Ratlarda roziglitazon’un telomer dinamiğine etkisinin moleküler yöntemlerle araştırılması

Year 2011, Volume: 28 Issue: 4, 162 - 167, 27.01.2012

Ali Okuyucu , Abdulkerim Bedir , Zeliha Özmen

https://doi.org/10.5835/jecm.omu.28.04.008

Abstract

Roziglitazon; Tip II diabet tedavisinde kullanılan tiazolidinedion grubu bir ilaçtır. Bu grup ilaçlar, karbonhidrat ve lipid metabolizmasını kontrol eden genlerin ekspresyonunun düzenlenmesinde görev alan peroksizom proliferatör aktive reseptör-gama (PPARgama)’nın ligandı olarak rol oynarlar. Telomer; kromozom uçlarında bulunan özelleşmiş DNA ve protein kompleksinden oluşmaktadır. Omurgalılarda telomerik DNA, TTAGGG gibi, guaninden zengin dizelerden oluşur. Normal hücrelerde, her DNA replikasyon peryodunda, telomer 50–150 baz kısalır. Tamir edilemeyen oksidatif DNA hasarı da bu telomer kısalmasına katkıda bulunabilir. Literatürde, roziglitazonun telomer uzunluğuna etkisini gösteren bir çalışma bulunmamaktadır. Bu nedenle, çalışmamızda roziglitazonun telomer uzunluğuna etkisini araştırmayı amaçladık. Çalışmada 7–8 haftalık, 248-275 g arasında, 16 tane erkek Sprague-Dawley cinsi rat kullanıldı. Ratlar; her grupta dört tane olacak şekilde dört gruba ayrıldı ve 14 gün boyunca 0, 0,5, 1,0 ve 2,0 mg/kg dozlarında roziglitazon oral yoldan uygulandı. Rat karaciğer, ince bağırsak, pankreas dokuları ve
lenfositlerinden genomik DNA izolasyonu yapıldı. Numunelerde genom sayısını belirleyebilmek için tek kopya gen olarak 36B4 geni seçildi. 36B4 genine spesifik primerlerin kullanıldığı Q-PCR ile bir rat dokusundaki genom kopya sayısı bulundu. Rat genom standardı olarak kullanılan bu numune tüm numunelerdeki genom sayısını bulmak için kullanıldı. Tüm numunelerde telomer (T) ve 36B4 (S) için kantitatif PCR yapıldı. İki çalışmada ile elde edilen ürünler birbirine oranlarak her numune için T/S oranı daha sonra da bir kalibratör numune kullanılarak relatif T/S oranı hesaplandı. Ayrıca 10,2 kb telomer standardı kullanarak numunelerin telomer uzunlukları kilobaz olarak da hesaplandı. Farklı roziglitazon dozları verilen numuneleri kontrol grubları ile karşılaştırıldığımızda telomer uzunluğu açısından istatistiksel olarak anlamlı bir fark bulamadık (p>0,05). Bu sonuç, roziglitazonun farklı rat dokularında telomer kısalmasına yol açmadığını göstermektedir

Keywords

PPAR-gama, Thiazolidinedion, Roziglitazon, Telomer, Oksidatif stres, Kantitatif PCR

