V79 Hücre Hattında ve İnsan Lenfositlerinde Öjenolün Antioksidant Aktivitesinin, Sitotoksisitesinin ve Genotoksisitesinin Değerlendirmesi

Abstract

Öjenol, Eugenia Caryophyllata’nın yaprak ve tomurcuklarından elde edilen, ka- ranfil yağının fenolik yapıdaki uçucu bir bileşenidir. İlaç, kozmetik, tarım ve gıda endüstrisinde kullanılmaktadır. Türevlerin lokal antiseptik ve anestezik olarak tıpta kullanımı bulunmaktadır. Öjenol genel olarak güvenli bir bileşik olarak de- ğerlendirilir ancak çok farklı uygulamaları ve yaygın kullanım alanları nedeniyle, toksisitesi son yıllarda ilgi odağı olmuştur. Öjenol’ün sitotoksisitesi ve genotok- sisitesi ile ilgili çalışmalar yetersiz ve çelişkilidir. Bu çalışmada, öjenolün V79 hücre hattında in vitro antioksidan aktivitesi ve si- totoksisitesi TEAC test ve nötral kırmızı alım yöntemiyle incelenmiştir. Sonuçla- ra göre, öjenol’un doza bağımlı olarak V79 hücrelerinde antioksidan aktivite ve sitotoksik etkiye sahip olduğu gösterilmiştir.Öjenol’un IC50 dozunun 341.5 μM olduğu bulunmuştur. Ayrıca, öjenol’un in vitro genotoksik etkileri, tek hücre jel elektroforez (Comet yöntemi) ve mikroçekirdek yön- temi ile insan periferal lenfositleri üzerinde incelennmiş- tir. Sonuçlarımıza göre, EUG negatif kontrol hücreleriyle karşılaştırıldığında, daha yüksek konsantrasyonlarda DNA hasarı yaratma potansiyeline sahiptir. Ayrıca bu çalışmada, a öjenol’un hücreleri H2O2 kaynaklı oksidatif DNA hasa- rına karşı koruduğunu göstermektedir.

Keywords

• Öjenol,Genotoksisite,Sitotoksisite,Tek hücre jel elektroforez (Comet) yöntemi,mikroçekirdek yöntemi.

Evaluation of Antioxidant Activity, Cytotoxicity and Genotoxicity of Eugenol in V79 cell line and Human Lymphocytes Respectively

Abstract

Eugenol (EUG) is a volatile phenolic constituent of clove essential oil obtained from Eugenia Caryophyllata buds and leaves. It has been used in pharmaceuti- cal, cosmetic, agriculture and food industry. The derivatives have been used in medicine as a local antiseptic and anesthetic. Although EUG is considered safe as a food additive, due to the vast range of different applications and extensive use, there has been a great concern about its toxicity in recent years. However, studies about cytotoxicity and genotoxicity of EUG are very limited. In the present study, we investigated the in vitro antioxidant activity and the cy- totoxicity of EUG on V79 cell line by the TEAC Assay and Neutral Red Uptake Assay (NRU). Our results demonstrated that EUG has antioxidant activity and cytotoxic effect in V79 cell line in a dose dependent manner. The IC50 value of EUG in V79 cell line was found to be 341.5 μM. The in vitro genotoxic effects of EUG are also studied on human peripheral lym- phocytes by Single Cell Gel Electrophoresis (Comet) assay and micronucleus assay. Results show that EUG is potent to induce DNA damage at higher con- centrations when compared with untreated control cells. Our study also indicated that EUG is potent to protect cells against H2O2-induced oxidative DNA damage.

Keywords

• Eugenol (EUG),Cytotoxicity,Genotoxicity,Single Gel Electrophoresis (Comet) Assay,Cytokinesis-block Micronucleus (CBMN) Assay

References

1. 1. Barceloux D. Clove and eugenol in: Medical Toxicology of Natural Substances in Foods, Fungi, Medicinal Herbs, Plants and Venomous Animals. NJ, USA Wiley: Hoboken,; 2008.
2. 2. Kamatou GP, Vermaak I, Viljoen AM. Eugenol--from the re- mote Maluku Islands to the international market place: a revi- ew of a remarkable and versatile molecule. Molecules (Basel, Switzerland). 2012;17 (6):6953-81.
3. 3. Nam H, Kim MM. Eugenol with antioxidant activity inhibits MMP-9 related to metastasis in human fibrosarcoma cells. Food and chemical toxicology : an international journal pub- lished for the British Industrial Biological Research Associati- on. 2013;55:106-12.
4. 4. Guidance for industry 150. Concerns related to the use of Clo- ve Oil as an anesthetic on fish. Food and Drug Administration Center for Veterinary Medicine. US2007.
5. 5. Chogo JB, Crank G. Chemical composition and biological ac- tivity of the Tanzanian plant Ocimum suave. Journal of natural products. 1981;44 (3):308-11.
6. 6. Nagababu E, Sesikeran B, Lakshmaiah N, . 1995 -. The protec- tive effects of EUG on carbon tetrachloride induced hepatoto- xicity in rats. Free Radical Research 1995;23[6):617-27.
7. 7.Kabuto H, Yamanushi T. Effects of zingerone [4-[4-hydroxy-3- methoxyphenyl)-2- butanone] and EUG [2-methoxy-4 [pro- penyl) phenol] on the pathological progress in the 6-hydroxy- dopamine-induced Parkinson’s disease mouse model. Neuroc- hemical Research. 2011;36:2244-9.
8. 8. Ken M, Riedl M, Hagermanry AE. Tannin-protein complexes as radical scavengers and radical sinks. . Journal of agricultu- ral and food chemist. 2001;949:4917-23.

