Christoph Bisig Adolphe Merkle Institute [email protected] [email protected] [1] Zhang, S. and McMahon, W.; SAE International Journal of Fuels and Lubricants 5(2), 637-646 (2012). [2] Reed, M.D. et al.; Inhalation toxicology 20(13), 1125-1143 (2008). [3] Bisig, C.J. et al.; CHIMIA International Journal for Chemistry 69(1), 68-68(1) (2015). [4] Bisig, C.J. et al. Emission Control Science and Technology 1.3 (2015): 237-246. This work was funded by the VERT Association, Swiss federal office for the environment, Swiss federal office for energy, Adolphe Merkle Foundation, Schweizer Erdölvereinigung, thank you very much. Conclusion o Coated and uncoated GPFs reduced PN significantly in comparison to unfiltered GDI exhaust, while the volatile emissions did not differ in the three GDI exhausts. o Diesel exhaust contained significant higher PN and NOx emissions compared to GDI exhaust, and effects in the 3D lung cell model such as oxidative stress, pro-inflamma- tion, and metabolic activation were measured. o The GDI vehicle used in the current study did not induce adverse effects in an acute exposure scenario for unfil- tered as well as filtered conditions. We have, however, shown recently that depending on the vehicle unfiltered GDI exhaust can induce oxidative stress and metabolic activation [4], therefore the conclusions cannot be gener- alized for all GDI vehicles. o In addition, long-term exposures are recommended for future studies to reveal chronic effects. Fig. 5 Picture of tail- pipe. A possible addi- tion of the particle filter. 1:10 dilution of exhaust. Fig. 6 Exhaust analy- sis directly at tail- pipe (particles, shown in this pic- ture) or in the CVS tunnel (CO, THC, NOx) Vehicle setup o Gasoline direct injection (GDI) passenger car (Euro5) Unfiltered (original) uncoated GPF coated GPF o Diesel passenger car (Euro2) unfiltered (original) o Worldwide Harmonized Light Vehicles Test Cycles (WLTC) Exposure setup o 6 hours (app 10 WLTC) exposure to filtered air or 10x diluted exhaust 3D human lung epithelial tissue model is composed of o Bronchial epithelial cells (16hbe, red) o Macrophages (from human monocytes, thiel) o Dendritic cells (from human monocytes, green) o The cells are grown at the air liquid interface (air on top, medium on bottom), (Fig.1b) and [3]. Endpoints (shown here) o Gene expression analysis (Fig. 3). All data has been nor- malized to filtered air and GAPDH, a standard gene whose expression is independent of a treatment. o Microscopy images (Fig. 4). DAPI stained the nucleus and Phalloidin Rhodamine F-actin cytoskeleton. Materials and Methods TNF IL8 0 2 4 6 8 Air GDI: unfiltered GDI: uncoated GPF GDI: coated GPF Diesel: unfiltered Oxidative stress. HMOX1: Induced after oxidative stress. SOD1/2: First line against ROS. GSR: Recovery of glutathione (one of the main detoxification molecule). Aryl hydrocarbon receptor (AhR) activation. NQO1: Detoxification of xenobiotics IDO1: Maintains stable condition in immune cells (homeostasis) NFE2L2: Regulates various other genes CYP1A2: Detoxification of xenobiotics (e.g. PAHs) Particles, gases, metals in the lungs can cause reactive oxygen species (ROS). Polyaromatic hydrocarbons (PAH) can bind to the aryl hydrocarbon receptor (AhR). Pro-inflammation. TNF: involved in acute, systemic inflammation IL8: recruits other immune cells Fig. 3 Gene expression analysis of 10 genes. All data has been normalised to filtered air control (Air line=1). n=4-8. GDI (both filtered and with GPF) No oxidative stress No pro-inflammation No increase in AhR activation Diesel (unfiltered) Oxidative stress Pro-inflammation Increased metabolisation (NQO1 upregulation) Fig. 4 Microscopy images. XZ cross-sections. Nucleus (green), F-Actin cytoskeleton (magenta). Confluent monolayer for all conditions Filtered air GDI unfiltered GDI uncoated GPF GDI coated GPF Diesel Results Fig. 2 Exhaust characterisation. Shown are emissions from the extra high part of the WLTC only. Both GPFs reduced particle number GDI emitted low amounts of THC and NOx Diesel emitted high amounts of PN and NOx Oxidative stress Pro-inflammation AhR activation NQO1 IDO1 NFE2L2 CYP1A2 0 2 4 6 8 Air HMOX1 SOD1 SOD2 GSR 0 2 4 6 8 Air Particle number 