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Hasan Bayram 1 * , Kaz u h i r o Ito 2 , K . Fan Chung 2 .

Effect of High Concentrations of Diesel Exhaust Particles on Human Lung Epithelial Cell Viability and Death. Hasan Bayram 1 * , Kaz u h i r o Ito 2 , K . Fan Chung 2 . 1 Department of Respiratory Medicine , School of Medicine,

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Hasan Bayram 1 * , Kaz u h i r o Ito 2 , K . Fan Chung 2 .

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  1. Effect of High Concentrations of Diesel Exhaust Particles on Human Lung Epithelial Cell Viability 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

  2. Background-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)

  3. Background-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)

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

  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 ofA549 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 ofA549 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 ofA549 Cells in 3.3% FCS following 48hrs’ incubation **p<0.01 vs 0g/ml DEP

  10. Effect of DEP on viability ofA549 Cells in 10% FCS following 48hrs’ incubation **p<0.01 vs 0g/ml DEP

  11. Effect of DEP on apoptosis/necrosis of A549 cells in 3.3% FCS following 48hrs’ incubation SF **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 DEPinduced cell apoptosis/necrosis.

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

  14. Thank you

  15. 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.

  16. Effect of DEP on viability ofA549 Cells in 0-10% FCS following 48hrs’ incubation

  17. 24 hrs Effect of DEP on viability of A549 Cells (MTT Assay) ***P<0.0001 vs 0g/ml DEP 72 hrs 48 hrs

  18. Effect of DEP (10g/ml)on cell cycle ofA549 cellsfollowing 48 hrs’ incubation *P<0.05, **P<0.01, ***P<0.0001 vs SF

  19. Effect of DEP on apoptosis of A549 cellsfollowing 48hrs’ incubation *P<0.05 and **P<0.005 vs SF

  20. Background • PM10 (10-50g/cm3) induce proliferation and apoptosis (25-50g/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 )

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

  22. 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 p21CIP1/WAF1 mRNA and protein expression of A549 cells.

  23. THANK YOU!

  24. 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

  25. Cell Signalling Pathways Associated with Apoptosis

  26. p21 and cell cycle pathways

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