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Role of organic compounds and metals in the resistance to cell
death conferred by urban fine
particles (PM2.5)Mélanie Leroux
Laboratory of Molecular and Cellular Responses to Xenobiotics
Supervisor: Dr. Karine Andréau
2nd European Doctoral College on Environment and Health
Composition (PM2.5) :
Organic compoundsBiological
compoundsMetals
CarbonCore
Coarse particles
(PM10)10 - 2,5µm
Fine particles(PM2.5)
2,5 - 0,1µm
Ultrafineparticles(PM0.1)
≤ 0,1µm
Health effects of atmospheric particles
Composition (PM2.5) :
Organic compoundsBiological
compoundsMetals
CarbonCore
Coarse particles
(PM10)10 - 2,5µm
Fine particles(PM2.5)
2,5 - 0,1µm
Ultrafineparticles(PM0.1)
≤ 0,1µm
o ↗ Cardiorespiratory morbidity and mortalityo Oxidative stresso Genotoxicity o Inflammatory responseo Modulation of apoptosis
Short term effects:
o Lung cancers o ↗ Chronic respiratory diseases : asthma, COPD (Chronic obstructive pulmonary disease)…o Accumulation of PM in the bronchial wall and airway remodeling
Long term effects:
Health effects of atmospheric particles
1 [PM] ≥ 100µg/cm² only found in high pollution periodsBUT …
2 Cancer cells = resistance to cell death
Atmospheric particles
Lung cancerEpidemiologicstudies
Previous in vitro studies
PM2.5 induce cell death (apoptosis)
Human Bronchial Epithelial cells
(16HBE)
1- INDUCTION of APOPTOSIS ?
Urban PM2.5 (10 µg/cm²)
2- REDUCTION of APOPTOSIS ?
Aim of our study
10µg/cm²=
5 days exposure to atmospheric
pollution (79µg/m3)
3- PM2.5 COMPOUNDS INVOLVED?
24H
Particles
Urban PM2.5Location of
samplingMain sources
identified
TrafficParis ring road
(2003) Car traffic
BackgroundParisian suburb
(2003)
Urban background
pollution
Wood burningParis
(Winter 2009)Wood-fired
heating
MixOuagadougou (Burkina-Faso
2009)
Wood-fired cooking,
Moped traffic
Methology: apoptosis measurement by flow cytometry
Urban PM2.5 particles are they toxic in human bronchial epithelial
cells ?
1.
Membrane permeabilization
Membrane bleebing
Mem
bra
ne
mod
ific
atio
ns
MMP
Condensation of the chromatin
Fragmentation of DNA and nucleus
Modification of the cellular granularity
Reduction of the cell size
Apoptosis
Oxydative stress
Methology: apoptosis measurement by flow cytometry
Urban PM2.5 particles are they toxic in human bronchial epithelial
cells ?
1.
Membrane permeabilization
Membrane bleebing
Mem
bra
ne
mod
ific
atio
ns
MMP
Condensation of the chromatin
Fragmentation of DNA and nucleus
Modification of the cellular granularity
Reduction of the cell size
Apoptosis
Oxydative stress
Propidium Iodide (PI)
HydroEtydium
(HE)
DiOC6(3)
1. Urban PM2.5 particles are they toxic in human bronchial epithelial cells ?
Questions
Human Bronchial Epithelial cells
(16HBE)
INDUCTION of APOPTOSIS ?
Urban PM2.5 (10 µg/cm²)
24 H
Urban PM2.5 particles are they toxic in human bronchial epithelial
cells ?
1.
* p< 0.001 vs Controln=3
Traffic Wood Burning
MixControl(Mediu
m)
Background
10 µg/cm²
Urban PM2.5 do not induce apoptosisin 16HBE cells
1. Urban PM2.5 particles are they toxic in human bronchial epithelial cells (16HBE) ?
Questions
Urban PM2.5 (10 µg/cm²) do not induce apoptosis of human bronchial epithelial cells
1. Urban PM2.5 particles are they toxic in human bronchial epithelial cells (16HBE)?
Questions
Urban PM2.5 (10 µg/cm²) do not induce apoptosis of human bronchial epithelial cells
2. Urban PM2.5 particles have they an antiapoptotic effect in 16HBE cells ?
INITIATION PROMOTION
APOPTOSISRESISTANCE
DNA adducts Cancer cell
Tumor formation :
Initiated cell
Urban PM2.5 particles have they an antiapoptotic effect in 16HBE cells ?
2.
Cell death
APOPTOSIS
Apoptosis inducer: A23 (Calcium ionophore)
↗CALCIUM
?
Methology: PM2.5 exposure before induction of apoptosis
20 H
16HBE
Particles(10µg/cm²)
4 H vvvvvvvvvvvvvvvvvvvvvvvv
Flow Cytometry
Apoptosis induction by A23
Urban PM2.5 particles have they an antiapoptotic effect in 16HBE cells ?
2.