9. 9. Repetto G, Peso Ad, Zurita JL. Repetto G, del Peso A, Zurita JL.. Neutral red uptake assay for the estimation of cell viabi- lity/cytotoxicity. Nat Protoc. 2008;3[7):1125-31.
10. 10. Wininger MT, Kulik FA, Ross WD. In vitro clonal cytotoxi- city assay for chemicals using Chinese hamster ovary cells [CHO-K1). Methods in Cell Science 1979;5[2]:1091-3.
11. 11. NP NS, McCoy M, Tice R, Schneider E. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988;175[1):184-91.
12. 12. UndegerU,BasaranN.UndegerU,BasaranN.Assessmentof DNA damage in workers occupationally exposed to pesticide mixtures by the alkaline comet assay. Archive of Toxicology. 2002;76:430-6.
13. 13. Boyum A. Boyum A. Isolation of lymphocytes, granulocytes and Isolation of lymphocytes, granulocytes and macrophages. Scandinavian Journal of Immunology. 1976;5:9-15.
14. 14. Fenech M. The in vitro micronucleus technique. Mutatation Research. 2000;455:81-95.
15. 15. Yoo C,Han K,Cho K,Ha J,Park H,Nam J,et al. Eugenoliso- lated from the essential oil of Eugenia caryophyllata induces a reactive oxygen species-mediated apoptosis in HL-60 human promyelocytic leukemia cells. Cancer Letter. 2005;225:41-52.
16. 16. Martins C, Doran C, Leiers A, Rodrigues A. Genotoxic and apoptotic activities of the food avorings myristicin and EUG in AA8 and XRCC1 deficient EM9 cells. Food and Chemical Toxicology. 2011;49:385-92.
17. 17. Markowitz K, Moynihan M, Liu M, Kim S. Biologic proper- ties of EUG and zinc oxide-EUG: a clinically oriented review. Oral Surgery, Oral Medicine. 1992;73[6):729-37.
18. 18. Yogalakshmi B, Viswanathan P, Anuradha C. Investigation of anti-oxidant, anti-in ammatory and DNA-protective properti- es of EUG in thioacetamide-induced liver injury in rats. Toxi- cology. 2010;268:204-12.
19. 19. ItoM,MurakamiK,YoshinoM.Antioxidantactionofeugenol compounds: role of metal ion in the inhibition of lipid peroxi- dation. Food and chemical Toxicology. 2005;43:461-6.
20. 20. AtsumiT,FujisawaS,TonosakiK.Acomparativestudyofthe antioxidant pro-oxidant activities of EUG and iso-EUG with various concentrations and oxidation conditions. Toxicology in Vitro. 2005;19[8):1025-33.
21. 21. Slameňová D, Horváthová E, Wsólová L, Šramková M, Navarová J. Investigation of anti- oxidative, cytotoxic, DNA- damaging and DNA-protective effects of plant volatiles EUG and borneol in human-derived HepG2, Caco-2 and VH1 ̊C ell lines. Mutatation Research. 2009;677[46-52).
22. 22. Ho Y, Haung F, Chang Y. Mechanism of cytotoxicity of EUG in human osteoblastic cells in vitro. International Endodontic Journal 2006;39:389-93.
23. 23. Jaganathan SK, Mazumdar A, Mondhe D, Mandal M. Jaga- nathan S.K, Mazumdar A, Mondhe D, Mandal M. Apoptotic effect of EUG in human colon cancer cell lines. Cell Biology International journal. 2011;35:607-15.
24. 24. Huang T, Lee H, Kao C. Evaluation of the genotoxicity of zinc oxide EUG-based, calcium hydroxide-based, and epoxy resin- based root canal sealers by comet assay. Journal of Endodon- tics. 2001;27: 744-8.
25. 25. BACANLI M, ANLAR HG, BAŞARAN AA, BAŞARAN N. Assessment of Cytotoxicity Profiles of Different Phytoc- hemicals: Comparison of Neutral Red and MTT Assays in Different Cells in Different Time Periods. Turk J Pharm Sci 2017;14[2]:95-107.
26. 26. Maralhas A, Monteiro A, Martins C, Kranendonk M, Laires A, Rueff J, et al. Maralhas A, Monteiro A, Martins C, Kra- nendonk M, Laires A, Rueff J, Rodrigues A.S. Genotoxicity and endoreduplication inducing activity of the food avoring EUG. Mutagenesis. 2006;21: 199–204.
27. 27. Ding W, Levy D, Bishop M, Lascelles EL-C, Kulkarni R, Chang C, et al. Methyl-EUG genotoxicity in the Fischer 344 rat using the comet assay and pathway-focused gene expressi- on profiling. Toxicological Sciences. 2011;123[1):103-12.