Pulmonary inflammation

Nanoparticles around us – traffic exhausts, forest
fires, volcanoes, accidents, disasters, and
engineered nanoparticles in workplaces
Nanotechnologies
- much ado about nothing
or risks to human health?
Harri Alenius
Research professor
Head, Unit of Immunotoxicology
Finnish Institute of occupational Health

Traffic is a major source of carbon nanoand
other particles

Forest fires release large amounts of carbon and
other nano- and larger particles

The eruption of the Eyjafjallajokull volcano in Iceland:
huge amounts of nanoparticles released

9/11 – WTC Twin Towers in New York

Engineered nanoparticles at workplaces –
the topic of this talk!

ENGINEERED
NANOMATERIALS

Special features of engineered
nanoparticles (ENP)
Small
Reactive
Surprising
Enabling
Huge technological
and financial potential
Risks unknown or
poorly known
1-100 nm

The essence of ENP: Unpredictable
➔ nano ≠ bulk
Bulk gold:
yellow
inert
1 200°C
1 nm gold NP:
blue
low reactivity
200°C
3 nm gold NP:
reddish
catalytic
200°C
Catalytic activity
Cluster diameter (nm)

CARBON NANOTUBES – CROWN JEWELS OF
NANOMATERIALS: EXAMPLES OF CHALLENGES FOR
HUMAN HEALTH PROTECTION
Single walled CNT
Multi walled CNT
The diameter of a SWCNT
about 1 nm, that of MWCNT
~ close to 100 nm, but their
length may be several μm's

Challenges of Nanotechnologies
• In 2007 ~ 7 billion € used for nanotechnology research
globally, 50/50 from public and private sources,
respectively
• Funding of research on health effects of ENP is
modest in many countries – in Europe and the US about
2-3 % of all nanotechnology research
• ENP and nanotechnologies are a challenge to the
EU new chemicals legislation REACH; REACH,
implemented since 2007, does not fully recognize ENP,
and the word 'nano' is not mentioned in REACH now
being modified to correspond the new needs – a new
ENP EU definition been proposed (Nov 19 deadline)

Nano-silver is an important example due to its
microbicidal properties
Cloth cleaner
containing
nanosilver
Cutting board
treated with
nanosilver
Teddy bear for
allergic children
treated with
nanosilver
Nanosilver treated children's
milk bottle brush
Nanosilver treated socks
http://www.nanotechproject.org/inventories/
Nanosilver
treated pillow

A NANOTECHNOLOGY PRODUCT CAN ALSO BE
LARGE – strengthening of concrete with
amorphous silica

Quality of life and social benefits of
nanotechnologies – how to regulate possible
benefits and risks already here?
From Andrew Maynard, Chief scientist of The Project on Emerging Nanotechnologies

ENTRY OF ENP INTO SYSTEMIC
CIRCULATION AND THE BODY
• Routes of ENP into the body:
– Lungs – rapid route to the circulation through
the alveoli, deposition in the lungs
– Nose – axons of olfactory nerve provide a
direct entry into the brain – effects?
– Gastrointestinal tract maybe important for
novel foods, not in the work environment
– Skin may be important in the case of skin
diseases and skin damages – the largest organ
in the body

INJECTION
UK Royal Society & Royal Academy of Engineering
Report on Nanoscience & nanotechnologies (2004)
Altistuminen: lähteet, kohteet, & reitit
DERM

The safety knowledge gap challenging
development of legislation, regulations
Schematic representation of emergence of nanotechnology products in comparison
to generated EHS data (Linkov and Satterstrom: Nanomaterial Risk Assessment and
Risk Management, 2008).

MODELLING
INFLAMMATORY
EFFECTS IN HEALTHY
SUBJECTS

Effects of exposure to metal
nanoparticles

Effects of airway exposure to TiO2 nanoparticles on
pulmonary inflammation in healthy mice
• Titanium dioxide (TiO2) nanoparticles are widely used in
the field of biotechnology, pharmaceuticals and cosmetics
and textile industries in applications such as paints,
coatings, UV protection and photocatalysis
• Characteristics of TiO2 nanoparticles are also altered in
several ways to improve their performance. Properties can
be altered by doping TiO2 with other elements, or by
modifying the TiO2 nanomaterials surface with other
semiconductors.
Aim of our study was to explore inflammatory
changes in the lungs induced by repeated airway
exposure to different type of TiO2 nanoparticles
using mouse and cell models
Rossi EM, et al. Toxicol Sci. 2010 113(2):422-433.
20

