Genotoksik hasarın belirlenmesinde mikronükleus testi

Year 2011, Volume: 68 Issue: 4, 241 - 252, 01.12.2011

Vedat Şekeroğlu Zülal Atlı Şekeroğlu

Abstract

Mikronükleus MN ’lar hücrenin mitoz bölünmesi sırasında ortaya çıkarlar ve esas çekirdeğe dâhil olmazlar. MN’ler tam kromozom veya asentrik kromozom parçalarından köken alan oluşumlardır.MN sayısındaki artış, somatik hücrelerdeki genomik kararsızlığın göstergesidir. MN testi, fiziksel ve kimyasal ajanların hücrelerde oluşturduğu genotoksik etkinin belirlenmesinde yaygın olarak kullanılan bir testtir. Bu test mitoz bölünme ile oluşan hemen hemen tüm hücre tipleri üzerinde in vitro ve in vivo olarak uygulanabilmekte ve kromozom anormallikleri testine göre daha kolay ve hızlı sonuç vermektedir. Kültürde bir kez bölünmesini tamamlamış binükleat hücrelerde MN frekansını saptayan ve sitokalasin-B ile sitokinezin bloklanmasına dayanan metodun gelişmesi ile MN testinin güvenilirliği ve geçerliliği artmıştır. MN testi aynı zamanda, in vitro çalışmalarda nükleer bölünme indeksi, in vivo çalışmalarda ise polikromatik eritrositler ile normokromatik eritrositler arasındaki oran kullanılarak sitotoksisitenin tahmin edilmesini de sağlamaktadır. İnsan ve diğer türler çevrelerinde bulunan çok sayıda farklı kimyasal ve fiziksel etkenlere maruz kalmaktadır. Bu nedenle kimyasal ve fiziksel faktörlerin potansiyel riskleri ve olumsuz etkilerini değerlendiren genotoksisite çalışmaları giderek daha çok önem kazanmaktadır. MN testi; fiziksel etkenlerin, ilaçların, çevresel kirleticiler ve gıda katkı maddeleri gibi günlük yaşamda sıklıkla maruz kaldığımız her türlü kimyasal maddenin genotoksik ve karsinojenik potansiyellerinin ve güvenilirliliklerinin araştırılmasını, kanser riskinin tahmin edilmesini ve kanserin izlenmesini sağlayan oldukça kullanışlı bir biyoizlem testidir. Basitliği, güvenirliliği, geçerliliği ve farklı hücre tiplerine uygulanabilirliği gibi avantajlara sahip olması nedeniyle yıllardır kullanılmakta olan MN testinin, gelecekte de mutajenitenin belirlenmesi ve önlenmesinde önemli bir rolü olacaktır. Bu derlemenin amacı hücrelerdeki genetik hasarın indirekt göstergesi olarak değerlendirilen MN tekniği hakkında bilgi vermektir

Keywords

Mikronükleus testi, kromozom hasarı, genotoksisite

Micronucleus test for determining genotoxic damage

Abstract

Micronuclei MN are formed during mitosis and do not integrate in the main nucleus. They may arise from a whole lagging chromosome or an acentric chromosome fragment. An increase of MN frequency indicates genomic instability. The MN test is commonly used to determine the genotoxic effects of chemical and physical agents on somatic cells. It is applied to all types of cells reproducing by mitosis in vitro and in vivo, and it is easier and faster to perform than the chromosome aberration assay. With the development of the in vitro cytochalasin-B block method for detecting MN frequency in binuclear cells, its reliability and validity has been increased. At the same time, the test enables an estimation of cytotoxicity using the frequency of nuclear division index in in vitro studies, and the ratio between polychromatic erythrocytes in in vivo studies. Human beings and other species are exposed to a large number of chemical and physical factors in their environment. Therefore, the genotoxic studies about the adverse effects and potential risks of those factors gain importance exponentially. The MN test is a practical bio-monitoring test that provides an investigation tool on genotoxic and carcinogenic potentials and reliability of physical agents, drugs and all types of other chemical such as pollutants, food additives to which people are exposed daily, and it helps to predict and monitor the cancer risk. The MN technique, kanserin izlenmesini sağlayan oldukça kullanışlı bir biyoizlem testidir. Basitliği, güvenirliliği, geçerliliği ve farklı hücre tiplerine uygulanabilirliği gibi avantajlara sahip olması nedeniyle yıllardır kullanılmakta olan MN testinin, gelecekte de mutajenitenin belirlenmesi ve önlenmesinde önemli bir rolü olacaktır. Bu derlemenin amacı hücrelerdeki genetik hasarın indirekt göstergesi olarak değerlendirilen MN tekniği hakkında bilgi vermektir

