Mechanistic Aspects of Nanoparticle Toxicology Particle Deposition and Clearance
What can we learn from the ultrafine particles?
Tobias Stöger
GSF-National Research Centre for Environment and Health
Institute of Inhalation Biology
Munich - Germany
GSF-National Research Centre
for Environment and Health
Member of the Helmholtz Association
IHB-Institute for
Inhalation Biology
Mechanistic Aspects of Nanoparticle Toxicology Particle Deposition and Clearance
Object: Draw parallels from NP to PM toxicity
¾
Deposition & Clearance Mechanism
¾
Acute Pulmonary Effects
¾
Translocation to Secondary Organs
¾
Extra-pulmonary Effects
modivied from Kevin Dreher, US EPA:
„Nanotechnology for Remediation Technical Nanotechnology for
Remediation Technical”
Workshop, Okt’05
What do we know about Particle-Toxicology?
PubMed
3000
ambient
model
nanomaterials
Organ systems exposed:
• Skin
surface area:1.5-2 m2
• Gastrointestinal Tract
surface area: 200 m2
1012-1014 fine particles are ingested per person/day
• Lung
surface area: 150 m2
1010-1012 fine particles deposit in the lung/day
thickness: 0.05 - 1.5 mm
Routes of Exposure to Ambient Particles:
“Ambient” Nanoparticles,
a Fraction of Particulate Matter (PM)
¾ mainly carbonaceous NPs, derived from combustion
processes
¾ low-solubility in aqueous/physiological solutions
¾ low-toxicity, no cytotoxic effects below 100 µg/ml
(“the dose makes the poison”)
Size distribution of fine ambient particles (PM2.5)
10 nm – 2.5 µm
(85%)
(14%)
-
13000 cm-3
(72 %)
-3
-3
(<1%)
74 µg/m3 12 µg/m3
daily mean:
-1
0.6 µg/m3
Teilchenanzahldichte
(cm µm
particle
number distribution
cm-3)]
number burden:
ultrafine particles < 0.1 µm
daily mean:
-1
Teilchenmassedichte
(µg m[µg/m
µm 3])
particle mass distribution
mass burden:
PM2.5: 0.1 µm – 2.5 µm
1000
100
10
1
0.1
0.01
0.1
0.5
2.5
Teilchendurchmesser
particle diameter [µm](µm)
5100 cm-3
60 cm-3
(27%)
( <1%)
1000000
100000
10000
1000
100
10
1
0.1
0.01
0.1
0.5
2.5
Teilchendurchmesser
particle diameter [µm](µm)
Tuch et al. 1997
Size Relation at Cellular Scale
Virus
exogen
Bacteria
1 nm
10 nm
100 nm
1 µm
10 µm
2
4
10 g/mol 10 g/mol
molar mass
6
10 g/mol
8
10 g/mol
Macrophage -
Erythrocyte -
Platelet -
LipoproteinComplexes /
Vesicles
Channels
- Myosin (6100aa)
- Insulin (51 aa)
endogen
- Hemoglobin (574 aa)
Proteins
100 µm
Size Relation at Cellular Scale
Virus
exogen
Bacteria
1 nm
10 nm
100 nm
1 µm
10 µm
2
4
10 g/mol 10 g/mol
molar mass
6
10 g/mol
proteins
PM2.5
ambient PM
8
10 g/mol
1-100 nm
> nanoparticles > vesicles
>
cells
Macrophage -
PM 2.5
Erythrocyte -
uf
Platelet -
uf
LipoproteinComplexes /
Vesicles
uf
Channels
- Myosin (6100aa)
- Insulin (51 aa)
endogen
- Hemoglobin (574 aa)
Proteins
100 µm
Shape of soot particles
Soot Particles
Diesel Soot (SRM1650a)
100 nm
Shape of soot particles
Diesel Soot (SRM1650a)
Soot Particles
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
25nm
Soot:
aggregates from carbonaceous
primary particles
100 nm
Deposition & Clearance
Particle Deposition in the Respiratory System
PM-mass
particle deposition
1.0
0.8
0.6
0.4
0.2
ultrafine particles cause the
highest burden to the small
bronchial and alveolar region!