References

• Adelfalk, C., 2001. Accelerated telomere shortening in Fanconi anemia fibroblasts a longitudinal study. FEBS Lett. 506, 22-26
• Bagi, Z., Koller, A., Kaley, G., 2004. PPARgamma activation, by reducing oxidative stress, increases NO bioavailability in coronary arterioles of mice with Type 2 diabetes. Am. J. Physiol. Heart Circ. Physiol. 286, 742-748.
• Bedir, A., Aliyazicioglu, Y., Bilgici, B., Yurdakul, Z., Uysal, M., Suvaci, D.E., Okuyucu, A., Kahraman, H., Hökelek, M., Alvur, M., 2008. As- sessment of genotoxicity in rats treated with the antidiabetic agent, pioglitazone. Environ. Mol. Mutagen. 49, 185-191.
• Bedir, A., Aliyazicioglu, Y., Kahraman, H., Yurdakul, Z., Uysal, M., Suvaci, D.E., Okuyucu, A., Hökelek, M., Alvur, M., 2006. Genotoxicity in rats treated with the antidiabetic agent, rosiglitazone. Environ. Mol. Mutagen. 47, 718-724.
• Brummendorf, T.H., 2001. Telomere length in leukocyte subpopulations of patients with aplastic anemia. Blood. 97, 895-900.
• Brunmair, B., Staniek, K., Gras, F., Scharf, N., Althaym, A., Clara, R., 2004. Thiazolidinediones, like metformin, inhibit respiratory complex I. Diabetes. 53, 1052-1059.
• Caballero, A.E., Saouaf, R., Lim, S.C., Hamdy, O., Abou-Elenin, K., O’Connor, C., Logerfo, F.W., Horton, E.S., Veves, A., 2003. The effects of troglitazone, an insulin-sensitizing agent, on the endothelial function in early and late type 2 diabetes: a placebo-controlled randomized clinical trial. Metabolism. 52, 173-180.
• Cadenas, E., Boveris, A., Ragan, C.I., Stoppani, A.O., 1997. Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch. Biochem. Biophys. 180, 248-257.
• Cawthon, RM., 2002. Telomere measurement by quantitative PCR. Nucleic Acids Res. 30, 47.
• Cawthon, R.M., Smith, K.R., O'Brien, E., Sivatchenko, A., Kerber, R.A., 2003. Association between telomere length in blood and mortality in people aged 60 years or older. Lancet. 361, 393-395.
• Ceolotto, G., Gallo, A., Papparella, I., Franco, L., Murphy, E., Iori, E., Pagnin, E., Fadini, G.P., Albiero, M., Semplicini, A, Avogaro, A., 2007. Rosiglitazone reduces glucose-induced oxidative stres mediated by NAD(P)H oxidasevia AMPK-dependent mechanism. Thromb. Vasc. Biol. 27, 2627-2633.
• Cherif, H., Tarry, J.L., Ozanne, S.E., Hales, C.N., 2003. Ageing and telomeres: A study into organ- and gender-specific telomere shortening. Nucleic Acids Res. 31, 1576-1583.
• Da Ros, R., Assaloni, R., Ceriello, A., 2004. The preventive antioxidant action of thiazolidinediones: A new therapeutic prospect in diabetes and insulin resistance. Diab. Med. 21, 1249-1252.
• De Lange, T., Shiue, L., Myers, R.M., Cox, D.R., Naylor, S.L., Killery, A.M., Varmus, H.E., 1990. Structure and variability of human chromo- some ends. Mol. Cell. Biol. 10, 518-527.
• Dumont, P., 2000. Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast. Free Radic. Biol. Med. 28, 361-373.
• Elısa, B.A., James, P.K., 2004. Dissociation of oxidant production by peroxisome proliferator-activated receptor ligands from cell death in hu- man cell linesfree. Radical Biol. Med. 37, 36-47.
• Evans, M.D., Cooke, M.S., 2004. Factors contributing to the outcome of oxidative damage to nucleic acids. Bio. Essays. 26, 533-542.
• Feinstein, D.L., Spagnolo, A., Akar, C., Weinberg, G., Murphy, P., Gavrilyuk, V. Dello, R.C., 2005. Receptor-independent actions of PPAR thi- azolidinedione agonists: Is mitochondrial function the key? Biochem. Pharmacol. 70, 77-188.
• Gale, EA., 2006. Troglitazone: The lesson that nobody learned? Diabetologia. 49, 1-6.
• Golubovskaya, V.M., Filatov, L.V., Behe, C.I., Presnell, S.., Hooth, M.J., Smith, G.J., Kaufmann, W.K., 1999. Telomere shortening, telomerase expression and chromosome instability in rat hepatic epithelial stem-like cells. Mol. Carcinog. 24, 209-217.
• Grommes, C., Landreth, G.E., Heneka, M.T., 2004. Antineoplastic effects of peroxisome proliferator-activated receptor gamma agonists. Lancet Oncol. 5, 419-429.
• Harley, C.B., Futcher, A.B., Greider, C.W., 1990. Telomeres shorten during ageing of human fibroblasts. Nature. 345, 458-460.
• Hastings, R., Li, N., Lacy, P.S., Patel, H., Herbert, K.E., Stanley, A.G., Williams, B., 2004. Rapid telomere attrition in cardiac tissue of the ageing wistar rat. Exp. Gerontol. 39, 855-857.
• Howarth, A.G., Wiehler, W.B., Pannirselvam, M., Jiang, Y., Berger, J.P., Severson, D., Anderson, T.J., Triggle, C.R., 2006. A nonthiazolidin- edione peroxisome proliferator-activated receptor gamma agonist reverses endothelial dysfunction in diabetic (db/db-/-) mice. J. Pharmacol. Exp. Ther. 316, 364-370.
• Iglarz, M., Touyz, R.M., Amiri, F., Lavoie, M.F., Diep, Q.N., Schiffrin, E.L., 2003. Effect of peroxisome proliferator-activated receptor-alpha and -gamma activators on vascular remodeling in endothelium-dependent hypertension. Arterioscler. Thromb. Vasc. Biol. 23, 45-51.
