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NAPIER UNIVERSITYEDINBURGH
The Cellular and Molecular Toxicity of Low Solubility
Nanoparticles
Prof Vicki StoneCentre for Health and Environment
Napier University, Edinburghhttp://www.lifesciences.napier.ac.uk/Research/CHE1.htm
Safety of nanomaterials Interdisciplinary Research Centrehttp://www.snirc.org/
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PM10
Carbon based particles•Traffic and industrial•Many ultrafine•Transition metals
Sulphates/Nitrates•Traffic/photochemical•Mainly ultrafine
Wind blown dust•Mainly coarse
Biological components•Spores•Pollen•Mainly fine and course
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500nm
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Lung Defence Mechanisms
Blood vesselGas exchange
Particles trapped and cleared on
mucociliaryescalator
Airway
AlveolusParticles
phagocytosedand cleared
mucus cilia
Macrophage••••
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Particles
Antioxidants
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The Ultrafine Particle Hypothesis
Seaton al. 1995 The Lancet 345: 176-178•
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Particles
Release of mediators by
macrophages and epithelial cells
Impaired movement
Blood vessel
INFLAMMATION
Particles cross the epithelial barrier
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Ultrafine particle toxicology
0
20
40
60
80
100
0 10 20 30 40 50 60 70Time from start of exposure (weeks)
Neutrophils in bronchoalveolar
lavage fluidX 105
21 nm TiO2
250 nmTiO2
Ferin et al 1992Am.J.Respir.Cell.Mol.Biol. 6: 535-542
‘Accidental’ versus ‘engineered’nanoparticles
Nanoparticles Source Exposure Toxicology
Accidental Fossil fuel combustionE.g. Traffic, cooking.
Low exposure to everyone
Lots e.g. diesel, CB, welding fume etc
Purposeful Bulk use of nanoparticles in industry e.g. carbon black
Nanotechnology
High exposure toworkers followed by low exposure to everyone
Virtually none
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Adapted from Ken Donaldson
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CB
100nm
100nm
14 nm254 m2/g
260 nm8 m2/g
Carbon black
535nm
202nm
64nm
500nm
Polystyrene beads
Model nanoparticles
Low solubility, low toxicity materials.
Factors involved in NP toxicity
..
10μm 1 μm 0.1μm
Bronchial epithelium
0.1μm1.0 μm
Cilia 0.25μm diameter
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1. SIZE
2. PARTICLE NUMBERThere are thousands more ultrafine particles / mg of dust than larger, respirable particles.
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Factors involved in NP toxicity
3. SURFACE AREAParticle surface area / mg is much greater for ultrafineparticles than for larger, respirable particles.
Fine carbon black 7.9 m2/gUltrafine carbon black 253.9 m2/g
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Particle surface area and surface reactivity
Low toxicity low solubility particles •straight line relationship between SA and neutrophils.
Highly pathogenic particles •highly reactive surface (eg quartz). •do not sit on same line as low toxicity particles.
0
5
10
0 200 400 600 800 1000Surface Area instilled (cm2)
Neutrophils in BAL (x106)
Relatively low toxicity particles
Quartz
Duffin et al. (2002) Ann.Occ.Hyg.46;242-245
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Reactive oxygen species production by carbon particles
Stone et al. 1998 Toxicol In Vitro 12: 649-659.
0
2
4
6
8
10
12
1 5 10Particle dose / 290 μl plasmid
Supercoiledband intensity x 103
260 nm CB14 nm CB
* *
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Reactive oxygen species produced by nanoparticles
Wilson et al. 2002 TAP 184: 172-179.
50
100
150
0 0.5 1.0 1.5 2.0 2.5
Particle dose (mg/ml)
DC
F Fl
uore
scen
ce In
tens
ity
260 nm CB
14 nm CB***
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**
-5
5
15
25
35
45
55
64 nm 202 nm 535 nmPolystyrene particle diameter
DC
F Fl
uore
scen
ce In
tens
ity
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Brown et al. 2001 TAP 175: 191-199.
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Effect of carbon particles on intracellular antioxidant content of A549 cells
Stone et al. 1998 TIV 12: 649-659.0.78 μg/mm2
14 nm CB260 nm CBControl
40
50
60
70
80
90
100
110
120
0 2 4 6 8 Time (hours)
GSH (% of control)
*
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Effect of particles on Ca2+ signalling in human MonoMac 6 cells
Stone et al. 2000 Eur Resp J
15: 297-303.
