1. Effect of High Concentrations of Diesel Exhaust Particles on Human Lung Epithelial Cell Viability and Death Hasan Bayram 1*, Kazuhiro Ito 2, K. Fan Chung 2. 1 Department of Respiratory Medicine, School of Medicine, Gaziantep University, Gaziantep, TURKEY; 2 Thoracic Medicine, National Heart & Lung Institute, Imperial College, London, UK. *Supported by an ERS Long-term Research Fellowship

2. Background-1 An association between particulate matter  10µm (PM 10 ), 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 PM 10 -induced respiratory morbidity DEP increase release of inflammatory mediators from airway epithelial cells (Bayram H et al, 1998)

3. Background-2 DEP induce A549 cell proliferation, and inhibit apoptosis through oxidative stress, inhibition of p21 CIP1/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)

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

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

6. Methods Culture A549 cells Incubation with 0, 10, 200 and 400  g/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)

7. Effect of DEP on viability of A549 Cells in 0% FCS following 48hrs’ incubation *p<0.05 and **p<0.001 vs 0  g/ml DEP

8. Effect of DEP on viability of A549 Cells in 1% FCS following 48hrs’ incubation *p<0.05 and **p<0.001 vs 0  g/ml DEP

9. Effect of DEP on viability of A549 Cells in 3.3% FCS following 48hrs’ incubation **p<0.01 vs 0  g/ml DEP

10. Effect of DEP on viability of A549 Cells in 10% FCS following 48hrs’ incubation **p<0.01 vs 0  g/ml DEP

11. SF Effect of DEP on apoptosis/necrosis of A549 cells in 3.3% FCS following 48hrs’ incubation **p<0.01 vs 0  g/ml DEP

12. Summary Under serum free condition, 10µg/ml DEP induced A549 cell viability, In the presence of 1-3.3%FCS, 200  g/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, 200-400  g/ml DEP induced cell apoptosis/necrosis.

13. Conclusion DEP may induce apoptosis/necrosis of airway epithelial cells Underlying mechanisms need to be investigated

14. Thank you

16. Conclusion 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.

17. Effect of DEP on viability of A549 Cells in 0-10% FCS following 48hrs’ incubation

18. Effect of DEP on viability of A549 Cells (MTT Assay) 24 hrs 48 hrs 72 hrs ***P<0.0001 vs 0  g/ml DEP

19. Effect of DEP (10  g/ml) on cell cycle of A549 cells following 48 hrs’ incubation *P<0.05, **P<0.01, ***P<0.0001 vs SF

20. Effect of DEP on apoptosis of A549 cells following 48hrs’ incubation *P<0.05 and **P<0.005 vs SF

21. PM 10 (10-50  g/cm 3 ) induce proliferation and apoptosis (25-50  g/cm 3 ) of murine alveolar epithelial cells (Timblin CR et al, 2002) PM 10 lead to cell death, DNA breakage and apoptosis in A549 cells (Moreno EA et al, 2002 ) Background

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

23. Summary DEP (5-200µg/ml) induced A549 cell viability, this was inhibited by NAC, SP600125 and SN50 DEP (10  g/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 p21 CIP1/WAF1 mRNA and protein expression of A549 cells.

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26. Effect of DEP on viability of A549 cells in the presence of 0-10% FCS after 48 hrs’ incubation ***P<0.0001 vs 0  g/ml DEP

27. Cell Signalling Pathways Associated with Apoptosis

28. p21 and cell cycle pathways