THE CELL Mr. King. Animal Cell Cell Membrane Animal Cell Cell Membrane Vacuole.
“Laboratory-generated soot with well-defined organic...
Transcript of “Laboratory-generated soot with well-defined organic...
“Laboratory-generated soot with well-defined organic coating for in-vitro cytotoxicity assessment”
Interdisciplinary project funded by the Federal Office for Environment (FOEN, N° 17.0094.PJ)
ZairaLeniKonstan'naVasilatou,MichaelaEss,AlejandroKeller,MarianneGeiser
ETHConference19June2019
# 6 Air pollution is the 6th highest-ranking risk
factor for death globally
Health Effects Institute. 2018. State of Global Air 2018. Boston MA
Global map of modelling annual median concentration of PM2.5 in µg/m3 http://maps.who.int/airpollution/
Source: WHO 2016
92% More than 9 out of 10 of the world’s population lives in areas
exceeding WHO guidelines for safe limits.
<1% In China and India, less than 1% of the population lives in areas
meeting the WHO guidelines
Adapted from “Recent advances in understanding SOA: implications for global climate forcing” M. Shrivastava et al., 2017.
Genera'onofwellcharacterizedagedsootpar'clescoatedwithoxidizedorganicma?er– Anthropogenicorigin(1,3,5-trimethylbenzene,urbansite)– Biogenicorigin(α-pinene,suburban)
Projectgoalsandexperimentalsetup(I)
miniCAST5201TypeBCM.EssandK.Vasilatou,AerosolScienceandTechnology(2019)
Dilu'on Photo-oxida'onreactor
Coated-sootpar'cles
Aethalometer,PAX(870nm)
TEOM
DiffusionSizeClassifier(minidisc)
EC/OCANALYSIS,Chromatography
Par*clesize&numberconcentra*on
Es*ma*onofcoa*ngthickness
Op*calproper*es
Massconcentra*on
Lung-depositedsurfacearea
Chemistry
1:10
Projectgoalsandexperimentalsetup(II)
54 × 49 × 48 cm (WDH), 30.5 kg
(Nano-)Aerosol Chamber for In-Vitro Toxicology, NACIVT (www.nacivt.ch)
Jeannet N. et al., Nanotoxicology , 2015; Geiser M. et al., Nanomaterials, 2017
Ø “All-in-one”,mobilesystemfordirectuseatanypar'clesource
Ø Mimicspar'cledeposi'oninlungs(T,RH,gascomposi'on,airflow,NP,Ndep)
Ø Simultaneousexposureof24cellcultures
Ø Controlled&stablecondi'onsallowlong-termexposures
Aerosol(externalproduc'on/source)
Aerosolcondi'oning(T;RH)
Aerosoldistribu'ontodeliverytubes
Par'cledeposi'onon24cellcultures
Customelectronics(aerosolcharger,integratedpump,
flowcontrol)
Objec'veandexperimentalsetup(III)The three experimental campaigns took place at the Federal Institute of Metrology (METAS)
1st: 13-16/08/2018 (TMB) 2nd: 03-17/12/2018 (TMB) 3rd: 13-24/05/2019 (α-pinene)
The equipment of University of Bern transferred to METAS:
• NACIVT chamber • Cell culture lab (e.g. sterile bench, cell incubator) • Light microscope
Exposureofrealis*ccellmodelstowell-controlled,chemicallydefinedsynthe*creferenceaerosols,toiden*fytheaerosolcomponents/proper*esresponsibleforadverserespiratoryeffects.