0
20
40
60
80
100
120
140
160
Control 14 nmCB
260 nmCB
[Ca2+]c
2000 sec
**
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Ca 2+
THAPSIGARGIN
Ca 2+
Ca 2+VERAPAMIL
0 200 400 600 800 1000 1200
Control
14 nmCB
260 nm CB
[Ca2+]c nM after treatment with thapsigargin
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Particles Particles + verapamil
The response to thapsigargin in human MM6 cells in the presence of CB and verapamil
Stone et al. 2000 Eur Resp J 15: 297-303.
2000 sec
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1500 sec Stone et al. 2000 Inhal Tox 12 (suppl 3): 345-351.
[Ca2+]c nM after treatment with thapsigargin0 100 200 300 400 500 600 700 800 900
Control
64 nm
202 nm
535 nm
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Pol
ysty
rene
bea
d di
amet
er
The effect of polystyrene nanoparticles on calcium signalling in macrophages
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[mM]
Ca 2+
Calciumchannel
[nM]
Ca 2+
Ca 2+BAPTACa chellator
Calmodulin
SignallingW-7
Endoplasmicreticulum
VerapamilCa2+
channel blocker
Calcium Signalling – Inhibitors
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TNFα pg/ml
0
50
100
150
200
250
Control 14nmCB
260nmCB
14nmCB
Verap
Verap 14nmCB
BAPTA
BAPTA
Rat alveolar macrophages4 hour treatments
***
Brown et al. 2004 AJP 286; L344-L353
The role of Ca2+ in the induction of TNFαexpression by carbon nanoparticles
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The role of Ca2+ in the induction of TNFαmRNA expression by CB nanoparticles
GAPDH
TNFαControl 14nm
CB260nm
CB14 CBVerap
Verap 14 CBBAPTA
BAPTA LPS
TNFα mRNA(% GAPDH)
05
1015202530
Control 14nmCB
260nmCB
14nm CBVerap
Verap 14nm CBBAPTA
BAPTA
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**
Brown et al. 2004 AJP 286; L344-L353
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Brown et al. 2004 AJP 286; L344-L353
The effect of antioxidants on CB induced TNFα expression
0
50
100
150
200
250
Control 14nmCB
14nmCB
Nacystelin
Nacystelin
TNFα pg/ml
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0
5
10
15
20
25
30
Control14nmCB
14nmCB
Nacystelin
Nacystelin
TNFα mRNA(% GAPDH)
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200
100
150
50
0Control 14 nm CB CB CB CB
CB + verap + BAPTA + W-7 + Trolox
TNFα pg/ml
Particle induced TNFα expression – role of calcium and oxidants
**Human monocyte derived macrophages
4 hour treatments
Brown et al. 2004 AJP 286; L344-L353
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Activation of NFκB and promoter binding
Phosphorylation
DNA
Promoter
Nuclear membrane
IκB
NFκB IκB
NFκB
PIκB
NFκB
P
NFκB
mRNA
Degraded
TRANSCRIPTION
P
U
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Control
14 nm CB
Immunofluorescent staining of the p65 subunit of NFκB in human monocyte/macrophages
Brown et al. 2004 AJP 286; L344-L353
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40
80
120
160
Control CB CB CB CB CB CB + verap + W-7 + BAPTA + Nac + Trolox
Nuclear p65 intensity(% control)
*
Brown et al. 2004 AJP 286; L344-L353
Nuclear localisation of the p65 subunit of NFκB in human monocytes
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Nanoparticles, calcium and oxidants
Ultrafine, nanoparticles
Macrophages
Oxidative Calcium stress signalling
Activation of NF-κB
Production of pro-inflammatory proteins
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J774 macrophages exposed to 14nm CB
CONTROL
14 nm CB
Wilson et al., manuscript in prep
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Twisting field BTW
Fluxgate sensor
Θ
Step twist
6 HzMagnetic particleswithin phagosomes Macrophage
•Magnetic twisting device•Aligned ferromagnetic microparticles ingested by macrophages,•Detection by an array of magnetic fluxgate sensors (Förster device).