respiratory tract
0.8
0.6
0.4
0.2
oral cavity
0.4
0.2
large bronchi
0.4
0.2
small bronchi
0.8
0.6
0.4
0.2
alveoli
Alveolar wall
- 2.5µm
- 20nm
0.01
0.1
1
10
particle diameter (µm)
uf. Particle
PM2.5
erythrocyte(7 µm)
Mechanisms of Defense – Alveolar Region
gas exchange
Phagocytosis of particles by
macrophages
alveole
Particles deposited on
epithelial cells
Pseudopodia
Morgenroth & Takenaka
Institute of Inhalation
Biology
Pathways of Particle Clearance
PM
mucociliary
escalator
Trachea
Bronchi
phagocytosis
by AMs
Oberdörster 2005
Impact of Particle Size
Biphasic Clearance
Kinetics
Particles lavaged or retained 24h post exposure
excreted or retained fraction
1.0
0.8
alv. macrophages
WKY rat, 192Ir NP, 1 hr exposure
15 nm CMD - 107 cm-3 – 200 µg/m³
epithelium +
interstitium
0.6
0.4
lung
cumm. feces
0.2
0.0
0
liver
2
4
days
6
8
Kreyling 2004
Oberdörster 2005
Small particles escape form AM clearance!
Impact of Particle Size
Biphasic Clearance
Kinetics
Particles lavaged or retained 24h post exposure
excreted or retained fraction
1.0
0.8
alv. macrophages
WKY rat, 192Ir NP, 1 hr exposure
15
nm CMD - 107 cm-3 – 200 µg/m³
mucociliary
epithelium +
interstitium
escalator
0.6
0.4
lung
AM
phagocytosis
cumm. feces
0.2
0.0
0
liver
2
4
days
6
8
Kreyling 2004
upper airways: fast clearance
alveolar region: slow clearance
Oberdörster 2005
Small particles escape form AM clearance!
Acute Pulmonary Effects
Acute Effects of Pulmonary Deposited
Particles?
Particle Exposition:
Intratracheal Instillation in Mice
Physical/Chemical Properties of Model Particles
25
In
sti
lla
tio
n
SootH
organic content [%]
UfCP
DEP
DEP
20
15
80-99% elemental carbon !
SootL
10
size: 10 – 50 nm
Ptx90
5
Ptx
G
PtxG
0
0
100
200
300
400
500
600
surface area [m2/g]
700
800
900
1000
BALB/cJ (20g)
Lung of a mouse 24h after intratracheal instillation
of 20 µg ufCP
20µm
Semithin section, Staining: toluidine blue, x 63,
© Sinji Takenaka (2005)
Carbonaceous nanoparticles induce acute
pulmonary inflammation 24h after instillation
PMN (x103)
Cytokine conc.
PMN-Influx
Determination of Inflammation Markers in Lung Lavages
Stoeger 2006, Environ Health Perspect 114:328
Carbonaceous nanoparticles induce acute
pulmonary inflammation 24h after instillation
PMN (x103)
Dose Response Relation:
Deposited mass is not a sufficient dose
metric for poorly soluble nanoparticles!
Cytokine conc.
PMN-Influx
Determination of Inflammation Markers in Lung Lavages
Stoeger 2006, Environ Health Perspect 114:328
In search of the most relevant parameter for
quantifying pulmonary inflammation ?
Primary Particle Size
Particle Surface Area (BET)
Stoeger 2006, Environ Health Perspect 114:328
In search of the most relevant parameter for
quantifying pulmonary inflammation ?
Primary particle size
size matters
Partikel Oberfläche (BET)
For the poorly soluble nanoparticles the deposited
particle surface area
serves as a suitable dose metric!
Stoeger 2006, Environ Health Perspect 114:328
Influence of Surface Reactivity
BAL Infux - Surface Area Correlation
60
PMN [%]
50
NSP-Carb_Munich
40
NSP-TiO_Rochester
30
fine-TiO_Rochester
20
10
Stoeger et al. 2006. Environ Health Perspect 114
Oberdorster et al. 2005. Environ Health Perspect.113
0
1
10
100
BET / gramm Lung [cm2/g]
1000
Influence of Surface Reactivity
BAL Infux - Surface Area Correlation
60
NSP-Edinburgh
50
NSP-Carb_Munich
PMN [%]
40
NSP-TiO_Rochester
fine-TiO_Rochester
30
20
10
Stoeger et al. 2006. Environ Health Perspect 114
Oberdorster et al. 2005. Environ Health Perspect.113
Dick et al. 2003. Inhalation Toxicology,15
0
1
10
100
BET / gramm Lung [cm2/g]
1000
Influence of Surface Reactivity
BAL Infux - Surface Area Correlation
60
high surface reactivity of transition metals
NSP-Edinburgh
50
NSP-Carb_Munich
PMN [%]
40
NSP-TiO_Rochester
- Ni
fine-TiO_Rochester
30
- Co
CB -
20
TiO2 -
10
Stoeger et al. 2006. Environ Health Perspect 114
Oberdorster et al. 2005. Environ Health Perspect.113
Dick et al. 2003. Inhalation Toxicology,15
0
1
10
100
BET / gramm Lung [cm2/g]
1000
Oxidative Surface Reactivity of CarbonNanoparticles
At equal mass, particles with larger surface areas showed higher
potency to cause methionine oxidation
In Situ (cell free system)
Oxidation of Methionine
100000
Me(O) [pMol/ml]
10000
oxidation
1000
tr .
