testing of aerosols for lung toxicity by in-vitro …
TRANSCRIPT
1Institute for Technical Chemistrywww.kit.edu
KIT – University of the State of Baden-Wuerttemberg and
National Research Center of the Helmholtz Association
TESTING OF AEROSOLS FOR LUNG TOXICITY BY IN-VITRO STUDIES AT THE
AIR-LIQUID INTERFACE FOR UP TO 24 HOURS
1S. Mülhopt, 2S. Diabaté, 2M. Dilger,3C. Schlager, 3M. Berger, 3T. Krebs, 1H.-R. Paur,2C. Weiss, 1D. Stapf
2 Institute of Toxicology and Genetics1 Institute for Technical Chemistry 3 VITROCELL Systems GmbH
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The Karlsruhe Exposure System
Mülhopt, S.; Paur, H.R.; Diabate, S.; Krug, H.F. In vitro testing of inhalable fly ash at the air liquid
interface. Kim, Y.J. [Hrsg.] Advanced Environmental Monitoring Dordrecht : Springer
Netherlands, 2007 S.402-14 ISBN 978-1-4020-6363-3
Paur, H.-R., Cassee, F.R., Teeguarden, J., Fissan, H., Diabate, S., Aufderheide, M., Kreyling,
W.G., Hänninen, O., Kasper, G., Riediker, M., Rothen-Rutishauser, B., Schmid, O. (2011)
Journal of Aerosol Science, 42, 10, 668-692
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VITROCELL® automated in-vitro exposure station
A lab scale measurementsystem for the air-liquidinterface exposure of humanlung cell cultures towardsairborne nanoparticles.
Direct aerosol sampling
Online dose measurement
Electrostatic deposition enhancement
Flow, temperature, and humidification control system
Data acquisition
Internal negative control using humidified synthetic air
Quality assurance by automatic leak test
Standard exposure protocol is automatically performed by the programmable controller
Mülhopt et al. (2016) Journal of Aerosol Science, 96, 18
Patents:Mülhopt, S.; Paur, H.R.; Wäscher, T.DE-OS 10 2007 013 938 (2008.09.25)Mülhopt, S.; Paur, H.R.; Schlager, C. DE 10 2014 118 846 B4 (2016.06.23)
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Aerosols
Manufactured materials
Metals/metal oxides:
Titania
- Aeroxide P25
Silica
- Amorph:
- Aerosil200
- Stöber
synthesised
- Quartz
Cerium oxide
Silver
Platinum
CNT
Combustion derived aerosols
- Ship diesel
- Diesel fuel
- Heavy fuel oil
- Wood combustion
- Log wood stoves
- Pellet boiler
- Log wood boiler
- Car emissions
- Diesel fuel
- Driving behaviour
- Municipal waste incinerator
- Nanocomposites of polymer and MNM
fillers
30.11.2
015
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Endpoints
Cell systems
Epithelial cells:
- A549 (alveolar)
- BEAS-2B (bronchial)
- 16HBE14o (bronchial)
Macrophages:
- RAW264.7 (mouse)
2D:
- Co-culture with THP-1
macrophages
3D:
- Triple cultures with THP-1
macrophages and HUVEC
endothelial cells
Primary cells:
- MucilAir (Epithelix)
Read out
- Oxidative stress:
- HMOX-1 (WB, RT-qPCR)
- GSH/GSSG
- Inflammation
- Cytokines (ELISA, RT-qPCR)
- Cytotoxicity
- LDH release, metabolic activity
(Alamar Blue, MTS)
- Genotoxicity
- DNA strand breaks (alkaline
unwinding, γ-H2Ax expression)
- high-throughput RT-qPCR with
selected gene sets1
- Metabolism of foreign substances
- Expression of CYP1A1
− Genome-wide RNA analysis
- RNA-seq (genome-wide analysis)
1 Fischer et al. Arch. Toxicol. (2016)
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Dosimetry at the Air-Liquid Interface
Airborne particles:
Number concentration cN [1/cm³]
Mass concentration cM [µg/cm³]
Particles on cell surface
Number concentration cN [1/cm²]
Mass concentration cM [µg/cm²]
Deposition efficiency
𝑊 =𝑑𝑒𝑝𝑜𝑠𝑖𝑡𝑒𝑑 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 𝑚𝑎𝑠𝑠/𝑛𝑢𝑚𝑏𝑒𝑟
𝑒𝑥𝑝𝑜𝑠𝑒𝑑 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 𝑚𝑎𝑠𝑠/𝑛𝑢𝑚𝑏𝑒𝑟[%]
Relevant in
vitro dose
Fluid: gas (air)
Viscosity ~ 1 µPa*s
Density ~ 10-3 g/cm³
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Dosimetry methods at the ALI