Keywords

Micronucleus technique, chromosome damage, genotoxicity

References

• 1. Vanparys P, Vermeiren F, Sysmans M, Temmerman R. The micronucleus assay as a test for the detection of aneugenic activitiy. Mutat Res, 1990; 244: 95-103.
• 2. Kirsch-Volders M, Elhajouji A, Cundari E, Van Hummelen P. The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. Mutat Res, 1997; 392(1-2): 19-30.
• 3. Stopper H, Müler OS. Micronuclei as a biological endpoint for genotoxicity: A Minireview. Toxicol In Vitro, 1997; 11: 661-7.
• 4. Choy WN. 2001. Genetic toxicology and cancer risk assessment. New York: Marcel Dekker, 2001: 163-86.
• 5. Demirel S, Zamani A. MN tekniği ve kullanım alanları. Genel Tıp Dergisi, 2002; 12(3): 123-7.
• 6. Yırtıcı Ü. Tartrazinin Cyprinus carpio’daki genotoksik etkisinin MN yöntemi ile araştırılması. Yüksek Lisans Tezi, Erciyes Üniversitesi Fen Bilimleri Enstitüsü, 2007.
• 7. Schmid W. The micronucleus test. Mutat Res, 1975; 31: 9-15.
• 8. Garewal HS, Ramsey L, Kaugars G, Boyle J. Clinical experience with the micronucleus assay. Cellular Biochem, 1993; 17: 206-12.
• 9. Cheng TJ, Christiani DC, Xu X, Wain JC, Wiencke JK, Kelsey KT. Increased micronucleus frequency in lymphocytes from smokers with lung cancer. Mutat Res, 1996; 349: 43-50.
• 10. Duffaud F, Orsiere T, Villani P, Pelissier AL, Volot F, Favre R et al. Comparison between micronucleated lymphocytes rates observed in healthy subject and cancer patients. Mutagenesis, 1997; 12: 227-31.
• 11. Fenech M. The in vitro micronucleus technique. Mutat Res, 2000; 455: 81-95.
• 12. Krishna G, Hayashi M. In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutat Res, 2000; 455: 155-66.
• 13. Widel M, Kolosza Z, Jedrus S, Lukaszczyk B, RaczekZwierzycka K, Swierniak A. Micronucleus assay in vivo provides significant prognostic information in human cervical carcinoma: The updated analysis. Int J Radiat Biol, 2001; 77: 631-6.
• 14. Von Ledebur MM, Schmid W. The micronucleus test: Methodological aspects. Mutat Res, 1973; 19: 109-17.
• 15. Heddle JA, Countryman RI. The production of micronuclei from chromosome aberration in irradiated cultures of human lymphocytes. Mutat Res, 1976; 41: 321-32.
• 16. Högstedt B, Karlsson A. The size of micronuclei in human lymphocytes varies according to inducing agent used. Mutat Res, 1985; 156: 229-32.
• 17. Eastmond DA, Tucker JD. Identification aneuploidy inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody. Environ Mol Mutagen, 1989; 13: 34-43.
• 18. Fenech M, Morley AA. Cytokinesis-block micronucleus method in human lymphocytes: Effect of in vivo agening and dose X-irradiation. Mutat Res, 1986; 161: 193-8.
• 19. Aardema JM, Kirsch-Volders M. The in vitro micronucleus assay. In Choy WN, eds. Genetic toxicology and cancer risk assesment. New York. Marcel Dekker, 2001; 163-86.
• 20. Titenko-Holland N, Windham G, Kolachana P, Reinisch F, Parvatham S, Osorio AM et al. Genotoxicity of malathion in human lymphocytes assessed using the micronucleus assay in vitro and in vivo: a study of malathion-exposed workers. Mutat Res, 1997; 388(1): 85-95.
• 21. Lorge E, Lambert C, Gervais V, Becourt-Lhote N, Delongeas L, Claude N. Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay. Toxicol Sci, 2007; 96(2): 214-7.
• 22. Surralles J, Xamena N, Creus A, Catalan J, Norppa H, Marcos R. Induction of micronuclei by five pyrethroid ınsecticides in whole-blood and isolated human lymphocyte cultures. Mutat Res, 1995; 341: 169-84.
• 23. Al-Sabti K. Comparative micronucleated erythrocyte cell induction in three cyprinids by five carcinogenicmutagenic chemicals. Cytobios, 1986; 47: 147-54.
• 24. Cotelle S, Masfaraud J, Ferard J. Assessment of the genotoxicity of contaminated soil with the Allium/Vicia-micronucleus and the Tradescantia micronucleus assays. Mutat Res, 1999; 426: 167-71.