10 11 10 12 10 13 10 14 Particle number [#/km] Normalized amount of mRNA Normalized amount of mRNA Normalized amount of mRNA Volatile exhaust CO THC NOx 0 10 20 30 500 1000 1500 2000 Volatile emission [mg/km] can lead to 40 μm ROS PAH Macrophages Bronchial lung cells Dendritic cells Introduction Gasoline direct injection (GDI) engines are increasingly used, due to their greater power, better fuel efficiency, and lower CO2-emissions, though with the drawback of emit- ting more (nano)particles than the older multipoint port fuel injection (MPI) [1]. The demand of implementing gas- oline particle filters (GPF) on GDI vehicles is therefore growing. Studies on possible toxicity from gasoline ex- haust are scarce, and most research so far has been per- formed with MPI engines (e.g. [2]). We therefore investi- gated the effects of whole diluted exhaust from a new GDI vehicle exposed to a sophisticated 3D human lung tissue model. In addition, changes of exhaust composition upon installation of two different GPFs and their effects on lung cell responses were compared. Fig. 1 Exposure setup. [A] An exposure box (blue) is directly connected to the exhaustpipe of a passenger car. The exposure box contains two chambers ([a] filtered air and exhaust). [B] Scheme of the human lung model composed of three different human cells. The cells are at the air-liquid interface, air (or exhaust) on top, and medium on the bottom [3]. A a B Exposure to complete exhaust 3D human lung model NOx CO HC CO HC NOx HC Christoph Bisig1, Pierre Comte2, Andreas Mayer3, Jan Czerwinski2, Alke Petri-Fink1, Barbara Rothen-Rutishauser1 Hazard assessment of gasoline direct injection engine exhaust directly exposed onto the surface of a 3D human lung model 1Adolphe Merkle Institute (AMI), University of Fribourg, Switzerland; 2Bern University for Applied Sciences (UASB), Switzerland; 3Technik Thermischer Maschinen (TTM), Switzerland
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Christoph BisigAdolphe Merkle [email protected] [email protected]
[1] Zhang, S. and McMahon, W.; SAE International Journal of Fuels and Lubricants 5(2), 637-646 (2012). [2] Reed, M.D. et al.; Inhalation toxicology 20(13), 1125-1143 (2008). [3] Bisig, C.J. et al.; CHIMIA International Journal for Chemistry 69(1), 68-68(1) (2015).[4] Bisig, C.J. et al. Emission Control Science and Technology 1.3 (2015): 237-246.This work was funded by the VERT Association, Swiss federal o�ce for the environment, Swiss federal o�ce for energy, Adolphe Merkle Foundation, Schweizer Erdölvereinigung, thank you very much.
Conclusiono Coated and uncoated GPFs reduced PN signi�cantly in comparison to un�ltered GDI exhaust, while the volatile emissions did not di�er in the three GDI exhausts.
o Diesel exhaust contained signi�cant higher PN and NOx emissions compared to GDI exhaust, and e�ects in the 3D lung cell model such as oxidative stress, pro-in�amma-tion, and metabolic activation were measured.
o The GDI vehicle used in the current study did not induce adverse e�ects in an acute exposure scenario for un�l-tered as well as �ltered conditions. We have, however, shown recently that depending on the vehicle un�ltered GDI exhaust can induce oxidative stress and metabolic activation [4], therefore the conclusions cannot be gener-alized for all GDI vehicles.
o In addition, long-term exposures are recommended for future studies to reveal chronic e�ects.
Fig. 5 Picture of tail-pipe. A possible addi-tion of the particle �lter. 1:10 dilution of exhaust.
Fig. 6 Exhaust analy-sis directly at tail-pipe (particles, shown in this pic-ture) or in the CVS tunnel (CO, THC, NOx)
Vehicle setupo Gasoline direct injection (GDI) passenger car (Euro5)
Un�ltered (original)uncoated GPFcoated GPF
o Diesel passenger car (Euro2)un�ltered (original)
o Worldwide Harmonized Light Vehicles Test Cycles (WLTC)
Exposure setupo 6 hours (app 10 WLTC) exposure to
�ltered air or 10x diluted exhaust
3D human lung epithelial tissue model is composed ofo Bronchial epithelial cells (16hbe, red)o Macrophages (from human monocytes, thiel)o Dendritic cells (from human monocytes, green)o The cells are grown at the air liquid interface (air on top, medium on bottom), (Fig.1b) and [3].