TEOM
EC/OC
PAX(870nm)AE33
IC
MSC
Mini-CAST
CellcultureequipmentNACIVTchamber
Par'cledeliverystrategy
EC/TC(%)99543126Size(nm)9188103122
Cytotoxicitydependsonorganiccoa?ng(experimentsrepeatedwithaddi%onalcoa%ngthickness)
EC(sootcore)ànotcytotoxic
Firstteststoevaluatecoa*ngeffectsoncytotoxicity
4h 24h
Exposure time (1h)
Par'clenumberconcentra'onremainsthesamebetween30nmand90nminallthreecampaigns:30nm∼4x105NP/cm3
90nm∼1.3x105NP/cm3
1stand2ndcampaign:TMBasSOA-precursor(I)
Ø Nosignificantdifferenceintermsofcytotoxicitybetweenincubatorcontrol(I.C),p-freeairandsynthe?cairexposedcells
Ø Sootpar%clesconsis%ngonlyofEC(90nmUnCoatedsoot)causenocytotoxiceffects
Ø Cytotoxicityincreaseswithincreasingcoa%ngthickness.Max-Coat90nminducesthehighestcytotoxicity,i.e.releaseofLDH(mean23.35%)
0 30 60 90 120 1500
10
20
30
40
CorrelationSize/Cytotoxicity
R2= 0.6061p-value= 0.0134
Size (nm)
Cyt
otox
icity
(%) L
DH
rele
ase
I.C
Synth.ai
r
p-free
air
UnCoat
Min-Coat
Med-C
oat
Max-C
oat
UnCoat
Min-Coat
Med-C
oat
Max-C
oat0
10
20
30
40
Ctrl Soot 90 nm Soot 30 nm
****
**
LDH
rele
ase
(%)
Mini-CASTSoot90nm
Mini-CASTSoot30nm
Ctrl
3rdcampaign:α-pineneasSOA-precursor
Ø Cytotoxicityincreaseswithcoa%ngthicknessasinthecasewithTMBasSOA-precursor
Ø General,slightlylowercytotoxiceffectwithα-pineneasprecursorcomparedtoTMB
0 30 60 90 120 1500
10
20
30
40
50
CorrelationSize/Cytotoxicity
R2= 0.5116p-value= 0.0303
Size (nm)
Cyt
otox
icity
(%) L
DH
rele
ase
I.C
Synth.ai
r
p-free
air
UnCoat
Min-Coat
Med-C
oat
Max-C
oat
UnCoat
Min-Coat
Med-C
oat
Max-C
oat0
10
20
30
40
Ctrl Soot 90 nm Soot 30 nm
**** *
* **
LDH
rele
ase
(%)
Mini-CASTSoot90nm
Mini-CASTSoot30nm
Ctrl
Ongoinganalysis:Secretome102cytokinesanalyzed
Outof102cytokinesandchemokinesanalyzed,35aredown-orup-regulatedincomparisontoincubatorcontrol
Adiponectin
Angiogenin
CD40 lig
and
Cystat
in CDkk
-1EGF
EMMPRIN
ENA-78
G-CSF
GDF-15
GM-CSF
GRO-alpha
ICAM-1
IGFBP-2
IGFBP-3
IL-1raIL-4IL-6IL-8
IL-11
IL-17AIL-23IL-24
Lipocalin
-2
MCP-1MIF
MIP-3alpha
MMP-9
Myelopero
xidas
e
Osteopontin
PDGF-AA
Serpin E1
SHBGuPAR
VEGF
p-free air
UnCoat
Min-Coat
Med-Coat
Max-Coat
Fold
cha
nge
rela
tive
to In
c. C
trl
5
10
15
20
25
30Soot90nm
Adiponectin
Angiogenin
CD40 lig
and
Cystat
in CDkk
-1EGF
EMMPRIN
ENA-78
G-CSF
GDF-15
GM-CSF
GRO-alpha
ICAM-1
IGFBP-2
IGFBP-3
IL-1raIL-4IL-6IL-8
IL-11
IL-17AIL-23IL-24
Lipocalin
-2
MCP-1MIF
MIP-3alpha
MMP-9
Myelopero
xidas
e
Osteopontin
PDGF-AA
Serpin E1
SHBGuPAR
VEGF
p-free air
UnCoat
Min-Coat
Med-Coat
Max-Coat
Fold
cha
nge
rela
tive
to In
c. C
trl
0
5
10
15
20
25
30Soot30nm
1stand2ndcampaign:TMBasSOA-precursor
ConclusionandoutlookPilotstudyfundedbyFOEN
Ø Sootpar'clesconsis'ngonlyofEC(90nmUnCoatedsoot)causenocytotoxiceffects.Ø Cytotoxicityincreaseswithcoa*ngthicknesswithTMBandα-pineneasSOA-precursors.
Max-Coat90nminducesthehighestcytotoxicity.Ø α-pineneasSOA-precursorcauseslightlylowercytotoxiceffectcomparedtoTMB.
Ongoingandfutureac'vi'es:Ø Transmissionelectronmicroscopy(TEM)ofcellculturesec'onsandofpar'clessampledonTEMgrids.Ø Secretomeanalysisforthe3rdcampaign
ThepilotstudyhasledtotheEUproject:EMPIRAeroTox(h?p://empir.npl.co.uk/aerotox/)Ø Confocalmicroscopyofsamplesfrom2ndand3rdcampaignattheNa'onalPhysicalLaboratory(NPL,UK).Ø Analysisofthedegreeofoxida'onoftheorganicma?erbyAerosolMassSpectrometry(NPL,UK).
