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Füsun FAKILI 1,2 , Bülent GÖĞEBAKAN 2 , Recep BAYRAKTAR 2 , Serdar ÖZTUZCU 2 , Hasan BAYRAM 1,2

D IESEL E XHAUST P ARTICLES and S ERUM M ODULATE A IRWAY E PITHELIAL C ELL V IABILITY by A FFECTING I NTRACELLULAR C ELL S IGNALING P ATHWAYS , WHICH ARE S ENSITIVE to O XIDATIVE S TRESS.

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Füsun FAKILI 1,2 , Bülent GÖĞEBAKAN 2 , Recep BAYRAKTAR 2 , Serdar ÖZTUZCU 2 , Hasan BAYRAM 1,2

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  1. DIESEL EXHAUST PARTICLESand SERUM MODULATE AIRWAY EPITHELIAL CELL VIABILITYby AFFECTING INTRACELLULAR CELL SIGNALING PATHWAYS, WHICHARE SENSITIVEto OXIDATIVE STRESS Füsun FAKILI1,2, Bülent GÖĞEBAKAN2, Recep BAYRAKTAR2,Serdar ÖZTUZCU2,Hasan BAYRAM1,2 Gaziantep University , School of Medicine, 1 Department of Respiratory Medicine 2 Cell Culture Laboratory The 13th Annual Congress of Turkish Thoracic Society 2010

  2. Introduction-1 • There is a close association between increases in particulate matter 10µm (PM10), 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 p21CIP1/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 DEPin the presence and absence ofN-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-2BCell 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 BronchialEpithelial 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) JNKinh. NAC ***p<0.0001 vs 0µg/ml DEP ♦p<0.0001 vs 200µg/ml DEP Φp<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) ERKinh. NF-kBinh. ***p<0.0001 vs 0µg/ml DEP

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

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

  15. Summary-1 • Although DEP induced A549 cell viabilityunder 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…

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