TESTING OF AEROSOLS FOR LUNG TOXICITY BY IN-VITRO …

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

2Institute for Technical Chemistry

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


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)


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


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)


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


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)


24 h exposure experiments with TiO2 aerosol

pump

TiO2 suspension

Synth. air

silicagel

drying

reactor

Two phase

nozzle

SMPS


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


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)


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.


Experimental set up


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


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


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


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


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


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.


Thank you!

Questions?

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TESTING OF AEROSOLS FOR LUNG TOXICITY BY IN-VITRO …