Fluorescein sodium dosimetry (FNA)spectroscopic measurement of deposited mass
Quartz crystal microbalanceOnline measurement of mass dose per area
TEM analysisImage analysis delivers information about
particle shape and homogeneity of distribution
Numerical simulationDeposition efficiencies in dependence of chosen
boundary conditions or geometries
cM [µg/cm²]
cM [µg/cm²]
f(t)
cN [µg/cm²]size
Shape
cN, cM
f(dP)
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24 h exposure experiments with TiO2 aerosol
pump
TiO2 suspension
Synth. air
silicagel
drying
reactor
Two phase
nozzle
SMPS
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Dosimetry I: Particle number size distribution
in the Exposure System determined by SMPS
10 100 1000
0.0
2.0x104
4.0x104
6.0x104
8.0x104
1.0x105
num
ber
concentr
ation d
N/d
lod(d
P)
[1/c
m³]
particle diameter dP [nm]
Modal value xM
Standard
Deviation sg
Total number
conc. cN
Estimated
diff. dose
Estimated
dose rate
[nm] [-/-] [1/cm³] [µg/cm²] [µg/(cm²*h)]
190 2.2 6.0E+04 0.235 0.01
Calculated using the
deposition efficiency
from fluorescein sodium
dosimetry f = 1.5 %:
Mass concentration
calculated with effective
density r = 0.638 g/cm³
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Dosimetry II: Image evaluation and QCM
50nm
5 µm
centre inner radius edge mean0.00
0.05
0.10
0.15
0.20
0.25
dose
ra
te [
µg/c
m²/
h]
position
0 2 4 6 8 10 12 14 16 18 20 22 24
-500
0
500
1000
1500
2000
2500
3000
Mass [
ng
/cm
²]
time [h]
Position 1
QCM measurement
Dose rate = 0.062 µg/(cm²*h)
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Cell viability (Alamar Blue assay) and cytotoxicity
(LDH assay) of TiO2 exposed A549 cells
• After 24 hours exposure of A549 towards TiO2 no viability loss is observed.
• After 24 hours exposure towards TiO2 no significant cytotoxicity or inflammatory
response can be observed.
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Experimental set up
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10 100
0,0
4,0x105
8,0x105
1,2x106
1,6x106
2,0x106
num
be
r co
nce
ntr
atio
n d
N/d
log
(dP)
[1/c
m³]
particle diameter dP [nm]
Particle number size distribution of CuO in the
Exposure System determined by SMPS
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TEM images of Cu NP
Effective density according Charvet et al. (2014), J. Nanopart Res:
reff = 15.528*dP-0.826, with dP = 30 nm reff = 0.9354 g/cm³
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Copper particle doses: comparison of methods
SMPS quartz microbalance image analysis0.000
0.005
0.010
0.015
0.020d
ose
ra
te [
µg/c
m²/
h]
dosimetry method
diffusional deposited
electrostatic deposited
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AlamarBlue assay
clean air diff. dep. Cu2O electr. dep.
Cu2O
incubator
control
0
20
40
60
80
100
120ce
ll vita
lity r
ela
tive
to c
ontr
ol [%
]
exposure conditions
Exp. #1
Exp. #2
Exp. #3172 ng/cm²42 ng/cm²
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Inflammatory response
clean air diff. dep.
Cu2O
electr.
dep.
Cu2O
incubator
control
lowest
standard
highest
standard
0.0
0.2
1.0
IL-8
re
lease
[p
g/m
l]
exposure conditions
Exp. #1
Exp. #2
Exp. #3
172 ng/cm²
42 ng/cm²
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Summary
Long term exposures towards clean air are performed
without viability loss or cytotoxic effects.
After 24 hours exposure of A549 towards TiO2 no viability
loss, significant cytotoxicity or inflammatory response can
be observed.
After 24 hours exposure of A549 towards Cu2O no viability
loss is observed.
After 24 hours exposure towards Cu2O no significant
cytotoxicity or inflammatory response can be observed.
Dose rate is a parameter for biological responses.
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Thank you!
Questions?