Mikronükleus (MN) ve Comet Assay testlerinin farklı hayvan türlerindeki uygulamalarına örnekler

Abstract

Farklı hayvanların dokularında biriken pek çok kirletici faktör canlıda genotoksik etki göstermektedir. Bu etkinin değerlendirilmesinde kullanılan genotoksisite testleri; kirlilik ajanlarının hayvanlarda DNA hasarına neden olup olmadığının belirlenmesine olanak sağlar. Bu faktörlerin etkilerini belirleyebilmek için farklı hayvan dokularında pek çok test sistemi geliştirilmiş ve genotoksisite testleri büyük önem kazanmıştır. Bu çalışmada, genotoksik hasarın belirlenmesinde çok yaygın olarak kullanılan iki test yöntemi olan Mikronükleus ve Comet Assay testleri karşılaştırılmış, avantaj ve dezavantajları belirlenerek farklı hayvan türlerinde yapılan çalışmaların değerlendirilmesi amaçlanmıştır.

Keywords

• Genotoksisite
• Genotoksik Test Yöntemleri
• MN Testi
• Comet Assay

Examples of the applications of Micronucleus (MN) and Comet Assay tests in different animal species.

Abstract

Many pollutants accumulate in the tissues of various animals and exhibit genotoxic effects in living organisms. Genotoxicity tests, used to assess this effect, allow for the determination of whether pollutants cause DNA damage in the organism. To determine the effects of these factors, various test systems have been developed in different animal tissues, and genotoxicity tests have gained significant importance. In this study, two widely used test methods for assessing genotoxic damage, Micronucleus and Comet Assay tests, were compared, their advantages and disadvantages were determined, and the evaluation of studies conducted in different animal species was aimed.

Keywords

• Genotoxicity
• Genotoxic Test Methods
• MN Test
• Comet Assay

References

1. Hutzinger, O., Tulp, M.T.M., ve Zitko, V., Chemicals with pollution potential. Aquatic Pollutants: Transformation and Biological Effects, 1, p.13 (2015).
2. Martins, M., Ferreira, A.M., Costa, M.H., ve Costa, P.M., Comparing the genotoxicity of a potentially carcinogenic and a noncarcinogenic PAH, singly, and in binary combination, on peripheral blood cells of the European sea bass. Environmental toxicology (2015).
3. Göney, G., Çevre Kirliliğinin Biyoizlenmesinde Balıklarda Genotoksisite Testleri. Türk Bilimsel Derlemeler Dergisi, (1), 43-49, (2016).
4. Wells, P.G., Biomonitoring the health of coastal marine ecosystems–the roles and challenges of microscale toxicity tests. Marine Pollution Bulletin, 39(1), 39- 47, (1999).
5. Al-Sabti, K., ve Metcalfe, C.D., Fish micronuclei for assessing genotoxicity in water. Mutation Research/Genetic Toxicology, 343(2), 121-135, (1995).
6. De Flora, S., Vigano, L., D’agostini, F., Camoirano, A., Bagnasco, M., Bennicelli, C., Melodia, F. ve Arillo, A., Multiple genotoxicity biomarkers in fish exposed in situ to polluted river water. Mutation Research/Genetic Toxicology, 319(3), 167-177, (1993).
7. Belpaeme, K., Delbeke, K., Zhu, L., Kirsch-Volders, M., Cytogenetic studies of PCB77 on brown trout (Salmo trutta fario) using the micronucleus test and the alkaline comet assay. Mutagenesis, 11(5), 485-492, (1996).
8. Klobučar, G.I., Štambuk, A., Pavlica, M., Erben, R., Genotoxicity monitoring of freshwater environment: comet and micronucleus assays. In Symposium Pollutant responses in marine organisms, 62; 306-316, (2006).