Measurement of cytoskeletal stiffness using ferromagnetic particles
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
14nmCB
EC90 DEP UD
Relaxation b5 (drug/control)
*
*
320 μg/million cells EC90
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6Relaxation b5 (drug/control)
EC90 Verap BAPTA W7
$ $ $*
Impact of particles on the macrophage phagosome transport
Moeller et al., 2003 Manuscript submitted
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Chemotaxis Chamber
Actual Filter
Filter (5 µm pore)
Chemoattractant
Macrophages (J774.2)
Direction of migration
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Chemotaxis of J774 macrophages in response to epithelial cell conditioned medium
50
100
150
200
ZAS 250 nmTiO2
20 nmTiO2
260 nmCB
14 nmCB
Chemotaxis% of Control Particles &
CellsParticles Only
*
Barlow et al. 2005 Particle Fibre Toxicol 2:11.
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Chemotactic response of J774 macrophages to epithelial cell conditioned medium
100
120
140
160
180
200
ZAS >30 10-30 5-10 <5
% of Control
Molecular Weight Fraction of Conditioned Medium kDa
Barlow et al. 2005 Particle Fibre Toxicol 2:11.
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Chemotactic response of J774 macrophages to serum treated with particles
260nm CB
90
110
130
150
170
190
210
ZAS 1.51 51.51 51.51 5 mg/ml1.51
% o
f Neg
ativ
e C
ontro
l
14nm CB 21nm TiO2 250nm TiO2
5
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Barlow et al. 2005 Toxicol Lett 155:397-401.
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Effect of TiO2 and CB on phagocytosis
Renwick et al. 2001 Toxicol Applied Pharmacol. 172: 119-127
% of cells that phagocytose >2 beads8h exposure
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Non-phagocytic cells
Renwick et al. 2001 Toxicol Applied Pharmacol. 172: 119-127
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Effects of nanotubes on cell viability
0
5
10
15
20
25
30
35
40
Control E21 3-01 PR19 1-03 9-01 UfCB LFA Triton TNF
Nanotube Treatment
% T
otal
Cel
ls
NecroticApoptotic
15.625ug/ml Nanotubes
0
5
10
15
20
25
30
35
40
Control E21 3-01 PR19 1-03 9-01 UfCB LFA Triton TNF
Nanotube treatment
% T
otal
Cel
ls
NecroticApoptotic
31.25ug/ml nanotubes
16μg/ml
31μg/ml
ApoptosisNecrosis
Nanotubes/fibres CB LFA Triton TNFαControl
% total cells40
30
20
10
0
40
30
20
10
0Nanotubes/fibres CB LFA Triton TNFαControl
% total cells
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Effect of nanotubes and nanofibres on phagocytosis
0102030405060708090
Medium E21
PR19Sam
ple 9-0
1Sam
ple 3-0
1Sam
ple 1-0
3
Uf CB
LFA
Treatment
Phag
ocyt
osis
(% T
otal
)
15.625ug/ml31.25ug/ml62.5ug/ml
All treatments induced a significant (p<0.05) inhibition
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Summary
Low toxicity, low solubility NP:
•Inflammation is related to surface area dose.
•Generate ROS leading to oxidative stress in epithelial and macrophage cells.
•Induce Ca2+ influx in macrophages in vitro resulting in; •activation of NFκB and AP1•up-regulation of pro-inflammatory cytokine expression•Inhibition of phagosome transport
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Summary
Low toxicity, low solubility NP:
•Stimulate production of chemotaxins by epithelial cells.
•Rapidly taken up into macrophages.
•Inhibit subsequent phagocytosis of larger particles
MWCNT:
•Morphology determines uptake by macrophages.
•Inhibit subsequent phagocytosis of E.Coli.
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Napier UniversityDr David BrownDr Martin WilsonMarieke TuinmanDr Suzanne PattersonJanis VamvakopolousDr Peter BarlowMartin Clift
University of EdinburghProf Ken Donaldson
University of CambridgeProf Alan WindleDr Ian Kinloch
UIF DusseldorfDr Rodger DuffinDr Roel SchinsDr Paul BormDr Markus Denhart
GSF MunichDr Winfried Moeller
Nottingham UniversityDan WaltersTHE COLT FOUNDATION