Tannik Asitin İnsan Lenfositlerinde DNA Hasarı Üzerine Etkisi

Yıl 2014, Sayı: 2, 133 - 146, 01.06.2014

Dilek Tokaç Sevtap Aydın Arif Ahmet Başaran Nurşen Başaran

Öz

Doğal ürünlerde bulunan fenolik maddeler reaktif oksijen türevlerinin zararlı etkilerine karşı organizmayı korumada önemli bir role sahiptir. Suda çözünen polifenolik tannik asit TA bitkisel çaylar, bira, ceviz, fındık ve üzümsü meyveler gibi pek çok bitkisel içecek ve gıdalarda bulunmaktadır. Tanenlerin kan pıhtılaşmasını hızlandırdığı, kan basıncını azalttığı, serum lipid düzeyini düşürdüğü, karaciğer nekrozuna neden olduğu ve immün yanıtları düzenlediği gibi bazı fizyolojik etkileri bildirilmektedir. TA konsantrasyonuna ve yapısında bulunan hidroksil grupların sayı ve bulunduğu yere göre antioksidan veya prooksidan olarak rol almaktadır. Ancak, TA’ca zengin gıdaların tüketiminin özofagus kanseri gibi belli kanser sıklıkları ile ilişkili olması nedeniyle TA’nın karsinojenik olabileceği de ileri sürülmektedir. Bu çalışmada TA’nın antioksidan kapasitesi troloks eşdeğer antioksidan kapasite deneyi ile ve insan lenfositlerinde H2O2 ile indüklenen DNA hasarı üzerine TA’nın etkisi standart comet yöntemi ve formamidoprimidin-DNA-glikosilaz Fpg ile adapte edilmiş comet yöntemi ile araştırılmıştır. Sonuçlarımız 0.5 μM konsantrasyon üzerinde TA’nın anlamlı olarak antioksidan kapasiteye sahip olduğunu göstermiştir. Çalışılan tüm konsantrasyonlarında 0.1-100 M TA’nın tek başına DNA hasarına neden olmadığı ve hatta H2O2 ile indüklenen oksidatif DNA hasarını anlamlı olarak azalttığı saptanmıştır. Ayrıca 0.1-100 M konsantrasyon aralığında tek başına TA’nın artmış okside pürin baz düzeylerinin göstergesi olan Fpg duyarlı bölgeleri indüklemediği, ancak TA’nın çalışılan tüm konsantrasyonlarında doz bağımlı olarak H2O2 ile indüklenen Fpg duyarlı bölgeleri anlamlı olarak azalttığı saptanmıştır. Sonuç olarak, TA’nın insan lenfositlerinde oksidatif DNA hasarı üzerine koruyucu bir etkiye sahip olduğu in vitro olarak görülmektedir. Diyetsel destek olarak uygun düzeylerde tannik asit doğal bir antioksidan olarak kullanılabilir, fakat tannik asitin insan sağlığı üzerine potansiyel etkilerini aydınlatmak için daha ileri in vivo ve in vitro çalışmalara gerek vardır.

Anahtar Kelimeler

Tannik asit, Hidrojen peroksit, Tek hücre jel elektroforez, Comet yöntemi, TEAK Troloks eşdeğer antioksidan kapasite yöntemi

