Effect of High Concentrations of Effect of High Concentrations of Diesel Exhaust Particles Diesel Exhaust Particles onon Human Lung Epithelial CellHuman Lung Epithelial Cell

Viability and DeathViability and Death

Hasan Bayram1*, Kazuhiro Ito2, K. Fan Chung2.

1Department of Respiratory Medicine, School of Medicine,

Gaziantep University, Gaziantep, TURKEY; 2Thoracic Medicine,

National Heart & Lung Institute, Imperial College, London, UK.

*Supported by an ERS Long-term Research Fellowship

BackgroundBackground-1-1• An association between particulate

matter 10µm (PM10), pollution and respiratory morbidity and cardio-pulmonary mortality(McConnell R et al, 2003, Pope CA et al, 2004 )

• Airway epithelial cells may play a role in PM10-induced respiratory morbidity

• DEP increase release of inflammatory mediators from airway epithelial cells (Bayram H et al, 1998)

BackgroundBackground-2-2

• DEP induce A549 cell proliferation, and inhibit apoptosis through oxidative stress, inhibition of p21CIP1/WAF1 and stimulation of JNK and NF-B under serum free condition(Bayram H et al, 2006)

• DEP may lead to apoptosis or necrosis of respiratory cells(Matsuo M et al, 2003; Li N et al, 2002; Hiura TS et al, 2000)

• Under normal culture conditions with serum, high concentrations of DEP lead to decreased A549 cell viability via inducing apoptosis/necrosis of these cells.

HypothesisHypothesis

ObjectivesObjectives

• To investigate effect of high concentrations of DEP on viability, apoptosis and necrosis of airway epithelial (A549) cells

MethodsMethods

• Culture A549 cells

• Incubation with 0, 10, 200 and 400g/ml DEP in the presence of 0-10% foetal calf serum (FCS) for 48 hours

• MTT Staining: Cell viability

• FACS: Evidence of apoptotic bodies (Annexin V/PI staining)

FCS Baseline 0 10 2000.00

0.25

0.50

0.75

1.00

1.25

1.50

DEP(g/ml) + 0% FCS(10%)

**

Optic

al D

ensi

ty

**

*

Effect of DEP on viability ofEffect of DEP on viability of A549 Cells A549 Cells inin0% FCS 0% FCS following following 48hrs’ 48hrs’ incubationincubation

*p<0.05 and **p<0.001 vs 0g/ml DEP

FCS Baseline 0 10 2000.00

0.25

0.50

0.75

1.00

1.25

1.50

DEP (g/ml) + FCS (1%)(10%)

*

Opt

ical

Den

sity

**

**

Effect of DEP on viability ofEffect of DEP on viability of A549 Cells A549 Cells inin1% FCS 1% FCS following following 48hrs’ 48hrs’ incubationincubation

*p<0.05 and **p<0.001 vs 0g/ml DEP

FCS Baseline 0 10 2000.00

0.25

0.50

0.75

1.00

1.25

1.50

DEP(g/ml) + FCS(3.3%)(10%)

**

Optic

al D

ensi

ty

**

Effect of DEP on viability ofEffect of DEP on viability of A549 Cells A549 Cells inin3.3% FCS 3.3% FCS following following 48hrs’ 48hrs’ incubationincubation

**p<0.01 vs 0g/ml DEP

FCS Baseline 0 10 2000.00

0.25

0.50

0.75

1.00

1.25

1.50

DEP(g/ml) + FCS(10%)(10%)

Opt

ical

Den

sity

**

Effect of DEP on viability ofEffect of DEP on viability of A549 Cells A549 Cells inin10% FCS 10% FCS following following 48hrs’ 48hrs’ incubationincubation

**p<0.01 vs 0g/ml DEP

LL(A -/P-) LR(A+/P-) UR(A+/P+) UL(A-/P+)0

10

20

30

40

5050

75

100

125

DEP (0g/ml)

DEP (10g/ml)

DEP(100g/ml)

DEP(200g/ml)DEP(400g/ml)

**

**

**

** **

Quadrants

% o

f cel

ls SF

Effect of DEP on Effect of DEP on apoptosis/necrosis of apoptosis/necrosis of A549A549 c cellsells in 3.3% FCS in 3.3% FCS following following 48hrs’ 48hrs’ incubationincubation

**p<0.01 vs 0g/ml DEP

SummarySummary

• Under serum free condition, 10µg/ml DEP induced A549 cell viability,

• In the presence of 1-3.3%FCS, 200g/ml DEP decreased cell viability

• In the presence of 10%FCS, DEP did not show any effect on cell viability

• In the presence of 3.3%FCS, 200-400g/ml DEP induced cell apoptosis/necrosis.

