Paraquat ile oluşturulmuş oksidatif stresin HepG2 hücrelerinde apoptozis üzerine etkisinin araştırılması

Bu çalışmada, paraquat ile oluşturulmuş oksidatif stresin HepG2 hücre hattında kaspaz bağımlı apoptozis üzerine etkilerini incelemek, sitokrom C düzeylerindeki değişimi ve DNA kırıklarının oluşumunu ortaya koymaktır. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) hücre canlılık testi, farklı paraquat yoğunluklarında ve zaman periodlarında uygulandı. HepG2 hücreleri için 24 saat inkubasyon süresinde Paraquatın IC50 10mM olarak belirlendi. Lipid peroksidasyonunun belirteci olarak malondialdehit düzeyleri analiz edildi. Kaspaz 3 ve kaspaz 9 düzeyleri, paraquat inkubasyonunun 6., 12. ve 24. saatlerinde kolorimetrik olarak tesbit edildi. Aynı zaman aralıklarında sitokrom C düzeyleri enzimimmunoassay ile belirlendi. DNA laddering testi, DNA fragmentasyonunu gösterebilmek amacıyla gerçekleştirildi. İnkubasyonun 6. ve 12. saatlerinde kaspaz 3 değerleri kontrol ve deneme grupları arasında istatistiksel olarak fark önemli bulunmazken, 24. saatte fark önemli bulundu. Diğer taraftan kaspaz 9 düzeyinde inkubasyonun 12. ve 24. saatlerinde kontrol ve deneme grupları arasında istatistik olarak önemli fark bulundu. Bütün gruplarda ve inkubasyon sürelerinde sitokrom C düzeyleri arasında fark bulunamadı. Ayrıca DNA fragmantasyon analizi sonucunda, kontrol ve deneme gruplarında DNA kırıkları belirlenemedi. HepG2 hücre hattında paraquat ile oluşturulan oksidatif stres kaspaz aktivasyonuna neden olmaktadır. Paraquat ile oluşturulan oksidatif stresin HepG2 hücrelerinde kaspaz bağımlı apoptosis için in vitro model olarak kullanılabileceğini göstermektedir.

The effect of oxidative stress induced by paraquat on apoptosis in HepG2 cells

The current study was undertaken to evaluate paraquat induced oxidative stress on caspase dependent apoptosis, cytochrom C levels and DNA laddering pattern in hepatocellular carcinoma cell line. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell viability and proliferation assay were performed different paraquat concentrations and time periods. 24h 10mM paraquat treatment was settled as IC50 for HepG2 cell line. Malondialdehyde, a marker of lipid peroxidation was estimated with TBA method. Caspase 3-9 were analyzed at 6., 12. and 24.h as apoptosis indicator with colorimetric assay. Cytochrom C levels were also estimated within the same time periods. DNA laddering assay performed to evaluate DNA fragmentation during apoptosis. The results indicated that there was no statistically significant difference after 6.h and 12.h but significant increase in 24.h for caspase 3. On the other hand statistically significant increase has been found after 12. and 24.h for caspase 9 compared to the control group. In all groups and time periods cytocrom C leves has been found at the same level. DNA fragmentation pattern has not been found in control and experiment groups. Paraquat induced oxidative stress were caused caspase activation. These observations suggested that oxidative stress caused by paraquat may be used as an in vitro model for caspase depended apoptosis pathways in HepG2 cell line.