Ox.S
Methionine
100
Particle
BET [m2/g]
ÛfCP
768
Ptx 90
Methionine
Ptx G
sulfoxide
320
DEP
128
10
1
1
10
100
1000
BET Surfacearea [cm2]
O=
Beck-Speier 2005. Free Radic Biol Med. 38(8):1080
Oxidative Stress
48
Hypothesis of Particle Induced Inflammation
(Particle – Cell Interaction)
tr.
Ox.S
Proinflammatory mediators:
Chemokines/Cytokines,
Prostaglandins …
?
Cell
Nucleus
particle
deposition
on cell
surface
induction of
oxidative
stress
signalling
Cascade
(NFkB/AP1)
proinflammatory
gene expression
release of
proinflammatory
mediators
Gene Expression Analysis at “threshold level”:
Inhalation of Carbon Nanoparticles Induces Proinflammatory Response
Biphasic reaction:
1. Stress response:
4h
Heat shock proteins (hsp70)
24
h
2. Proinflammatory response:
- Interleukin 1 beta
- Cyclooxygenase 1
- Serum amyloid A3
- Osteopontin
- TIMP 1
- Lipocalin 2
- Galectin 3
- Amphiregulin
- Coagulation factor III
- Thrombospondin 1
-…
Andre and Stoeger 2006, Eur Respir J.
Translocation to Secondary Organs
Do inhaled nanoparticles penetrate the tissue of the lung?
Experimental design
Intubated ventilated WKY rat,
TiO2 NP, 1 hr exposure
22 nm CMD, 107 cm-3, 0.1 mg/m³
Lung morphometry 1 & 24 h after inhalation
TiO2
____
50 nm
Particle profiles (% of total lungs)
Volume density V(v) of lung compartment
•
•
•
•
90
V(v)
UFP TiO2
1h
24h
mean
80
70
60
50
20
epithelial cell
15
10
5
vessel
0
UFP
Air spaces
Intracellular,
on epithelium
epithelium
GSF-Forschungszentrum
für Umwelt und Gesundheit
Connective
tissue
Capillary
tissue
GMC
Geiser et al. 2005,Holger
Environ
Health Perspect. 113
Schulz
endothelial cell
IHB-Institut für
Inhalationsbiologie
Do inhaled nanoparticles penetrate the tissue of the lung?
Experimental design
TiO2
Intubated ventilated WKY rat,
TiO2 NP, 1 hr exposure
22 nm CMD, 107 cm-3, 0.1 mg/m³
Lung morphometry 1 & 24 h after inhalation
____
50 nm
Particle profiles (% of total lungs)
Volume density V(v) of lung compartment
•
•
•
•
90
V(v)
UFP TiO2
1h
24h
mean
80
70
60
50
20
epithelial cell
15
10
5
vessel
0
UFP
Air spaces
Intracellular,
Connective
Capillary
ÎFast
translocation
for ~20%
of deposited NP
on epithelium
epithelium
tissue
tissue
endothelial cell
ÎVolume proportional: stochastic process (“diffusion”)?
GSF-Forschungszentrum
für Umwelt und Gesundheit
GMC
Geiser et al. 2005,Holger
Environ
Health Perspect. 113
Schulz
IHB-Institut für
Inhalationsbiologie
Translocation into secondary target organs
WKY Rats exposed via endotracheal intubation,
single 1- to 1.5-h inhalation of 192Ir UFP,
mass conc.: 0.2 mg m−3, CMD: 15 nm
Lung
Extra Pulmonary Organs
Pulmonary retention and excretion during 6
mo after inhalation of 192IrUFP.
Translocated fractions into secondary target organs
during 6 mo after inhalation of 192IrUFP.