• Inmaculada, G.R., Rodrıguez-Juan, C., Diaz-Sanjuan, T., Martınez, M.A., Munoz-Yague, T., Solıs-Herruzo, J.A., 2007. Effects of rosiglitazone on the liver histology and mitochondrial function in ob/ob Mice. Hepatology. 46, 414-423.
• Jennings, B.J., Ozanne, S.E., Dorling, M.W., Hales, C.N., 1999. Early growth determines longevity in male rats and may be related to telomere shortening in the kidney. FEBS Lett. 448, 4-8
• José, M.P., Tranque, P., Burgos, M., Vaquero, C.F., Llopis, J., 2007. Glitazones induce astroglioma cell death by releasing reactive oxygen spe- cies from mitochondria: Modulation of cytotoxicity by nitric oxide. Mol. Pharmacol. 72, 407-417.
• Kawanishi, S., Oikawa, S., 2004. Mechanism of telomere shortening by oxidative stress. Ann. N.Y. Acad. Sci. 1019, 278-284.
• Kipling, D., Cooke, H.J., 1990. Hypervariable ultra-long telomeres in mice. Nature. 347, 400-402.
• Makarov, VL., Hirose, Y., Langmore, JP., 1997. Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell. 88, 657-666.
• Makino, H., Miyamoto, Y., Sawai, K., 2006. Altered gene expression related to glomerulogenesis and podocyte structure in early diabetic neph- ropathy of db/db mice and its restoration by pioglitazone. Diabetes. 55, 2747–2756.
• Manning, P.J., Sutherland, W.H., Walker, R.J., Williams, S.M., de Jong, S.A., Berry, E.A., 2008. The effect of rosiglitazone on oxidative stress and insulin resistance in overweight individuals. Diabetes Res. Clin. Pr. 81, 209–215.
• Moyzis, R.K., Buckingham, J.M., Cram, L.S., Dani, M., Deaven, L.L., Jones, M.D., Meyne, J., Ratliff, R.L., Wu. J.R., 1988. A highly conserved repetitive DNA sequence, (TTAGGG) n, present at the telomeres of human chromosomes. Proc. Natl. Acad. Sci. 85, 6622–6626.
• Narayanan, P.K., Hart, T., Elcock, F., Zhang, C., Hahn, L., McFarland, D., Schwartz, L., Morgan, D.G., Bugelski, P., 2003. Troglitazone-induced intracellular oxidative stress in rat hepatoma cells: A flow cytometric assessment. Cytometry A. 52, 28-35.
• Oikawa, S., Kawanishi, S., 1999. Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening. FEBS Lett. 453, 365–368.
• Oikawa, S., Tada-Oikawa, S., Kawanishi, S., 2001. Site-specific DNA damage at the GGG sequence by UVA involves acceleration of telomere shortening. Biochemistry. 40, 4763–4768.
• Perez-Ortiz, J.M., Tranque, P., Vaquero, C.F., Domingo, B., Molina, F., Calvo, S., Jordan, J., Cena, V., Llopis, J., 2004. Glitazones differentially regulate primary astrocyte and glioma cell survival. Involvement of reactive oxygen species and peroxisome proliferator-activated receptor- gamma. J. Biol. Chem. 279, 8976-8985.
• Petersen, S., Saretzki, G., Von Zglinicki, T., 1998. Preferential accumulation of single-stranded regions in telomeres of human fibroblasts. Exp. Cell Res. 239, 152–160.
• Prowse, K.R., Greider, C.W., 1995. Developmental and tissue-specific regulation of fare telomerase and telomere length. Proc. Natl. Acad. Sci. 92, 4818-4822.
• Rubelj, I., Vondracek, Z., 1999. Stochastic mechanism of cellular aging – abrupt telomere shortening as a model for stochastic nature of cellular aging. J. Theor. Biol. 197, 425–438.
• Samani, N.J., 2001. Telomere shortening in atherosclerosis. Lancet. 358, 472–473
• Son, N.H., Murray, S., Yanovski, J., Hodes, R.J., Weng, N., 2000. Lineage-specific telomere shortening and unaltered capacity fortelomerase expression in human T and B lymphocytes with age. J. Immunol. 165, 1191-1196.
• Suzui, N., Yoshimi, N., Kawabata, K., Mori, H., 1999. The telomerase activities in several organs and strains of rats with ageing. Lab. Animals. 33, 149-154.
• Von Zglinicki, T., 1995. Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: A model for senescence? Exp. Cell Res. 220, 186–193.
• Von Zglinicki, T., 2000. Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts. Free Radic. Biol. Med. 28, 64–74.
• Von Zglinicki, T., 2000. Short telomeres in patients with vascular dementia: an indicator of low antioxidative capacity and a possible risk factor? Lab. Invest. 80, 1739–47
• Von Zglinicki, T., 2002. Oxidative stress shortens telomeres. Trends Biochem. Sci. 27, 339–344.
• Wai, L.K., 2004. Telomeres, telomerase, and tumorigenesis. Med.Gen.Med. 6, 19.
• Yamaguchi, Y., Nozawa, K., Savoysky, E., Hayakawa, N., Nimura, Y., Yoshida, S., 1998. Change in telomerase activity of rat organs during growth and aging. Exp. Cell Res. 242, 120-127.
• Young, T., Cunningham, C.C., Bailey, S.M., 2002. Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: Studies using myxothiazol. Arch. Biochem. Biophys. 405, 65-72.
• Zijlmans, J.M., Martens, U.M., Poon, S.S., Raap, A.K., Tanke, H.J., Ward, R.K., Lansdorp, P.M., 1997. Telomeres in the mouse have large inter- chromosomal variations in the number of TA2AG3 repeats. Proc. Natl. Acad. Sci. 94, 742.