Characteristics of particles used in the
present study
6 different particle types were tested
- 3 different commercially available TiO 2
nanoparticles
- 1 on-site generated TiO 2
nanoparticle
- 1 commercial coarse TiO 2
- 1 SiO 2
nanoparticles (coating material)
Characteristics identified:
- Particle size, agglomeration size, surface area, phase structure,
radical formation capacity, Z-potential
Name
Coated nanosized rutile
TiO 2
Nanosized anatase TiO 2 Coarse rutile TiO 2
Nanosized SiO 2 Nanosized rutile TiO 2
Nanosized
anatase/brookite TiO 2
Vendor Sigma-Aldrich Sigma-Aldrich Sigma-Aldrich
Product number 637262 637254 224227
Particle size 10x40nm

Inhalation exposure procedures
Dust generator
(brush)
On-site reactor
Constant production of TiO2 aerosol at the concentration of 10mg/m3
22

Pulmonary inflammation in mouse lungs after TiO2
exposure
12
9
Neutrophils
A= 2 hours exposure
B= 4 days exposure
C= 4 weeks exposure
% of BAL cells
6
3
0
Exposure time
- A B A B C A B A B A B C A B C
nanoSiO2
amorphous
nanoTiO2
+SiO2
rutile
nanoTiO2
rutile
nanoTiO2
anatase
nanoTiO2
anatase
(generated)
coarseTiO2
rutile
• None of the nanoTiO2 preparation tested, other than SiO2 coated
TiO2, elicited pulmonary inflammation in mice !!!
• Exposure to nanoSiO2 did not induce any inflammatory effects !!!
23

Nanoparticles accumulate in pulmonary
macrophage phagosomes in murine lungs
TiO2
TiO2 + SiO2
24

Conclusions
• Nanoparticle induced lung inflammation could not
be explained by the surface area of the particles,
their primary or agglomerate particle size or radical
formation capacity, but is rather explained by the
surface coating.
Our findings emphasize that it is vitally
important to take into account in the risk
assessment that alterations of nanoparticles,
e.g. by surface coating, may drastically
change their toxicological potential.
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Effects of exposure to carbon
nanotubes

Asbestos exposure induced disorders
Asbestosis is a breathing disorder
caused by inhaling asbestos
fibers. Prolonged accumulation of
these fibers in lungs can lead to
scarring of lung tissue and
diminished breathing capacity.
Signs and symptoms of
asbestosis usually don't appear
until years after exposure.
27

Background
• Needle-like fiber shape of carbon nanotubes have has
been compared to asbestos, raising concerns that
widespread use of carbon nanotubes may lead to
mesothelioma, cancer of the lining of the lungs caused
by exposure to asbestos.
• Mesothelioma is almost exclusively found following
asbestos exposure and mesothelioma is a particle
response unique to fibre-shaped particles.
• Authors hypothesize that the most important
acute response to long MWCNTs, if they were to
behave like asbestos, would be mesothelial injury.
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Recruitment of inflammatory cells in the peritoneal
cavity after introduction of fibers
Inflammatory response
24 h post -installation
Granuloma response
7 days post -installation
Short fibers
Long fibers
Short fibers
Long fibers
Female C57Bl/6 mice were intraperitoneally instilled with 50 µg of vehicle control, nanoparticulate
carbon black (NPCB), short-fibre amosite (SFA), long-fibre amosite (LFA), two curled/tangled MWNT
samples of different lengths (NTtang1, NTtang2) and two samples containing long MWNTs (NTlong1,
NTlong2).
29

Morphological features of diaphragms of after
particle/fiber exposure after 7 days
Short MWCNT
Sample 1
Short MWCNT
Sample 2
Long amosite
(brown asbestos)
Long MWCNT
Sample 1
Long MWCNT
Sample 2
Poland et al. Nature Nanotechnology. 2008 Jul;3(7):423-8.
30