GSF-Forschungszentrum
für Umwelt und Gesundheit
Semmler et Holger
al.GMC
2004,
Inhal Toxicol.
Schulz
IHB-Institut für
Inhalationsbiologie
Translocation into secondary target organs
WKY Rats exposed via endotracheal intubation,
single 1- to 1.5-h inhalation of 192Ir UFP,
mass conc.: 0.2 mg m−3, CMD: 15 nm
Lung
Extra Pulmonary Organs
Long-term translocation of ultrafine 192Ir particles from lungs to secondary target organs
does not steadily increase the particle burden in those organs but is superimposed by
clearance from these organs, yielding a constant but very slow fraction of about 0.001 of
the deposited amount of particles.
Pulmonary retention and excretion during 6
mo after inhalation of 192IrUFP.
GSF-Forschungszentrum
für Umwelt und Gesundheit
Translocated fractions into secondary target organs
during 6 mo after inhalation of 192IrUFP.
Semmler et Holger
al.GMC
2004,
Inhal Toxicol.
Schulz
IHB-Institut für
Inhalationsbiologie
Extra-pulmonary Effects
Which kind of extra-pulmonary PM- effects have been described?
Cardio Vascular System (CVS)
- Disturbance of vegetative balance – stress response
- Disturbance of vasomotor function – vasoconstriction
- Cardiac Arrhythmia
- Systemic/endothelial inflammation
- Procoagulative state
- Endothelial dysfunktion
- Aggravation of atherosclerotic process
=> Infarction – Coronary failure
Central Nervous System (CNS)
- Inflammation / neurodegeneration (activation of microglia cells)
ApoE-Modell + Mexico City Daten
GSF-Forschungszentrum
für Umwelt und Gesundheit
GMC
Holger Schulz
IHB-Institut für
Inhalationsbiologie
Which kind of extrapulmonary PM- effects have been described?
Cardio Vascular System (CVS)
- Disturbance of vegetative balance – stress response
- Disturbance
Harder
2005:of vasomotor function – vasoconstriction
Rats
exposed
to UfCP (180µg/m3) for 24h
- Cardiac
Arrhythmia
- Systemic/endothelial inflammation
⇒ increased heart rate
- Procoagulative
⇒
reduced heart state
rate variability
- Endothelial dysfunktion
sympathetic stress response ?
- Aggravation of atherosclerotic process
=> Infarction – Coronary failure
Central Nervous System (CNS)
- Inflammation / neurodegeneration (activation of microglia cells)
ApoE-Modell + Mexico City Daten
GSF-Forschungszentrum
für Umwelt und Gesundheit
GMC
Holger Schulz
IHB-Institut für
Inhalationsbiologie
Which kind of extrapulmonary PM- effects have been described?
Cardio Vascular System (CVS)
- Disturbance of vegetative balance – stress response
- Disturbance of vasomotor function – vasoconstriction
Harder
2005:
(Telemetric study)
- Cardiac
Arrhythmia
Hypertensive rats exposed to UfCP (180µg/m3) for 24h
- Systemic/endothelial
⇒
increased heart rate inflammation
⇒
increased diastolic
blood pressure
- Procoagulative
state
Elder
2004: dysfunktion
- Endothelial
On-Road Exposure of Aged Rats
- Aggravation
of atherosclerotic
process
⇒
Increased plasma
levels of Endothelin
=> Infarction
Dvonch
2004: – Coronary failure
Rats exposed to PM2.5 (Detroit, 354µg/m3, 3 days, 8h/day)
⇒ Increased plasma levels of ADMA
(asymmetric
dimethylarginie,
eNOS
inhibitor)
Central
Nervous
System
(CNS)
! vasoconstriction,
endothelial(activation
dysfunktion
- Inflammation
/ neurodegeneration
of!microglia cells)
ApoE-Modell + Mexico City Daten
GSF-Forschungszentrum
für Umwelt und Gesundheit
GMC
Holger Schulz
IHB-Institut für
Inhalationsbiologie
Which kind of extrapulmonary PM- effects have been described?
Cardio Vascular System (CVS)
- Disturbance of vegetative balance – stress response
- Disturbance of vasomotor function – vasoconstriction
- Cardiac Arrhythmia
- Systemic/endothelial inflammation
- Procoagulative state
Khandoga
2004:
- Endothelial
dysfunktion
intra-arterial infusion of UfCP in healthy mice (107/ mouse)
- Aggravation
of atherosclerotic
process
⇒
induced platelet
adhesion
⇒
deposition
and vWF expression on the endothelial
=>increased
Infarctionfibrin
– Coronary
failure
surface
! Prothrombotic effect !