9. Raisuddin, S., ve Jha, A.N., Relative sensitivity of fish and mammalian cells to sodium arsenate and arsenite as determined by alkaline single cell gel electrophoresis and cytokinesis block micronucleus assay. Environmental and molecular mutagenesis, 44(1), 83-89, (2004).
10. Obiakor, M., Okonkwo, J., Nnabude, P., ve Ezeonyejiaku, C., Eco-genotoxicology: micronucleus assay in fish erythrocytes as in situ aquatic pollution. Journal of Animal Science Advances, 2(1), pp.123-133, (2012).
11. Kutoğlu, S., Tatlarin Baraj Gölündeki su kirliliğinin bazı Cyprinidae türlerine genotoksik etkisinin mikronükleus testi ile belirlenmesi, Yüksek Lisans Tezi, Nevşehir Hacı Bektaş Veli Üniversitesi, Fen Bilimleri Enstitüsü, Nevşehir, (2017).
12. Bulut, B., Titanyum Dioksit’in (TİO2) Ergin Labıdochromıs caeruleus (Fryer, 1956) Türüne Genotoksik Etkileri: Mikronükleus Testi, Yüksek Lisans Tezi, Aydın Adnan Menderes Üniversitesi, Fen Bilimleri Enstitüsü, Aydın, (2021).
13. Güner, U. ve Gökalp Muranlı, FD., Balıklarda tek hücre jel elektroforezi (comet assay). Karadeniz Fen Bilimleri Dergisi, 3(9), 103-114, (2013).
14. Ma, TH., Tredescantia micronucleus bioassay and polen tube chromatidaberration test for in situ monitoring and mutagen screening, Environmental Health Perspective, 37, 85-90, (1981).
15. Scarpato, R., Migliore, L. Ve Barale, R., The micronucleus assay in Anadontacygnea for the detection of drinking water mutagenity. Mutation Research, 245, 231-237, (1990).
16. Düzel, S., Sentetik Piretroit Deltametrin’in Pseudorasbora parva (Temminck & Schlegel, 1846) Üzerindeki Akut ve Genotoksik Etkileri, Yüksek Lisans Tezi, Gazi Üniversitesi, Fen Bilimleri Enstitüsü, Ankara, (2013).
17. Jha, A.N., Ecotoxicological applications and significance of the comet assay. Mutagenesis, 23(3), 207- 221, (2008).
18. Kurucu, G., Curi Deresi (Ordu) Su, Sediment ve Alburnus chalcoides Türünde Ağır Metal Birikimi ve Genotoksik Etkilerinin Araştırılması, Doktora Tezi, Ordu Üniversitesi Fen Bilimleri Enstitüsü, Ordu, (2020).
19. Fenech, M. ve Crott, JW., Micronuclei, nucleoplasmic bridges and nuclear buds induced in folic acid deficient human lymphocytes-evidence for breakagefusion- bridge cycles in the cytokinesis-block micronucleus assay. Fundamental and Molecular Mechanisms of Mutagenesis, Mutation Research, 504, 131-136, (2002).
20. Heddle, JA., Hite, M., Kirkhart, B., Mavournn, K., Macgregor, JT., Newell, GW. ve Salamone, MF., The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency GeneTox Program, Mutation Research, 123, 61-118, (1983).
21. Yıldız, Y. ve Önen, Ö., Bazı kirleticilerin Teleostlar üzerindeki genotoksik etkileri. Kafkas Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 9(1):63-74, (2016).
22. Heddle, JA., Cimino, MC., Hayashi, M., Romagna, F., Shelby, MD., Tucker, JD., Vanparys, P., MacGregor, JT., Micronuclei as an index of cytogenetic damage: past, present, and future. Environmental and Molecular Mutagenesis, 18(4), 277-291, (1991).
23. Fenech, M., The in vitro micronucleus technique. Mutation Research, 455, 81- 95, (2000).
24. Şekeroğlu, V. ve Şekeroğlu, ZA., Genotoksik hasarın belirlenmesinde mikronükleus testi. Türk Hijyen ve Deneysel Biyoloji Dergisi, 68(4), 241-252, (2011).
25. Schiffmann, D. ve De Boni, U., Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechanism by which micronuclei can arise. Mutation Research, 246(1), 113-122, (1991).
26. Yıldırım, A. ve Yıldırım, MS., Matbaa sanayinde çalışan işçilerin bukkal mukoza hücrelerinde mikronükleus ve binükleotid sıklığının belirlenmesi. Tıp Araştırmaları Dergisi, 9(1), 25-28, (2011).