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1. Stratton M, Campbell P, Futreal A. The cancer genome. Nature 2009; 458: 719–724.
2. Anonim. “Stop the poisonings! Stop paraquat!”. www.panap.net/11.03-2011.
3. Mollace V, Lannone M, Muscoli C, et al. The role of oxidative stress in paraquat induced neurotoxicity in rats: protection by non peptydyl superoxide sismutase mimetic. Neurosci Lett 2003; 335:163-166.
4. Mercan U. Toksikolojide serbest radikallerin önemi. YYU Vet Fak Derg 2004; 15(1-2): 91-96.
5. Cuthberst C, Wang Z, Zhang X, Tam S. Regulation of
human apolipoprotein A-I gene expression by gramoxane. J Bio Chem 1997; 272: 14957-14960.
6. Finkel T, Holbrook J. Oxidants, oxidative stress and the biology of ageing. Nature 2000; 408:239-247.
7. Lau T, Chiu F. Proteomic and biochemical analyses of in vitro carcinogen-induced lung cell transformation: synergism between arsenic and benzo(a)pyrone. Proteomics 2006; 6: 1619-1630.
8. Lau T, Wang Y, Chiu J. Reactive oxygen species: current knowledge and applications in cancer research and therapeutic. J Cell Biochem 2008; 104(2): 657–667.
9. Molinari BL, Tasat DR, Palmieri MA, O'Connor SE, Cabrini RL. Cell-based quantitative evaluation of the MTT assay. Anal Quant Cytol Histol 2003; 25(5): 254-262.
10. Plumb JA. Cell sensitivity assays: the MTT assay. Methods Mol Med 2004; 88: 165-169.
11. Dypbukt JM, Ankarcrona M, Burkitt M, et al. Different prooxidant levels stimulate growth, trigger apoptosis, or produce necrosis of insulin-secreting RINm5F cells. The role of intracellular polyamines. J Biol Chem 1994; 269: 30553-30560.
12. Burkitt MJ, Milne L, Nicotera P, Orrenius S. 1,10- Phenanthroline stimulates internucleosomal DNA fragmentation in isolated rat-liver nuclei by promoting the redox activity of endogenous copper ions. Biochem J 1996; 313: 163-169.
13. Yoshioka T, Kawada K, Shimada T, Mori M. Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxygen toxicity in the blood. Am J Obstet Gynecol 1979; 135(3): 372-376.
14. Sümbüloğlu K, Sümbüloğlu V. Biyoistatistik. 9.Baskı, No.53, Ankara: Hatipoğlu yayınları, 2000.
15. Nakamura T, Liptan A. Molecular mechanisms of nitrosative stres-mediated protein missfolding in neurodegenerative diseases. Cell and Mol L Sci 2007; 64(13): 1609-1620.
16. Fabisiak J, Kagan V, Tyurina Y, Tyurin V, Lazo J. Paraquat induced phosphatidylserine oxidation and apoptosis are independent of activation of PLA2. Am J Physiol Lung Cell Mol Physiol 1998; 274: 793-802.
17. Cappelletti G, Maggioni M, Maci R. Apoptosis in human epithelial cells: trigersing by paraquat and modulation by antioxidants. Cell Biology Internationale 1998; 22: 671–678
18. Niso-Santano M, Morán JM, García-Rubio L, et al. Low concentrations of paraquat induces early activation of extracellular signal-regulated kinase 1/2, protein kinase B, and c-Jun N-terminal kinase 1/2 pathways: role of c-Jun Nterminal kinase in paraquat-induced cell death. Toxicol Sci 2006; 92(2): 507-515.
19. Lei K, Nimnual A, Zong W, et al. The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c- Jun NH(2)-terminal kinase. Mol Cell Biol 2002; 22 (13): 4929–4942.
20. Zou H, Li Y, Lıu X, Wang X. An APAF-1.cytochom C multimeric coplex is a functional apoptosime that activates procaspase 9. J Bio Chem 1999; 274: 11549-11556.
21. Lopez E, Ferrer I. Staurosporine and H-7 induced cell death in SH-SY%Y nuroblastoma cells is associated with caspase-2 and caspase-3 activation but not with activation of Fas/Fas-L- caspase 8 signaling pathway. Mol Brain Res 2000; 85: 61-67.
22. Xu Y, Zhao L, Li Y. Alisol B asetate induces apoptosis of SGC7901 cells via mitochondrial and phoshatidylinositol 3- kinases / Akt signaling pathways. World J Gastroentero 2009; 15: 2870-2877.
23. Lavrik N, Golks A, Krammer P. Caspases: Pharmacologycal manipulations of cell death. J Cinic Invest 2005; 115: 2665-2672.
24. Green D, Kroemer G. Pharmacologycal manipulation of cell death: Clinical applications in sight? J Clinic Invest 2005; 115: 2610-2617.
25. Meidan Y, Tu Chongxing T, Ying H, et al. MSFTZ, a flavanone derivative, induces human hepatoma cell apoptosis via a reactive oxygen species and caspase dependet mitochondrial pathway. J Pharmacol Exp Ther 2008; 325:758-765
26. Janıcke UR, Sprengart M, Wati R, Porter A. Caspase-3 is required for DNA fragmentation on morphologycal changesassociated with apoptosis. J Bio Chem 1998; 273: 9357-9360
27. Sun HB, Zhang B, Zhao PX, et al. Analysis of in vivo patterns if caspase 3 gene expression in primary hepatocellular carcinoma and its relationship to p21Waf1 expression and hepatic apoptosis. World J Gastroentero 2000; 6(3): 356-360
28. Reshef A, Shirvan A, Shohami E, et al. Targeting cell death in vivo in experimental traumatic brain injury by a novel molecular probe. J Neurotrauma 2008; 25: 1-12.
29. Fei Q, Mccormac A, Monte D, Ethell D. Paraquat neurotoxicity is mediated by a Bak-dependet mechanism. J Bio Chem 2007; 4: 1-8.