Acknowledgments
Institute of Anatomy
Group Geiser
Ø Prof. M. Geiser Ø B. Kupferschmid
Former members: Ø M. Delaval Ø H. Jonsdottir
METAS & FHNW
Laboratory Particles and Aerosols
Ø K. Vasilatou Ø M. Ess
Laboratory Aerosol Measurement
Technology
Ø A. Keller
BAFU
Air quality section Ø Richard Ballaman
Traffic section
Ø Giovanni D’Urbano
Projectfunding
AeroToxPartners
Ongoinganalysis:TEMinserts2ndEXPERIMENTALCAMPAIGN:TMBasSOA-precursor
Inc.Ctrl
p-free
air
Synth. a
ir0
5
10
15
20 ***ns
**
LDH
rele
ase
(%)
KS normality test
Passed normality test (alpha=0.05)? Yes Yes Yes P value summary ns ns ns D'Agostino & Pearson normality test Passed normality test (alpha=0.05)? Yes Yes Yes P value summary ns ns ns Shapiro-Wilk normality test Passed normality test (alpha=0.05)? Yes Yes Yes P value summary ns ns ns
3rdcampaign:α-pinene as SOA-precursor
0
10
20
30
40
Ctrl
I.C
Synt
. Air
Soot 90 nm
UnCoated
Exp
p-fr
ee Exp
p-fr
ee Exp
p-fr
ee Exp
p-fr
ee
Min-Coat Med-Coat Coated
Soot 30 nmEx
p
p-fr
ee Exp
p-fr
ee Exp
p-fr
ee Exp
p-fr
ee
UnCoated Min-Coat Med-Coat Coated
** * *** **** ns *** *
LDH
rele
ase
(%)
Cytotoxicityuponexposuretocoatedsootmuchhigherthanthatofincubatorcontrolandpar?cle-freeairtests->Theincreaseincytotoxicityisindeedcausedbycoatedsootpar%cles
1stand2ndcampaign:TMBasSOA-precursor(II)
Goodreproducibilitybetweenthe1stand2ndcampaign
0
10
20
30
40
Ctrl
I.C
Synt
h. A
ir
Soot 90 nm
UnCoated
Exp
p-fr
ee Exp
p-fr
ee Exp
p-fr
ee Exp
p-fr
ee
Min-Coat Med-Coat Max-Coated
Soot 30 nmEx
p
p-fr
ee Exp
p-fr
ee Exp
p-fr
ee Exp
p-fr
ee
1st Campaign
UnCoated Min-Coat Med-Coat Max-Coated
**ns ns *** ****** * ns ***
2nd Campaign
LDH
rele
ase
(%)
Current project EMPIR project
Model aerosols: internal mixture of soot core particles with coating from secondary organic matter Properties tested: uncoated vs. coated particles, role of soot core size, coating thickness and coating chemistry
Further tests to clarify the role of: - primary vs. secondary organic coating - secondary inorganic coating - internal metal admixtures - externally mixed dust particles (e.g. from
brake abrasion) Biomedical analyses will be complemented with toxicogenomics and particle uptake studies
metals different coatings
Spring 2018 - Spring 2019 June 2019 - May 2022
Genera'onofagedsootaerosol
Oxida'onflowreactor
ozone,OH
UVClamp
UVClamp
0.0E+00
5.0E+04
1.0E+05
1.5E+05
2.0E+05
2.5E+05
3.0E+05
3.5E+05
4.0E+05
4.5E+05
10 100 1000
Num
bercon
centra*o
n[#/cm3]
Mobilitydiameter[nm]
0.00.10.20.30.40.50.60.70.80.9
90 110 130 150 170 190
SSA
(870
nm) [
-]
Mobilitydiameter[nm]
SSA= 𝑙𝑖𝑔ℎ𝑡 𝑠𝑐𝑎𝑡𝑡𝑒𝑟𝑖𝑛𝑔/𝑙𝑖𝑔ℎ𝑡 𝑒𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛
PrototypeminiCASTsootgeneratorModel5201BC
(JingLtd.,Bern,Switzerland)
0102030405060708090100
Par*clecompo
si*o
n[%
]
EC/TC[%]
OC/TC[%]
organicvaporconcentra*on(ppm)
Elementalcarbon(EC)
Organiccarbon(OC)