Inhalation exposure to carbon nanotubes
• Male C57BL6 mice were
exposed (nose-only) to an
aerosol of MWCNT or carbon
black (CB) nanoparticles
(30 mg/m 3 ) for 6 hours
– MWCNT length 0.3-50um
• Lung tissues were collected
and carefully examined 1
day, 2 weeks, 6 weeks and
14 weeks post exposure
31

Subpleural fibrosis in mice after CNT
inhalation
saline aerosol-exposed
Subpleural fibrotic lesion
showing MØ with CNT
CNT exposed mice
2 weeks after inhalation
Ryman-Rasmussen et al. Nature Nanotech. 4(11):747-751 (2009)
32

Discussion
• MWCNT behave as asbestos as they...
– Are deposited at the alveolar level
– Reach the subpleural level after inhalation
– Remain in the subpleura intact for weeks and
stimulate fibrosis (scar formation)
33

Movement of particles through the lung
Long nanotubes
lodge at the
drainage points
and block them
→ inflammation,
and pathological
changes over
time
Donaldsson and Poland, 2009
pleura = red lines
pleural space containing fluid = between the two membranes
small particles = blue circles
short or tangled nanotubes = black lines
34

EFFECTS IN DISEASE
MODELS

Background
• Evidence exits that individuals with airway allergies
may be more pronounced to show respiratory
symptoms compared to healthy people when exposed
to particulate matter.
• It is important to examine whether the
pulmonary inflammatory responses against
nanomaterials vary depending on the disease
status.
36

Effects of titanium dioxide
particle exposure on allergic
asthma
Rossi E, Pylkkänen L, Koivisto A, Nykäsenoja H, Wolff H, Savolainen
K, and Alenius H. Particle and Fiber Toxicology (revised)

Airway reactivity and airway inflammation is
suppressed after inhalation of TiO2 particles
8
Airway reactivity
to inhaled metacholine
40
30
Eosinophils
*
50
40
IL-13 mRNA
** ***
Penh
6
4
OVA
OVA + nTiO2
PBS
cells / HPF
20
10
0
- nTiO2 fTiO2 - nTiO2 fTiO2
RU
30
20
10
0
- nTiO2 fTiO2 - nTiO2 fTiO2
2
PBS
OVA
PBS
OVA
3
30
MCh (mg/ml)
100
Lymphocytes
IL-4 mRNA
PBS = healthy mice
OVA = allergic mice
cells / HPF
8
6
4
2
**
**
RU
15
10
5
*** **
nTiO2 = nanoTiO2
fTiO2 = fineTiO2
0
- nTiO2 fTiO2 - nTiO2 fTiO2
PBS
OVA
0
- nTiO2 fTiO2 - nTiO2 fTiO2
PBS
OVA
38

Conclusions
• Mice with allergic asthma are not more pronounced to
show respiratory symptoms and airway inflammation
compared to healthy mice when exposed to TiO2
particles.
• Rather, Th2 type pulmonary inflammation and airway
hyperreactivity is dramatically suppressed in mice which
are simultaneously exposed to TiO2 particles.
39

Inhaled Multiwalled Carbon
Nanotubes Potentiate Airway
Fibrosis in Murine Allergic
Asthma
Ryman-Rasmussen P et al. Am J Resp Cell Mol
Biol. Vol. 40, pp. 349-358, 2009

Combined Ovalbumin Allergen Challenge and
MWCNT Inhalation Cause Airway Fibrosis
SAL
OVA
SAL+MWCNT
OVA+MWCNT
PBS = healthy mice
OVA = allergic mice
14 days after exposure
41

Levels of platelet-derived growth factor (PDGF)
and transforming growth factor (TGF)-β1 protein
in BALF 1 day after MWCNT inhalation.
PDGF+TGFΒ1=FIBROSIS
42

Conclusions
• Study suggests that airway fibrosis resulting from
combined ovalbumin sensitization and MWCNT inhalation
requires PDGF, a potent fibroblast mitogen, and TGF-β1,
which stimulates collagen production.
Individuals with pre-existing allergic inflammation
may be susceptible to airway fibrosis from inhaled
MWCNT.
43

General Conclusions
• Different type of particles may elicit drastic
differences in the inflammatory response
• Surface coating may have critical effect to the
toxicity of the particles
• Underlying disease influence significantly to
the health effects of particles. We can not study
only healthy people
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THANK YOU FOR
YOUR ATTENTION!!
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