Hen’s Egg Test For Micronucleus Induction (HET-MN)

Öz

One of widely used methods in genotoxicity tests is micronucleus technique. Hen’s
Egg Test for Micronucleus Induction (HET-MN) is a member of relatively new of micronucleus tests.
HET-MN is an extremely simple, inexpensive and rapid genotoxicity assay which is positioned between
pure in vitro and in vivo analysis, strictly in line with animal protection regulations and ethical aspects.
Furthermore the high metabolic competency provided by this model enables metabolic activation of
xenobiotics including mutagens and promutagens. In this article, recent publications about the kinds of
micronucleus tests and then the application of HET-MN have been compiled  

Anahtar Kelimeler

• Chicken embryo,erythrocyte,genotoxicity,micronucleus

Tavuk Yumurtası Mikronükleus Testi

Öz

Genotoksisite testleri içinde çok yaygın kullanılan yöntemlerden biri mikronükleus tekniğidir.
Mikronükleus testleri içerisinde ise diğerlerine kıyasla daha yeni bir teknik Tavuk Yumurtası Mikronükleus
Testi (Hen’s Egg Test for Micronucleus Induction, HET-MN)’dir. HET-MN hayvan hakları ve etik
açıdan kesinlikle uygun, in vitro ve in vivo deneyler arasına yerleştirebileceğimiz son derece basit, ucuz
ve hızlı bir genotoksisite testidir. Ayrıca bu modelin sağladığı yüksek metabolik yetkinlik mutajen ve
promutajenleri içeren ksenobiyotiklerin metabolik aktivasyonuna da imkân tanımaktadır. Bu makalede
önce mikronükleus testlerinin çeşitleri, ardından HET-MN’nin uygulamasıyla ilgili güncel yayınlar
derlenmiştir  

Anahtar Kelimeler

• Eritrosit,genotoksisite,mikronükleus,tavuk embriyosu

Kaynakça

1. Alakoç C, 2010. Gazaltı kaynağından çıkan gazlara maruz kalan kişilerin periferal kan lenfositlerindeki mitotik indeks, replikasyon indeksi ve mikronükleus parametrelerinin değerlendirilmesi. Bozok Üniversitesi Fen Bilimleri Enstitüsü, Yüksek Lisans Tezi, Yozgat.
2. Al-Sabti K, Metcalfe CD, 1995. Fish micronuclei for assessing genotoxicity in water. Mutation Research, 343: 121-135.
3. Al-Sabti K, 1986. Comparative micronucleated erythrocyte cell induction in three cyprinids by fve carcinogenic-mutagenic chemicals. Cytobios, 47: 147-154.
4. Aydın SA, 2007. Fluniksin megluminin genotoksisitesinin in vitro ve in vivo/in vitro mikronükleus testleriyle değerlendirilmesi. İstanbul Üniversitesi Sağlık Bilimler Enstitüsü, Doktora Tezi, İstanbul.
5. Ayllon F, Garcia-Vazquez E, 2000. Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fsh micronucleus test. Mutation Research, 467: 177-186.
6. Aymak C, 2010. Mersin bölgesinde yaşayan Rana ridibunda pallas, 1771 (Ranidae, Ampibia)’nın mikronükleus test yöntemi ile genotoksik etkilerinin ve ağır metal kirliliğinin tespit edilmesi. Mersin Üniversitesi Fen Bilimleri Enstitüsü, Doktora Tezi, Mersin.
7. Bhunya SP, Jena GB, 1993. Studies on the genotoxicity of monocrotophos, an organophosphate insecticide, in the chick in vivo test system. Mutation Research, 292: 231-239.
8. Bhunya SP, Jena GB, 1996. Clastogenic effects of copper sulphate in chick in vivo test system. Mutation Research, 367 (2): 57- 63.

