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