• 25. Djomo JE, Ferrier V, Bekaert C. Amphibian micronucleus test in vivo (Jaylet Test) to evaluate the genotoxicity of petrochemical waste waters. Bull Environ Contam Toxicol, 2000; 65: 168-74.
• 26. Stich HF, Stich W, Parida BB. Elevated frequency of micronucleated cells in the buccal mucosa of individuals at high risk for oral cancer: Betel quid chewers. Cancer Lett, 1982; 17: 125-34.
• 27. Stich HF, Rosin MP. Micronuclei in exfoliated human cells as a tool for studies in cancer risk and intervention. Cancer Lett, 1984; 22: 241-53. 28. Rosin MP, Gilbert AM. Modulation of genotoxic effects in humans: Mutation and the environment. NewYork. Wiley-Liss, 1990; 51-9.
• 29. Rothfuss A, Schutz P, Bochum S, Volm T, Eberhardt E, Kreienberg R et al. Induced micronucleus frequencies in peripheral lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families. Cancer Res, 2000; 60: 390-4.
• 30. Kirsch-Volders M, Sofuni T, Aardemac M, Albertini S, Eastmond D, Fenech M et al. Report from the in vitro micronucleus assay working group. Mutat Res, 2003; 540: 153-63.
• 31. Rojas E, Herrera LA, Sordo M, Gonsebatt ME, Montero R, Rodriguez R et al. Mitotic index and cell proliferation kinetics for the identification of antineoplastic activity. Anti-Cancer Drug, 1993; 4: 637-40.
• 32. Anderson D, Jenkinson PC, Dewdeney RS, Franis AJ, Godbert P, Butterworth KR. Chromosome aberrations, mitogen-induced blastogenesis and proliferative rate index in peripheral lymphocytes from 106 control individuals of U.K. population. Mutat Res, 1988; 204: 407-20.
• 33. Lopez Nigro MM, Palermo AM, Mudry MD, Carballo MA. Cytogenetic evaluation of two nitroimidazole derivatives. Toxicol In Vitro, 2003; 17: 35-40.
• 34. Seligmann IC, Lima PD, Cardoso PC, Khayat AS, Bahia MO, Buchi DF et al. The anticancer homeopathic composite “Canova Method” is not genotoxic for human lymphocytes in vitro. Genet Mol Res, 2003; 2(2): 223-8.
• 35. Albert RE, Magee PS. The tumorigenicity of mutagenic contact sensitizing chemicals. Risk Anal, 2000; 20(3): 317-25.
• 36. Fenech M. The Advantages and disadvantages of the cytokinesis-block micronucleus method. Mutat Res, 1997; 392: 11-18.
• 37. Yavuz Kocaman A, Topaktaş M. In vitro evaluation of the genotoxicity of acetamiprid in human peripheral blood lymphocytes. Environ Mol Mutagen, 2007; 48: 483-90.
• 38. Heddle J. A rapid in vivo test for chromosome damage. Mutat Res, 1973; 18: 187-90.
• 39. Hayashi M, Mac Gregor JT, Gatehouse DG, Adler ID, Blakey DH, Dertinger S et al. In vivo rodent erythrocyte micronucleus assay: Aspects of protocol desing including repeated treatments, integration with toxicity testing, and automated scoring. A report from the international work shop on genotoxicity test procedures (IWGTP). Environ Mol Mutagen, 2000; 35: 234-52.
• 40. Üstün F. Albendazol'ün olası genotoksisitesi üzerine askorbik asitin etkisi. Doktora Tezi, İstanbul Üniversitesi Sağlık Bilimleri Enstitüsü, 2007.
• 41. Lambert IB, Singer TM, Boucher SE, Douglas GR. Detailed review of transgenic rodent mutation assays. Mutat Res, 2005; 590: 1-280.
• 42. MacGregor TJ, Heddle AJ, Hite M, Margolin HB, Ramel C, Salamone MF et al. Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes. Mutat Res, 1987; 189: 103-12.
• 43. Paulsson B, Kotova N, Grawé J, Henderson A, Granath F, Golding B et al. Induction of micronuclei in mouse and rat by glycidamide, genotoxic metabolite of acrylamide. Mutat Res, 2003; 535: 15-24.
• 44. Yener Y, Dikmenli M. Increased micronucleus frequency in rat bone marrow after acrylamide treatment. Food Chem Toxicol, 2009; 47 (8): 2120-3.
• 45. Cicchetti R, Bari M, Argentin G. Induction of micronuclei in bone marrow by two pesticides and their differentiation with CREST staining: an in vivo study in mice. Mutat Res, 1999; 439: 239-48.