The Effect of Tannic Acid on DNA Damage in Human Lymphocytes

Öz

Phenolic substances in natural products play an important role in protecting the organism against harmful effects of reactive oxygen species. Tannic acid TA , water-soluble polyphenol, is present in many plant beverages and foods, such as herbal teas, beer, walnut, hazelnut, and berries. Tannins have been reported to exert some physiological effects, such as to accelerate blood clotting, reduce blood pressure, decrease the serum lipid level, cause liver necrosis, and modulate immunoresponses. It acts as an antioxidant or a prooxidant, depending on the concentrations of TA and the number and position of hydroxyl groups on TA. It has been suggested that tannins might be carcinogenic, as the incidences of certain cancers, such as esophageal cancer, are related to consumption of tannins-rich foods. In the present study, the antioxidant capacity of TA was determined by the trolox equivalent antioxidant capacity assay and the effect of TA on DNA damage induced by H2O2 in human lymphocytes were investigated by the standard comet assay and the formamidopyrimidine-DNA-glycoslase Fpg modified comet assay. Our study showed that above the concentrations of 0.5 μM TA showed significant antioxidant capacity. It was determined that at all the concentrations studied 0.1-100 M , TA alone did not cause DNA damage and even significantly reduced oxidative DNA damage induced by H2O2. It was also determined that within the concentration of 0.1-100 M TA alone did not induce Fpg sensitive sites indicating the increased oxidized purine base levels, but TA treatment at all the concentrations studied significantly reduced the Fpg sensitive sites induced by H2O2 in a dose dependent. In conclusion, it seems that TA exerts a protective effect on oxidative DNA damage in human lymphocytes in vitro. In appropriate levels as a dietary supplement, TA may be used as a natural antioxidant however it is necessary to perform the further in vivo and in vitro studies to clarify the potential effects of TA on human health.

Anahtar Kelimeler

Tannic acid, Hydrogen peroxide, Single cell gel electrophoresis, Comet assay, TEAC Trolox equivalent antioxidant capacity assay