Traffic PM2.5 have an antiapoptotic effect towards A23-induced apoptosis
p< 0.001 vs Controln=3
*
Milieu Périf 2003
10 µg/cm²
Vitry 2003
10 µg/cm²
Paris 2009
10 µg/cm²
Ouaga 2009
10 µg/cm²
0
20
40
60
80
100
120
Traffic10µg/cm²
Background
10µg/cm²
Wood burning10µg/cm²
Mix10µg/cm²
+ A23
Control(Mediu
m)
*
A2
3-i
nduce
d a
popto
sis
(%)
1. Urban PM2.5 particles are they toxic in human bronchial epithelial cells (16HBE) ?
Questions
Urban PM2.5 (10 µg/cm²) do not induce apoptosis of human bronchial epithelial cells
2. Urban PM2.5 particles have they an antiapoptotic effect in 16HBE cells ?YES : Only Traffic PM2.5
1. Urban PM2.5 particles are they toxic in human bronchial epithelial cells (16HBE) ?
Questions
Urban PM2.5 (10 µg/cm²) do not induce apoptosis of human bronchial epithelial cells
2. Urban PM2.5 particles have they an antiapoptotic effect in 16HBE cells ?YES : Only Traffic PM2.5
Possible link with calcium
3. Which Traffic PM2.5 components are involved in the antiapoptotic effect ?
↗CALCIUM
?
Organic compounds
(PAH)
Water-solubleComponents
(metals)
Composition (PM2.5) :
Organic compoundsBiological
compoundsMetals
CarbonCore
Which Traffic PM2.5 components are involved in the antiapoptotic
effect ?
3.
?
Apoptosis inducer: A23 (Calcium ionophore)
deso
rbtio
n
Contro
l
Traffi
cOrg
. Ex.
Aq. E
x.W
ashe
d
*
0
20
40
60
BC
+ A23
p<0.001 vs controln=3
Ferecatu et al. Particle and Fibre Toxicology 2010,7:18
Organic and Aqueous components contribute
to the antiapoptotic effect of Traffic PM2.5
Which Traffic PM2.5 components are involved in the antiapoptotic
effect ?
3.
* * **A
23
-induce
d a
popto
sis
(%)
Composition (PM2.5) :
Organic compoundsBiological
compoundsMetals
CarbonCore
Ferecatu et al. Particle and Fibre Toxicology 2010,7:18
Which Traffic PM2.5 components are involved in the antiapoptotic
effect ?
3.
a) Organic compounds : PAH
↗CALCIUM
Organic compounds
PAH
Apoptosis inducer: A23 (Calcium ionophore)
Ferecatu et al. Particle and Fibre Toxicology 2010,7:18
Heavy PAHs partially contribute to the antiapoptotic effect of Traffic PM2.5 via the AhR
Which Traffic PM2.5 components are involved in the antiapoptotic
effect ?
3.
a) Organic compounds : PAH
PAH
Organic compounds
AhR
beta NF
alpha NFsiRNA AhR
XRE
*
**
* p< 0.05 vs Mediumn=3
A23-i
ndu
ced
apopto
sis
(%)
Medium
Which Traffic PM2.5 components are involved in the antiapoptotic
effect ?
3.
b) Aqueous compounds : MetalsMetals (mg/g) : Fer Aluminium Cuivre
Antiapoptotic effect
Traffic 13,2 3,4 0,86 YES
Background 21,6 20,4 0,40 NO
Which Traffic PM2.5 components are involved in the antiapoptotic
effect ?
3.
b) Aqueous compounds : Metals
Series10
2
4
6
8
10
12 Traffic Background p< 0.05 vs Controln=6
*
* * * * *
Control (Medium)
PM2.5 Fe2+ Fe3+ Al3+ Cu2+qs 10 µg/cm²
% a
popto
sis
ind
uct
ion
(D
iOC
6(3
) lo
w H
E
hig
h)
+ A23
Metals (mg/g) : Fer Aluminium Cuivre
Antiapoptotic effect
Traffic 13,2 3,4 0,86 YES
Background 21,6 20,4 0,40 NO
Which Traffic PM2.5 components are involved in the antiapoptotic
effect ?
3.
b) Aqueous compounds : Metals
Series10
2
4
6
8
10
12 Traffic Background p< 0.05 vs Controln=6
*
* * * * *
Control (Medium)
PM2.5 Fe2+ Fe3+ Al3+ Cu2+qs 10 µg/cm²
% a
popto
sis
ind
uct
ion
(D
iOC
6(3
) lo
w H
E
hig
h)
+ A23
The content of transition metals in particles
is involved in the antiapoptotic effect
1. Urban PM2.5 particles are they toxic in human bronchial epithelial cells (16HBE) ?
Conclusions and Prospects
Urban PM2.5 (10 µg/cm²) do not induce apoptosis of human bronchial epithelial cells
2. Urban PM2.5 particles have they an antiapoptotic effect in 16HBE cells ?YES : Only Traffic PM2.5
Possible link with calcium
3. Which Traffic PM2.5 components are involved in the antiapoptotic effect ? Heavy PAHs via AhR Metals (Fe, Al, Cu)
PM2.5
PAH desorption
AhR
XRE
A23
Metalliccompounds
MMP
1. Role of oxydative stress triggered by metals ?
2. Effect of metals on intracellular calcium ?
3. Role of mitochondria ?
4. Role of Caspases and Calpains ?
ROS
Ca2+
Caspasescalpains
Ferecatu et al. Particle and Fibre Toxicology 2010,7:18
Conclusions and Prospects
Nicole Boggetto
The « Apoptotic » RMCX team:Ioana Ferecatu
Aïda BouharrourEmile Petit
Armelle BaezaKarine Andreau
Francelyne MaranoJean-Marie Dupret
Thank you for your attentionThe « Apoptotic » RMCX team:Ioana Ferecatu
Aïda BouharrourEmile Petit
Armelle BaezaKarine Andreau
Francelyne MaranoJean-Marie Dupret
Nicole Boggetto
BbF : Benzo[b]fluoranthrene
iP : Indeno[1,2,3-cd]pyrene
BghiP : Benzo[g,h,i]perylene
DBahA : Dibenzo[a,h]anthracene
BaP: Benzo[a]pyrene