• 46. Uma Devi P, Satish Rao BS, Kamath R. A method to score micronuclei in vivo using cytochalasin B-induced cytokinesis block. Mutat Res, 1998; 401: 33-7.
• 47. Şekeroğlu V. Thiacloprid ve deltamethrin insektisitlerinin tek başlarına ve karışım halinde kullanıldıkları zaman sıçan kemik iliği hücrelerinde in vivo genotoksik etkileri. Doktora Tezi, Ondokuz Mayıs Üniversitesi Fen Bilimleri Enstitüsü, 2010.
• 48. Lehucker-Michel MP, Di-Giorgio C, Amara YA, Laget M, Botta A. The micronucleus assay in human exfoliated urothelial cells: Effects of smoking. Mutagenesis, 1995; 10: 329-32.
• 49. Moore LE, Warner ML, Smith AH, Kalman D, Smith MT. Use of fluorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells. Environ Mol Mutagen, 1996; 27: 176-84.
• 50. Jagetia GC, Jayakrishnan A, Fernandes D, Vidyasagar MS. Evaluation of micronuclei frequency in the cultured peripheral blood lymphocytes of cancer patients before and after radiation treatment. Mutat Res, 2001; 491: 9-16.
• 51. Guzman P, Sotelo-Regil RC, Mohar A, Gonsebatt ME. Positive correlation between the frequence of micronucleated cells and dysplasia in papanicolaou smears. Environ Mol Mutagen, 2003; 41: 339-43.
• 52. Olaharski A, Sotelo R, Solorza-Luna G, Gonsebatt ME, Guzman P, Mohar A et al. Tetraploidy and chromosomal instability are early events during cervical carcinogenesis. Carcinogenesis, 2006; 27: 3317-43.
• 53. Wang Y, Hopwood VL, Hu P, Lennon A, Osterberger J, Glassman A. Determination of secondary chromosomal aberrations of chronic myelocytic leukemia. Cancer Genet Cytogenet, 2004; 153: 53-6.
• 54. Oliveira NG, Castro M, Rodrigues AS, Gonçalves IC, Cassapo R, Fernandes AP et al. Evaluation of the genotoxic effects of the boron neutron capture reaction in human melanoma cells using the cytokinesis block micronucleus assay. Mutagenesis, 2001; 16: 369-75.
• 55. Koyama S, Nakahara T, Wake K, Taki M, Isozumi Y, Miyakoshi J. Effects of high frequency electromagnetic fields on micronucleus formation in CHO-K1 cells. Mutat Res, 2003; 541: 81-9.
• 56. Simi S, Ballardin M, Casella M, De Marchi D, Hartwig V, Giovannetti G et al. Is the genotoxic effect of magnetic resonance negligible? Low persistence of micronucleus frequency in lymphocytes of individuals after cardiac scan. Mutat Res, 2008; 645: 39-43.
• 57. Hayashi M, Kishi M, Sofuni T, Ishidate MJr. Micronucleus tests in mice on 39 food additives and eight miscellaneous chemicals. Food Chem Toxicol, 1988; 26(6): 487-500.
• 58. Rosin MP, Anwar W. Chromosomal damage in urothelial cells from Egyptians with chronic Schistosoma haematobium infections. Int J Cancer, 1992; 50: 539-43.
• 59. Cruz AD, McArthur AG, Silva CC, Curado MP, Glickman BW. Human micronucleus counts are correlated with age, smoking, and cesium-137 dose in the Goiania (Brazil) radiological accident. Mutat Res, 1994; 313: 57-68.
• 60. Özkul Y, Dönmez H, Erenmemişoğlu A, Demirtaş H, İmamoğlu N. Introduction of micronuclei by smokeless tobacco on buccal smears of habitual users. Mutagen, 1997; 12: 470-9.
• 61. Çelik A, Mazmancı B, Çamlıca Y, Aşkın A, Çömelekoğlu Ü. Induction of micronuclei by lambda-cyhalothrin in Wistar rat bone marrow and gut epithelial cells. Mutagenesis, 2005; 20: 125-9.
• 62. Demsia G, Vlastos D, Goumenou M, Matthopoulos DP. Assessment of the genotoxicity of Imidacloprid and Metalaxyl in cultured human lymphocytes and rat bone-marrow. Mutat Res, 2007; 634(1–2): 32-9.
• 63. Huang Y, Gao H, Gou M, Ye H, Liu Y, Gao Y, et al. Acute toxicity and genotoxicity studies on poly (ε-caprolactone)-poly (ethyleneglycol)-poly (ε-caprolactone) nanomaterials. Mutat Res, 2010; 696: 101-6.
• 64. Al-Sabti K. Chlorotriazine reactive azo red 120 textile dye induces micronuclei in fish, Ecotox Environ Safe, 2000; 47: 149-55.