Endpoints (shown here)o Gene expression analysis (Fig. 3). All data has been nor-malized to �ltered air and GAPDH, a standard gene whose expression is independent of a treatment.o Microscopy images (Fig. 4). DAPI stained the nucleus and Phalloidin Rhodamine F-actin cytoskeleton.
Materials and Methods
TNF IL80
2
4
6
8
Air
GDI: un�lteredGDI: uncoated GPF
GDI: coated GPFDiesel: un�ltered
Oxidative stress. HMOX1: Induced after oxidative stress.SOD1/2: First line against ROS.GSR: Recovery of glutathione (one of the main detoxi�cation molecule).
Aryl hydrocarbon receptor (AhR) activation.NQO1: Detoxi�cation of xenobioticsIDO1: Maintains stable condition in immune cells (homeostasis)NFE2L2: Regulates various other genes CYP1A2: Detoxi�cation of xenobiotics (e.g. PAHs)
Particles, gases, metals in the lungs can cause reactive oxygen species (ROS).Polyaromatic hydrocarbons (PAH) can bind to the aryl hydrocarbon receptor (AhR).
Pro-in�ammation.TNF: involved in acute, systemic in�ammationIL8: recruits other immune cells
Fig. 3 Gene expression analysis of 10 genes. All data has been normalised to �ltered air control (Air line=1). n=4-8.
GDI (both �ltered and with GPF)No oxidative stressNo pro-in�ammationNo increase in AhR activation
Diesel (un�ltered)Oxidative stress Pro-in�ammationIncreased metabolisation (NQO1 upregulation)
Fig. 4 Microscopy images. XZ cross-sections. Nucleus (green), F-Actin cytoskeleton (magenta).
Con�uent monolayer for all conditions
Filtered air
GDI un�ltered
GDI uncoated GPF
GDI coated GPF
Diesel
Results
Fig. 2 Exhaust characterisation. Shown are emissions from the extra high part of the WLTC only.
Both GPFs reduced particle numberGDI emitted low amounts of THC and NOx Diesel emitted high amounts of PN and NOx
Oxidative stress Pro-in�ammation
AhR activation
NQO1 IDO1 NFE2L2 CYP1A20
2
4
6
8
Air
HMOX1 SOD1 SOD2 GSR0
2
4
6
8
Air
Particle number
1011
1012
1013
1014
Part
icle
num
ber [
#/km
]
Nor
mal
ized
am
ount
of m
RNA
Nor
mal
ized
am
ount
of m
RNA
Nor
mal
ized
am
ount
of m
RNAVolatile exhaust
CO THC NOx0
10
20
30
500
1000
1500
2000
Vola
tile
emis
sion
[mg/
km]
can lead to
40 µm
ROS
PAH
MacrophagesBronchial lung cellsDendritic cells
IntroductionGasoline direct injection (GDI) engines are increasingly used, due to their greater power, better fuel e�ciency, and lower CO2-emissions, though with the drawback of emit-ting more (nano)particles than the older multipoint port fuel injection (MPI) [1]. The demand of implementing gas-oline particle �lters (GPF) on GDI vehicles is therefore growing. Studies on possible toxicity from gasoline ex-haust are scarce, and most research so far has been per-formed with MPI engines (e.g. [2]). We therefore investi-gated the e�ects of whole diluted exhaust from a new GDI vehicle exposed to a sophisticated 3D human lung tissue model. In addition, changes of exhaust composition upon installation of two di�erent GPFs and their e�ects on lung cell responses were compared.
Fig. 1 Exposure setup. [A] An exposure box (blue) is directly connected to the exhaustpipe of a passenger car. The exposure box contains two chambers ([a] �ltered air and exhaust). [B] Scheme of the human lung model composed of three di�erent human cells. The cells are at the air-liquid interface, air (or exhaust) on top, and medium on the bottom [3].
A
a
BExposure to
complete exhaust
3D human lung model
NOxCO
HCCO
HCNOx
HC
Christoph Bisig1, Pierre Comte2, Andreas Mayer3, Jan Czerwinski2, Alke Petri-Fink1, Barbara Rothen-Rutishauser1
Hazard assessment of gasoline direct injection engine exhaust directly exposed onto the surface of a 3D human lung model
1Adolphe Merkle Institute (AMI), University of Fribourg, Switzerland; 2Bern University for Applied Sciences (UASB), Switzerland; 3Technik Thermischer Maschinen (TTM), Switzerland
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