27. Stopper, H. ve Müller, SO., Micronuclei as a biological endpoint for genotoxicity: a minireview. Toxicology in Vitro, 11, 661-667, (1997).
28. Fenech, M., Holland, N., Chang, WP., Zeiger, E., ve Bonassi, S., The human micronucleus project-an ınternational collaborative study on the use of micronucleus technique for measuring DNA damage in humans. Mutation Research, 428, 271-283, (1999).
29. Gül, G., Endokrin Bozucu Kirleticilerin Bazı Ekonomik Balık Türlerinde Gonad Histopatolojisine ve Vitellogeninlerine Etkileri, Yüksek Lisans Tezi, Gazi Üniversitesi, Fen Bilimleri Enstitüsü, Ankara, (2014).
30. Anbumani, S. ve Mohankumar, M.N., Nuclear and cytoplasmic abnormalities in the fish Catla catla (Hamilton) exposed to chemicals and ionizing radiation. Research Journal of Environmental Sciences, 5(12), p.867, (2011).
31. Hayashi, M., Ueda, T., Uyeno, K., Wada, K., Kinae, N., Saotome, K., Tanaka, N., Takai, A., Sasaki, Y.F., Asano, N., ve Sofuni, T., Development of genotoxicity assay systems that use aquatic organisms. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 399(2), 125-133, (1998).
32. Bolognesi, C., ve Hayashi, M., Micronucleus assay in aquatic animals. Mutagenesis, 26(1), 205-213, (2011).
33. Schmidt, W., The micronucleus test, Mutation Research, 31, 9-15, (1975).
34. Ali, F., El-Shehawi, A.M., ve Seehy, M.A., Micronucleus test in fish genome: A sensitive monitor for aquatic pollution. African journal of biotechnology, 7(5), 606-612, (2008).
35. Ayllon, F., ve Garcia-Vazquez, E., Micronuclei and other nuclear lesions as genotoxicity indicators in rainbow trout Oncorhynchus mykiss. Ecotoxicology and Environmental Safety, 49(3), 221-225, (2001).
36. Çavaş, T., ve Ergene‐Gözükara, S., Micronucleus test in fish cells: a bioassay for in situ monitoring of genotoxic pollution in the marine environment. Environmental and molecular mutagenesis, 46(1), 64-70, (2005).
37. Obiakor, M.O., Okonkwo, J.C., ve Ezeonyejiaku, C.D., Genotoxicity of freshwater ecosystem shows DNA damage in preponderant fish as validated by in vivo micronucleus induction in gill and kidney erythrocytes. Mutation Research/Genetic Toxicology and Environmental Mutagenesis,775, 20-30, (2014).
38. Singh, N.P., McCoy, M.T., Tice, R.R., ve Schneider, E.L., A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental cell re search, 175(1), 184-191, (1988).
39. Cotelle, S. ve Ferard, JF., Comet assay in genetic ecotoxicology: a review. Environmental and Molecular Mutagenesis, 34, 246-255, (1999).
40. Rajaguru, P., Suba, S., Palanivel, M., ve Kalaiselvi, K., Genotoxicity of a polluted river system measured using the alkaline comet assay on fish and earthworm tissues. Environmental and Molecular Mutagenesis, 41(2), 85-91, (2003).
41. Frenzilli, G., Scarcelli, V., Del Barga, I., Nigro, M., Förlin, L., Bolognesi, C. ve Sturve, J., DNA damage in eelpout (Zoarces viviparus) from Göteborg harbour. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 552(1), 187-195, (2004).
42. Frenzilli, G., Nigro, M., ve Lyons, B.P., The Comet assay for the evaluation of genotoxic impact in aquatic environments. Mutation Research/Reviews in Mutation Research, 681(1), 80-92, (2009).
43. Nagarani, N., Devi, V.J., ve Kumaraguru, A.K., Identification of DNA damage in marine fish Therapon jarbua by comet assay technique. Journal of Environmental Biology, 33(4), p.699, (2012).
44. Schnurstein, A., ve Braunbeck, T., Tail moment versus tail length—application of an in vitro version of the comet assay in biomonitoring for genotoxicity in native surface waters using primary hepatocytes and gill cells from zebrafish (Danio rerio). Ecotoxicology and Environmental Safety, 49(2), 187-196, (2001).
