1. DIESEL EXHAUST PARTICLES and SERUM MODULATE AIRWAY EPITHELIAL CELL VIABILITY by AFFECTING INTRACELLULAR CELL SIGNALING PATHWAYS, WHICH ARE SENSITIVE to OXIDATIVE STRESS Füsun FAKILI 1,2, Bülent GÖĞEBAKAN 2, Recep BAYRAKTAR 2, Serdar ÖZTUZCU 2,Hasan BAYRAM 1,2 Gaziantep University, School of Medicine, 1 Department of Respiratory Medicine 2 Cell Culture Laboratory The 13 th Annual Congress of Turkish Thoracic Society 2010

2. Introduction-1 There is a close association between increases in particulate matter  10µm (PM 10 ), and respiratory morbidity and cardiopulmonary mortality - McConnell R et al, AJRCCM 2003, - Pope CA et al, NEJM 2004) Diesel exhaust particles (DEP) increase release of inflammatory mediators from airway epithelial cells - Bayram H et al, AJRCMB 1998

3. Introduction-2 DEP induce A549 cell proliferation, and inhibit apoptosis through oxidative stress, inhibition of p21 CIP1/WAF1 and stimulation of JNK and NF-  B pathways under serum free condition - Bayram H et al, Eur Respir J 2006

4. Introduction-3 c-Jun N-Terminal Kinaz (JNK), ‘Extra Cellular Regulated Kinase’ (ERK) and Nuclear Factor (NF)-kB pathways are activated in inflammatory airway diseases such as asthma and chronic obstructive pulmonary diseases (COPD) - Hoshino S et al, Biochemical and Biophysical Research Com. 2005 - Rahman I. Journal of Biochemistry and Molecular Biology 2003 - Lee YC at al, Am J Physiol Lung Cell Mol Physiol 2008 - Edwards MR et al, Pharmacology & Therapeutics 2009

5. Objectives To create a model of inflamed airways by adding serum to cell culture media in vitro To investigate the role of oxidative stress and cell signalling pathways including c-jun N Terminal Kinase (JNK), Extra Regulated Kinase (ERK) and Nuclear Factor (NF)-κB To investigate the effects of DEP on airway epithelial cell viability, apoptosis and p21, p27 and p53 expression

6. Methods-1 A549, BEAS-2B and primary bronchial epithelial cell cultures were incubated with 0, 50, 100, 200, 400 ve 1000  g/ml DEP in the presence and absence of N-acetylcysteine (NAC), an inhibitor of JNK (SP600125), an inhibitor of ERK (PD 98059) and NF-  B inhibitor (SN50) under 0%, 1, 3.3 ve 10 FCS condition for 48 hours. Cells viability was evaluated by MTT assay

7. Methods-2 Cells were double stained by Annexin V-PE and 7AAD dyes and analyzed by flow cytometry to assess apoptotic cells and necrotic cells p21, P27 and p53 mRNA expression was studied by means of real-time (PCR)

8. Effect of DEP on A549 Cell Viability of in the Presence of Different Concentrations of FCS *p<0.05, **p<0.001, ***p<0.0001 vs 0µg/ml DEP

9. Effects of DEP on BEAS-2B Cell Viability in the Presence of Different Concentrations of FCS *p<0.05, **p<0.001, ***p<0.0001 vs 0µg/ml DEP

10. Effects of DEP on Primary Bronchial Epithelial Cell Viability in the Presence of Different Concentrations of FCS *p<0.05, **p<0.001, ***p<0.0001 vs 0µg/ml DEP

11. Effect of NAC and JNK inh. on A549 Cell Viability Modified by Serum (3.3%) and DEP (200µg/ml) NAC JNKinh. ***p<0.0001 vs 0µg/ml DEP ♦ p<0.0001 vs 200µg/ml DEP Φ p<0.01 vs 200µg/ml DEP + DMSO

12. Effect of Inhibitors of ERK and NF-κB on A549 Cell Viability Modified by Serum (3.3%) and DEP (200µg/ml) ***p<0.0001 vs 0µg/ml DEP ERKinh.NF-kBinh.

13. Effect of DEP(200µg/ml) on A549 Cell Apoptosis in the Presence of FCS (3.3%)

14. Effect of DEP on mRNA Expression of p21, P27 and p53 by A549 Cells in the Presence of FCS(3.3%) p21 p27 p53

15. Summary-1 Although DEP induced A549 cell viability under serum free condition, they reduced cell viability in the presence of 3.3%FCS The role of oxidative stress and the oxidative stress pathways (JNK and ERK) and NF-κB in this process looks limited. NAC and inhibitors of JNK, ERK and NF-κB inhibit A549 cell viability in the presence of serum DEP do not affect A549 cell apoptosis/necrosis in the presence of serum DEP induce mRNA expression of p53 in the presence of 3.3% serum

16. Summary-2 In BEAS-2B cells, although lower concentrations of DEP induced cell viability under 0-3.3 % FCS condition, relatively higher doses decreased cell viability. In the presence of 10% FCS, higher concentrations suppressed cell viability In primary bronchial epithelial cells, higher DEP concentrations reduced cell viability under 0-10% FCS condition.

17. Conclusion The activated status of JNK, ERK, and NF-kB in inflamed airways that can be seen in respiratory disorders such as asthma and COPD may, at least in part, be due to the leakage of serum to airway mucosa Under such conditions, the toxic effects of pollutants such as DEP may be enhanced at cellular level. Airway epithelial cells from different origins may be affected by the toxic effects of DEP at different levels The underlying mechanisms need to be investigated further

18. This study funded by the Research Fund of Gaziantep University. Thank you…