Central
Nervous System (CNS)
- Inflammation / neurodegeneration (activation of microglia cells)
ApoE-Modell + Mexico City Daten
GSF-Forschungszentrum
für Umwelt und Gesundheit
GMC
Holger Schulz
IHB-Institut für
Inhalationsbiologie
Which kind of extrapulmonary PM- effects have been described?
Cardio Vascular System (CVS)
- Disturbance of vegetative balance – stress response
- Disturbance of vasomotor function – vasoconstriction
- Cardiac Arrhythmia
- Systemic/endothelial inflammation
- Procoagulative state
- Endothelial dysfunktion
- Aggravation of atherosclerotic process
Sun
2005: – Coronary failure
=>
Infarction
ApoE-/- mice (+ high fat diet) exposed to 85 µg/m3 PM2.5 for 6 month
⇒ increased atherosclerotic plaque development
⇒ increased
vascular
inflammation
Central
Nervous
System
(CNS)
⇒ increased vasomotor tone
- Inflammation / neurodegeneration (activation of microglia cells)
ApoE-Modell
+ Mexico
City Daten
! potentiated
atherosclerosis
GSF-Forschungszentrum
für Umwelt und Gesundheit
GMC
Holger Schulz
!
IHB-Institut für
Inhalationsbiologie
Which kind of extrapulmonary PM- effects have been described?
Cardio Vascular System (CVS)
- Disturbance of vegetative balance – stress response
- Disturbance of vasomotor function – vasoconstriction
- Cardiac Arrhythmia
- Systemic/endothelial inflammation
- Procoagulative state
- Endothelial dysfunktion
- Aggravation of atherosclerotic process
=> Infarction – Coronary failure
Central Nervous System (CNS)
- Inflammation / neurodegeneration (activation of microglia cells)
ApoE-Modell + Mexico City Daten
GSF-Forschungszentrum
für Umwelt und Gesundheit
GMC
Holger Schulz
IHB-Institut für
Inhalationsbiologie
Epidemiological data
PM and frequency in heart attacks (Augsburg 1995-2000)
60
40
80
PartikelParticle
masse
mass
(PM10))
(PM
10
Veränderung in %
Veränderung
Change (%)in %
80
20
0
-20
60
40
PartikelParticle
anzahl
number
(geschätzt)
(estimate)
20
0
-20
Erst-Infarkt
Re-Infarkt
Todesfall
Erst-Infarkt
Re-Infarkt
Todesfall
Association of particle mass - or number-concentration with infarct rate
GSF-Forschungszentrum
für Umwelt und Gesundheit
GMC
Peters
et al.
Holger Schulz
IHB-Institut für
Inhalationsbiologie
Mechanism of cardiovascular effects
Ambient PM
Pulmonary Reflexes
Translocation
Pulmonary Inflammation
Autonomic Nervous
System
Heart
Vascular System
Systemic Inflammation
Oxidative stress
Electrophysiological Instability
Conduction / Repolarization
Heart Rate
Endothelial Cell
Dysfunction
GSF-Forschungszentrum
für Umwelt und Gesundheit
Acute phase
Response Coagulation
Factors
Progression of Atherosclerosis
Plaque Instability
Cardiac Rhythm
Brachycardia
Tachycardia
Arrhythmia
Ventricular Fibrillation
Leucocyte,
Platelet
Activation
Thrombosis - Plaque rupture
Cardiac Events
Sudden Cardiac Death
GMC
Holger Schulz
CNS
IHB-Institut für
Inhalationsbiologie
Mechanistic Aspects of Nanoparticle Toxicology Particle Deposition and Clearance
1.
Deposition & Clearance Mechanism
- high alveolar deposition
- ineffective clearance
2. Acute Pulmonary Effects
- surface area as dose metric
- NP cause oxidative stress to cellular structures
3. Translocation to Secondary Organs
- translocation efficiency ~0.1%
4. Extra-pulmonary Effects
- aggravation of inflammatory & cardiovascular diseases
Mechanistic Aspects of Nanoparticle Toxicology Particle Deposition and Clearance and Effects
Thank you for your attention!
And special thanks to:
Prof. Holger Schulz
Dr. Wolfgang Kreyling
GSF-National Research Center
for Environment and Health
member of the Helmholtz Association
IHB-Institute for
Inhalation Biology