’étude de processus impliqués en toxicologie cérébrale a été entreprise 24 heures après exposition. Des modifications régionalisées d’un point de vue anatomique et dépendantes de la concentration d’exposition pour les acteurs impliqués dans ces processus cibles ont été observées. Au niveau du bulbe olfactif, les résultats suggèrent un potentiel effet précoce sur la survie et la morphologie des cellules microgliales qui pourrait témoigner d’une réactivité au contaminant. Selon les modes d’exposition, ces cellules présentent une diminution de leur densité associée à une augmentation de marqueur apoptotique. Un phénotype anormal des noyaux de certains neurones matures, potentiellement signe d’une souffrance neuronale, a également été observé au niveau du cortex frontal et serait en potentiellement lié avec l’implication du stress oxydant. Les effets différentiels observés selon les schémas d’exposition pourraient impliquer plusieurs composantes : locale (propre au cerveau) et/ou systémique. En effet, le tungstène, en plus d’être retrouvé dans les poumons et les reins, est présent dans le cerveau des animaux exposés à la forte concentration, et semble modifier les processus inflammatoires au niveau du plasma. Nos données questionnent la relative innocuité du tungstène et soulèvent plusieurs hypothèses concernant de possibles mécanismes adaptatifs ou neurotoxiques qui pourraient finalement altérer l’intégrité neuronale selon les schémas d’exposition.
... The development and steady introduction of new engineered nanomaterials (NMs) to the market has raised awareness about potential adverse health effects resulting from long-term exposures. The health risk concerns for NMs originated from inhalation toxicology studies that could substantiate the role of ultrafine particles in the epidemiological link between ambient air pollution exposure and cardiopulmonary diseases (reviewed by [1]). Likewise, the awareness about potential adverse effects of NMs on the central nervous system came from inhalation studies in more recent years. ...
Article
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Background There is an increasing concern about the neurotoxicity of engineered nanomaterials (NMs). To investigate the effects of subchronic oral exposures to SiO 2 and CeO 2 NMs on Alzheimer’s disease (AD)-like pathology, 5xFAD transgenic mice and their C57BL/6J littermates were fed ad libitum for 3 or 14 weeks with control food pellets, or pellets dosed with these respective NMs at 0.1% or 1% (w/w). Behaviour effects were evaluated by X-maze, string suspension, balance beam and open field tests. Brains were analysed for plaque load, beta-amyloid peptide levels, markers of oxidative stress and neuroinflammation. Results No marked behavioural impairments were observed in the mice exposed to SiO 2 or CeO 2 and neither treatment resulted in accelerated plaque formation, increased oxidative stress or inflammation. In contrast, the 5xFAD mice exposed to 1% CeO 2 for 14 weeks showed significantly lower hippocampal Aβ plaque load and improved locomotor activity compared to the corresponding controls. Conclusions The findings from the present study suggest that long-term oral exposure to SiO 2 or CeO 2 NMs has no neurotoxic and AD-promoting effects. The reduced plaque burden observed in the mice following dietary CeO 2 exposure warrants further investigation to establish the underlying mechanism, given the easy applicability of this administration method.
... Not much is known about the neurotoxic effects of engineered NPs in humans, but we know that nano-sized air pollutants may also affect the brain (Genc et al. 2012). Ambient ultrafine particles (UFPs, < 0.1 μm) are thought to be a possible source of brain NP exposure (Stone et al. 2017). Epidemiological and animal studies have shown that air pollution can induce neurotoxicity (Morris-Schaffer et al 2019; Allen et al 2017). ...
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... In addition to pulmonary toxicity, significant genotoxicity and reproductive/developmental toxicity are also reported through direct and indirect mechanisms [21][22][23]. Moreover, CB is widely used as a model for environmental ultrafine particulate exposures [24]. ...
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... Similarly, the genotoxic and/or carcinogenic impacts may be known and understood for some of the current macro-sized analytes used in ENMs; however, the probability for increased adverse impacts due to the decreased size is under continuous research and investigation. Nevertheless, respiratory issues and cardiovascular disease are among the common health impacts associated with exposures to ultrafine particulate matter (Seaton et al. 2010;Stone et al. 2017). ...
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