9. Bhunya SP, Jena GB, 1992. Genotoxic potential of the organochlorine insecticide lindane (γ-BHC): An in vivo study in chicks. Mutation Research, 272: 175-181.
10. Bruns GAP, Ingram VM, 1973. The erythroid cells and haemoglobins of the chick embryo. Philosophical Transactions of the Royal Society, 266: 225-305.
11. Campana MA, Panzeri AM, Moreno VJ, Dulout FN, 2003. Micronuclei induction in Rana catesbeiana tadpoles by the pyrethroid insecticide lambda-chalothrin. Genetics and Molecular Biology, 26: 99- 103.
12. Choy WN, 2001. Genetic Toxicology and Cancer Risk Assessment, Marcel Dekker, New York, 406 p.
13. Cunny H, Hodgson E, 2004. Toxicity Testing, In: Hodgson, E. (ed.), A Textbook of Modern Toxicology, John Wiley & Sons, Inc., USA, p. 353-397.
14. Çakır Ş, 2004. Bazı organik fosforlu insektisitlerin Drosophila melanogaster’in yaşama yüzdesi üzerine etkisi. Gazi Üniversitesi Gazi Eğitim Fakültesi Dergisi, 24(3): 71-80.
15. Çavaş T, Ergene-Gözükara S, 2003. Micronuclei, nuclear lesions and interphase silver-stained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill effluent. Mutation ResearchGen Tox En, 538: 81-91.
16. Çavaş T, 2004. Endüstriyel atıkların genotoksik etkilerinin mikronükleus ve AgNOR analiz teknikleri kullanılarak insitu ve laboratuvar koşulları altında araştırılması. Mersin Üniversitesi Fen Bilimleri Enstitüsü, Doktora Tezi, Mersin.
17. Çavuşoğlu K, Yalçın E, Dönmez S, Kaymaz K, Özdemir G, Özgörür Z, Balcı D, Aslan B, Çakır M. 2008. Vicia faba L. (Fabaceae) kök ucu hücrelerinde fenol tarafından teşvik edilen sitotoksisitenin belirlenmesi. SDÜ Fen Dergisi (E-Dergi) 3(2): 139-148.
18. Demirel S, Zamani A. 2002. MN tekniği ve kullanım alanları. Genel Tıp Dergisi, 12(3): 123-127.
19. Duffaud F, Orsiere T, Villani P, Pelissier AL, Volot F, Favre R, 1997. Comparison between micronucleated lymphocytes rates observed in healthy subject and cancer patients. Mutagenesis, 12: 227-231.
20. Ergene Gözükara S, Çavaş T, Aymak C, 2001. Cypermethrin’in Oreochromis niloticus (L.,1758) üzerindeki genotoksik etkilerin mikronükleus testi ile araştırılması. XI. Ulusal Su Ürünleri Sempozyumu, 2: 765-771.
21. Ergene S, Kaya Ş, Dürgen A, Uçar A, Erkek M, Ergene M, Könen AS, Özbaba O, Önder H, 2012. Mersin rehabilitasyon merkezinde tedavi altında bulunan yaralı deniz kaplumbağalarının hematolojik ve biyokimyasal özelliklerinin incelenmesi, araştırma makalesi. Anadolu Doğa Bilimleri Dergisi 3(2): 34- 40.
22. Fenech M, Chang WP, Kirsch-Volders M, Holland N, Bonassi S, Zeiger E, 2003. Human Micronucleus Project. HUMN Project: Detailed description of the scoring criteria for the cytokinesis– block micronucleus assay using isolated human lymphocyte cultures. Mutation Research-Gen Tox En, 534: 65-75.
23. Fenech M, 2000. The in vitro micronucleus technique. Mutation Research-Fund Mol, 435: 81-95.
24. Fenech M, Crott J, Turner J, Brown S, 1999. Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: Description of the method and results for hydrogen peroxide. Mutagenesis, 14(6): 605-612.
25. Fenech M, Morley AA, 1985. Measurement of micronuclei in lymphocytes. Mutation Research, 147: 29-36.
26. Giri S, Sharma GD, Giri A, Prasad SB, 2002. Genotoxic effects of malathion in chick in vivo micronucleus assay. Cytologia, 67: 53-59.
27. Gomez-Arroyo S, Díaz-Sánchez Y, Meneses-Pérez MA, VillalobosPietrini R, De León-Rodríguez J, 2000. Cytogenetic biomonitoring in a mexican floriculture worker group exposed to pesticides. Mutation Research, 466: 117-124.