ConclusionConclusion

• DEP may induce apoptosis/necrosis of airway epithelial cells

• Underlying mechanisms need to be investigated

Thank you

ConclusionConclusion

• DEP effect of A549 cell viability and apoptosis/necrosis depends on its dose and presence of serum in cell culture condition.

• DEP effect cell viability and apoptosis/necrosis; an effect dependant on concentration of DEP or serum.

0.0

0.5

1.0

1.50g/ml10g/ml200g/ml

Baseline 3.3 100 1

***

***

Fetal Calf Serum (%)

Opt

ical

Den

sity

Effect of DEP on viability ofEffect of DEP on viability of A549 Cells A549 Cells inin0-10% FCS 0-10% FCS following following 48hrs’ 48hrs’ incubationincubation

Effect of DEP on viability ofEffect of DEP on viability ofA549 Cells (MTT Assay) A549 Cells (MTT Assay)

24 hrs

48 hrs 72 hrs

FCS 0 5 10 50 100 2000.0

0.5

1.0

1.5

2.0

Diesel Exhaust Particles (g/ml)10%

Opt

ical

Den

sity

FCS 0 5 10 50 100 200 4000.0

0.5

1.0

1.5

2.0

******

*** ***

***

***

10% Diesel Exhaust Particles (g/ml)

Opt

ical

Den

sity

FCS 0 5 10 50 100 2000.0

0.5

1.0

1.5

2.0

***

***

***

*** ***

***

10% Diesel Exhaust Particles (g/ml)

Opt

ical

Den

sity

***P<0.0001 vs 0g/ml DEP

Effect of DEP (10Effect of DEP (10g/ml)g/ml) on on cell cycle cell cycle ofof A549 A549 ccellsells following following 48 hrs’ 48 hrs’ incubationincubation

G0/1 S G2/M0

25

50

75

100Serum Free

DEP (10g/ml)

FCS (10%)***

***

*

**

*****

Cell cycle compartments

Per

cent

of

cells

*P<0.05, **P<0.01, ***P<0.0001 vs SF

Effect of DEP on Effect of DEP on apoptosis of apoptosis of A549A549ccellsells following following 48hrs’ 48hrs’ incubationincubation

LL(A-/P-) LR(A+/P-) UR(A+/P+) UL(A-/P+)0

10

20

30

40

70

100 **

*

FCS(10%)SF

DEP (10g/ml)

*

*

Quadrants

% o

f cel

ls

*P<0.05 and**P<0.005 vs SF

• PM10 (10-50g/cm3) induce proliferation and apoptosis (25-50g/cm3) of murine alveolar epithelial cells(Timblin CR et al, 2002)

• PM10 lead to cell death, DNA breakage and apoptosis in A549 cells

(Moreno EA et al, 2002 )

BackgroundBackground

• Diesel exhaust particles (DEP) induce respiratory cell proliferation and hypertrophy in mice(Koike E et al, 2002; Ichinose T et al, 1998)

• DEP cause oxidative stress and higher concentrations may lead to apoptosis or necrosis of respiratory cells

BackgroundBackground

(Matsuo M et al, 2003; Li N et al, 2002; Hiura TS et al, 2000)

SummarySummary

• DEP (5-200µg/ml) induced A549 cell viability, this was inhibited by NAC, SP600125 and SN50

• DEP (10g/ml) induced cell cycle progression, which was inhibited by NAC and SP600125

• DEP (10 g/ml) prevented A549 cell apoptosis• DEP (10 g/ml) decreased p21CIP1/WAF1 mRNA

and protein expression of A549 cells.

THANK YOU!THANK YOU!

0.0

0.5

1.0

1.50 g/ml DEP

10 g/ml DEP

Baseline 3.3 100 1

***

Fetal Calf Serum (%)

Opt

ical

Den

sity

Effect of DEP on viability of A549 cells in theEffect of DEP on viability of A549 cells in thepresence of 0-10% FCS after 48 hrs’ incubationpresence of 0-10% FCS after 48 hrs’ incubation

***P<0.0001 vs0g/ml DEP

Cell Signalling Pathways Associated with Apoptosis

p21 and cell cycle pathwaysp21 and cell cycle pathways