45. Kumaravel, T. S., ve Jha, A. N., Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutation Research, 605, 7-16, (2006).
46. Çavaş, T., In vivo genotoxicity evaluation of atrazine and atrazine–based herbicide on fish Carassius auratus using the micronucleus test and the comet assay. Food and Chemical Toxicology, 49(6), 1431-1435, (2011).
47. Tice, RR., Agurell, E., ve Anderson, D., Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environmental and Molecular Mutagenesis, 35(3), 206-221, (2000).
48. Çavaş, T. ve Könen, S., Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis, 22(4), 263-268, (2007).
49. Çavaş, T. ve Könen, S., In vivo genotoxicity testing of the amnesic shellfish poison (domoic acid) in piscine erythrocytes using the micronucleus test and the comet assay. Aquatic Toxicology, 90(2), 154-159, (2008).
50. Bopp, KS., Abicht, HK., ve Knauer, K., Copper-induced oxidative stress in rainbow trout gill cells. Aquatic Toxicology, 86(2), 197-204, (2008).
51. Kontaş, S., ve Bostancı, D., Genotoxic effects of environmental pollutant heavy metals on Alburnus chalcoides (Pisces: Cyprinidae) inhabiting lower Melet River (Ordu, Turkey). Bulletin of Environmental Contamination and Toxicology, 104, 763-769, (2020).
52. Rodriguez-Cea, A., Ayllon, F., ve Garcia-Vazquez, E., Micronucleus test in freshwater fish species: an evaluation of its sensitivity for application in field surveys. Ecotoxicology and Environmental Safety, 56(3), 442-448, (2003).
53. Buschini, A., Martino, A., Gustavino, B., Monfrinotti, M., Poli, P., Rossi, C., Santoro, M., Dörr, A.J.M. ve Rizzoni, M., Comet assay and micronucleus test in circulating erythrocytes of Cyprinus carpio specimens exposed in situ to lake waters treated with disinfectants for potabilization. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 557(2), 119-129, (2004).
54. De Andrade, V.M., Da Silva, J., Da Silva, F.R., Heuser, V.D., Dias, J.F., Yoneama, M.L. ve De Freitas, T.R., Fish as bioindicators to assess the effects of pollution in two southern Brazilian rivers using the Comet assay and micronucleus test. Environmental and molecular mutagenesis,44(5), 459-468, (2004).
55. Çavaş, T., In vivo genotoxicity of mercury chloride and lead acetate: micronucleus test on acridine orange stained fish cells. Food and Chemical Toxicology, 46(1), 352-358, (2008).
56. Kılıçle, P. A., ve Doğan, A., Akrilamidin Capoeta capoeta (Guldenstaedt 1773) Üzerindeki Genotoksik Etkileri. Kafkas Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 9(1), 75-81, (2016).
57. Özkurnaz, G., Çanakkale Boğazı Umurbey Kıyılarında Yetişen Bazı Çift Kabuklu Yumuşakçaların (Bivalvia) Solungaçlarındaki Ağır Metal Birikimleri ve Genotoksisite Üzerine Etkileri. Yüksek Lisans Tezi, Çanakkale Onsekiz Mart Üniversitesi, Fen Bilimleri Enstitüsü, Çanakkale, (2012).
58. Yalçın, E., Taşlı, B., Çıçek, F., Demırtaş, G., Yapar, K., ve Çavuşoğlu, K., Albino farelerde dinikanazol toksisitesine karşı üzüm çekirdeği özütünün koruyucu rolünün araştırılması. Erciyes Üniversitesi Fen Bilimleri Enstitüsü Fen Bilimleri Dergisi, 32(1), 1-6, (2016).
59. Hasanbašić, D., ve Rukavina, D., Micronuclei in lymphocytes of horses and pigs after in vitro irradiation. Acta veterinaria, 57(4), 341-350, (2007).
60. Saleh, K., Mikronükleus Testi Ile Bazı Kimyasal Maddelerin ve Çevre Kirleticilerin Neden Olduğu Klastojenik Etkilerin Araştırılması, Doktora Tezi, Anadolu Üniversitesi, (2015).