Kaynakça

• Basaga, H.S.: Biochemical aspects of free radicals, Biochem Cell Biol, 68, 989-998 (1990).
• Negre-Salvayre, A., Auge, N., Ayala, V., Basaga, H., Boada, J., Brenke, R., Chapple, S., Cohen, G., Feher, J., Grune, T., Lengyel, G., Mann, G.E,, Pamplona, R., Poli, G., Portero- Otin, M., Riahi, Y., Salvayre, R., Sasson, S., Serrano, J., Shamni, O., Siems, W., Siow, R.C., Wiswedel, I., Zarkovic, K., Zarkovic, N.: Pathological aspects of lipid peroxidation, Free Radic Res, 44, 1125-1175 (2010).
• Sutherland, B.A., Rahman, R.M.A., Appleton, I.: Mechanisms of action of green tea catechins, with a focus on ischemia- induced neurodegeneration, J Nutr Biochem, 17, 291-306 (2006).
• Anon., Occupational Safety and Health Administration (OSHA) issues tentative carcinogen list, C & EN., 56, 20 (1978).
• Cooke, M.S., Evans, M.D., Dizdaroglu, M., Lunec, J.: Oxidative DNA damage: mechanisms, mutation, and disease, FASEB J, 17, 1195-1214 (2003).
• Evans, M.D., Cooke, M.S.: Factors contributing to the outcome of oxidative damage to nucleic acids, Bioessays, 26, 533-542 (2004).
• Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F.: Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ Mol Mutagen, 35, 206-221 (2000).
• Collins, A.R., Duthie, S.J., Dobson, V.L.: Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA, Carcinogenesis, 14, 1733-1735 (1993).
• Collins, A.R., Dusinska, M., Gedik, C.M., Stetina, R.: Oxidative damage to DNA: do we have a reliable biomarker? Environ Health Perspect, 104, 465-469 (1996).
• Collins, A.R., Dobson, V.L., Dusinka, M., Kennedy, G., Stetina, R.: The comet assay: what can it really tell us? Mutat Res, 375,183-193 (1997).
• Cemeli, E., Baumgartner, A., Anderson, D.: Antioxidants and comet assay, Mutat Res, 681, 51-57 (2009).
• Cabrera, C., Artacho, R., Gimenez, A.: Beneficial effects of green tea-A Review, J Am Coll Nutr, 25(2), 79-99 (2006).
• Zhao, B., Guo, Q., Xin, W.: Free radical scavenging by green tea polyphenols, Method Enzymol, 335, 217-231 (2001).
• Boyum, A.: Isolation of lymphocytes, granulocytes and macrophages, Scand J Immunol, 5, 9-15 (1976).
• Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L.: A simple technique for quantitation of low levels of DNA damage in individual cells, Exp Cell Res, 175, 184-191 (1988).
• Chung, K.T., Wong, T.Y., Wei, C.I., Huang, Y.W., Lin, Y.: Tannins and Human Health: A Review, Food Sci Nutr, 38, 421-464 (1998).
• Yang, C.S., Landau, J.M., Huang, M.T., Newmark, H.L.: Inhibition of carcinogenesis by dietary polyphenolic compounds. Annu Rev Nutr, 21, 381-406 (2001).
• Hatano, T., Yasuhara, T., Yoshihara, R., Yoshida, T., Agata, I., Noro, T., and Okuda, T.: Inhibitory effects of tannins and related polyphenols on xanthine oxidase, Chem Pharm Bull, 38, 1224-1229 (1990).
• Bhat, R., Hadi, S.M.: DNA breakage by tannic acid and Cu(II): generation of active oxygen species and biological activity of the reaction, Mutat Res, 313, 49-55 (1994).
• Ferguson, LR.: Role of plant polyphenols in genomic stability, Mutat Res, 475, 89-111 (2001).
• Noguchi, N., Niki, E.: Phenolic antioxidants: a rationale for design and evaluation of novel antioxidant drug for atherosclerosis, Free Radical Biol Med, 28, 1538-1546, (2000).
• Cao, G., Sofic, E., Prior, R.L.: Antioxidant and prooxidant behavior of flavonoids: structure- activity relationships, Free Radic Biol Med, 22, 749-760 (1997).
• Kondo, K., Kurihara, M., Miyata, N., Suzuki, T., Toyoda, M.: Scavenging mechanisms of (-)-epigallocatechin gallate and (-)-epicatechin gallate on peroxyl radicals and formation of superoxide during the inhibitory action, Free Radic Biol Med, 27, 855-863 (1999).
• Langley-Evans, S.C.: Antioxidant potential of green and black tea determined using the ferric reducing power (FRAP) assay, Int J Food Sci Nutr, 51, 181-188 (2000).
• Song, L.L., Liang, R., Li, D.D., Xing, Y.D., Han, R.M., Zhang, J.P., Skibsted, L.H.: -carotene radical cation addition to green tea polyphenols. Mechanism of antioxidant antagonism in peroxidizing liposomes, J Agric Food Chem, 59, 12643-12651 (2011).
• Sueishi, Y., Ishikawa, M, Yoshioka, D., Endoh, N., Oowada, S., Shimmei, M,, Fujii, H., Kotake, Y.: Oxygen radical absorbance capacity (ORAC) of cyclodextrin-solubilized flavonoids, resveratrol and astaxanthin as measured with the ORAC-EPR method, J Clin Biochem Nutr, 50, 127-132 (2012).
• Lopes, G.K., Schulman, H.M., Hermes-Lima, M.: Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions, Biochim Biophys Acta, 1472, 142-152 (1999).
• Srivastava, R.C., Husain, M.M., Hasan, S.K., Athar, M.: Green tea polyphenols and tanniv acid act as potent inhibitors of phorbol ester-induced nitric oxide generation in rat hepatocytes independent of their antioxidant properties, Cancer Letters, 153, 1-5 (2000).
• Vance, R.E., Teel, R.W.: Effect of tannic acid on rat liver S9 mediated mutagenesis, metabolism and DNA binding of benzo [a]pyrene, Cancer Letters, 47, 37-44 (1989).
• Fedeli, D., Berrettini, M., Gabryelak, T., Falcioni, G.: The effect of some tannins on trout erythrocytes exposed to oxidative stres, Mutation Research, 563, 89-96 (2004).
• Labieniec, M., Gabryelak, T.: Measurement of DNA damage and protein oxidation after the incubation of B14 Chinese hamster cells with chosen polyphenols, Toxicology Letters, 155, 15-25 (2005).
• Wua, L. T., Chub, C.C., Chunga, J.G., Chenc, C.-H., Hsud, L.-S., Liub, J.-K., Chena, S.C.: Effects of tannic acid and its related compounds on food mutagens or hydrogen peroxide- induced DNA strands breaks in human lymphocytes, Mutation Research, 556, 75-82 (2004).