61. Silveira, E. D. R., Benvindo-Souza, M., Assis, R. A., Dos Santos, C. G. A., de Lima Amorim, N. P., Borges, R. E., ve de Souza Santos, L. R., Micronucleus and different nuclear abnormalities in wild birds in the Cerrado, Brazil. Environmental Science and Pollution Research, 1-9, (2022).
62. Özparlak, H., Arslan, A., ve Güler, G. Ö., Organik insektisit Fipronil’in genotoksik etkilerinin civciv mikronukleus test sisteminde belirlenmesi. Selçuk Üniversitesi Fen Fakültesi Fen Dergisi, 2(37), 1-8, (2011).
63. Türkez, H., Tatar, A., Geyikoğlu, F., Togar, B., ve Keleş, M., Bakır Asetoarsenit'in İn Vivo Genotoksik Etkilerinin Değerlendirilmesi. Süleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 13(2), 132-136, (2014).
64. Gürhan, B., Şenel, E., Dakılır, G., ve Öztürkmen, H., Şap Aşılı Sığırlarda Mikronötralizasyon ve ELİSA ile Antikor Düzeylerinin Saptanması. Etlik Veteriner Mikrobiyoloji Dergisi, 7(5), 99-108, (1994).
65. Maria, V. L., Correia, A. C., ve Santos, M. A., Genotoxic and biochemical responses in caged eel (Anguilla anguilla L.) after short-term exposure to harbour waters. Environment International, 29, 923–929, (2003).
66. Villela I. V., de Oliveira I. M., Silveira J. C., Dias J. F., Henriques J. A. P. ve da Silva J., Assessment of Environmental Stress by the Micronucleus and Comet Assays on Limnoperna fortunei Exposed to Guaíba Hydrographic Region Samples (Brazil) Under Laboratory Conditions. Mutation Research, 628: 76-86, (2007).
67. Turna Demir, F., Farklı Kirlilik Potansiyellerine Sahip Lokalitelerde Yaşayan Mıcrotus guentherı Örneklerindeki Ağır Metal Birikim Seviyelerinin Saptanması ve Genetik Hasarın Araştırılması. Doktora Tezi, Akdeniz Üniversitesi, Fen Bilimleri Enstitüsü, Antalya, (2018).
68. Lourenço, J., Pereira, R., Gonçalves, F. ve Mendo, S., Metal bioaccumulation, genotoxicity and gene expression in the European wood mouse (Apodemus sylvaticus) inhabiting an abandoned uranium mining area. Science of the Total Environment, 443: 673-680, (2013).
69. Mitkovska, V., Chassovnikarova, T., Atanasov, N. ve Dimitrov, H., DNA damage detected by Comet assay in Apodemus flavicollis (Melchior, 1834) from Strandzha Natural Park. Acta Zoologica Bulgarica, 4: 155-158, (2012).
70. Kontaş, S., ve Bostancı, D., Balıklarda genotoksik hasarın belirlenmesine yönelik bir araştırma: Capoeta banarescui örneği. Journal of Limnology and Freshwater Fisheries Research, 6(2), 144-152, (2020).
71. Baos R, Jovani R, Pastor N, Tella JL, Jiménez B., ve Gómez G., Evaluation of genotoxic effects of heavy metals and arsenic in wild nestling white storks (Ciconia ciconia) and black kites (Milvus migrans) from southwestern Spain after a mining accident. Environmental Toxicological Chemical, 25(10):2, 794-803, (2006).
72. Mouchet F, Gauthier L, Baudrimont M, Gonzalez P, Mailhes C., ve Ferrier V. Comparative evaluation of the toxicity and genotoxicity of cadmium in amphibian Larvae (Xenopus laevis and Pleurodeles waltl) using the Comet assay and the micronucleus test, Environmental Toxicology, 22(4):422– 35, (2007).
73. Møller, P., The comet assay: ready for 30 more years. Mutagenesis, 33(1), 1-7, (2018).
74. Jiang, N., Naz, S., Ma, Y., Ullah, Q., Khan, M. Z., Wang, J., Lu, X., Luosang, D. Z., Tabassum, S., Cahtha, A. M. M., ve Basang, W. D., An Overview of Comet Assay Application for Detecting DNA Damage in Aquatic Animals. Agriculture, 13(3), 623, (2023).
75. Cordelli, E., Bignami, M., ve Pacchierotti, F., Comet assay: a versatile but complex tool in genotoxicity testing. Toxicol Research 10: 68–78, (2021).
76. Azqueta, A., Slyskova, J., Langie, S. A., O’Neill Gaivão, I., ve Collins, A., Comet assay to measure DNA repair: approach and applications. Frontiers in genetics, 5, 288, (2014).