28. GPT, 2006. Micronucleus assay. GenPharmTox. http://www. genpharmtox.com/ services/ Toxicology/micro_advanced_ test.html (28.05.2012)
29. Greywe D, Kreutz J, Banduhn N, Krauledat M, Scheel J, Schroeder KR, Wolf T, Reisinger K, 2012. Applicability and robustness of the hen’s egg test for analysis of micronucleus induction (HET-MN): Results from an inter-laboratory trial. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 747(1): 118-134.
30. Griffths AJF, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM, 2000. An Introduction to Genetic Analysis. W.H. Freeman and Company, New York, USA.
31. Hamilton JV, Denison MS, Bloom SE, 1983. Development of basal and induced aryl hydrcarbon (benzo(a)pyrene) hydroxylase activity in the chick embryo in ovo. Proceedings of the National Academy of Sciences of the United States of America, 80: 3372-3376.
32. Heddle JA, Cimino MC, Hayashi M, Romagna F, Shelby MD, Tucker JD, Vanparys P, MacGregor JT, 1991. Micronuclei as an index of cytogenetic damage: Past, Present, and, future. Environmental and Molecular Mutagenesis, 18: 277-291.
33. Hothorn LA, Reisinger K, Wolf T, Albrecht P, Fieblinger D, Liebsch M, Pirow R, 2013. Statistical analysis of the hen’s egg test for micronucleus induction (HET-MN assay). Mutation Research, 757: 68-78.
34. Jelinek R, 1977. The chick embryotoxicity screening test (CHEST). In: Methods in Prenatal Toxicology. Neubert, D., Merker, H.J. and Kwasigrooh, T.E. (eds.), Georg Thieme, Stutgart, pp. 381-386.
35. Jena GB, Bhunya SP, 1994. Mutagenicity of an organophosphate insecticide acephate-an in vivo study in chicks. Mutagenesis 9 (4): 319-324.
36. Jena GB, Bhunya SP, 1992. Thirty day genotoxicity study of an organophosphate insecticide, monocrotophos, in a chick in vivo test system. In vivo 6(5): 527-530.
37. Jena GB, Kaul CL, Ramarao P, 2002. Genotoxicity testing, a regulatory requirement for drug discovery and development: impact of ICH guidelines. Indian Journal of Pharmacology, 34: 86-99.
38. Kemper FH, Luepke NP, 1986. Toxicity testing by the hen’s egg test (HET). Food Chemical Toxicology, 24(6/7): 647-648.
39. Kirsch-Volders M, Fenech M, 2001. Inclusion of micronuclei in non-divided mononuclear lymphocytes and necrosis/apoptosis may provide a more comprehensive cytokinesis block micronucleus assay for biomonitoring purposes. Mutagenesis, 16(1): 51-58.
40. Kirsch-Volders M, Elhajouji A, Cundari E, Van Hummelen P, 1997. The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction. Mutation Research, 392(1-2): 19-30.
41. Könen S, 2007. Trifluralin ve askorbik asit kombinasyonlarının Oreochromis niloticus üzerindeki genotoksik ve antigenotoksik etkilerinin mikronükleus testi kullanılarak araştırılması. Mersin Üniversitesi Fen Bilimleri Enstitüsü, Yüksek lisans Tezi, Mersin.
42. Laffon B, Pasaro E, Mendez J, 2001. Genotoxic effects of styrene- 7,8- oxide in human white blood cells: comet assay in relation to the induction of sister-chromatid exchanges and micronuclei. Mutation Research, 491:163-172.
43. Lucas AM, Jamroz C, 1961. Atlas of Avian Hematology. Agriculture Monograph 25, United States Department of Agriculture, U.S. Government Printing Offce, Washington, DC. Luepke NP, 1985. Hen’s egg chorioallantoic membrane test for irritation potential. Food Chemical Toxicology, 23: 287-291.
44. MacGregor TJ, Heddle AJ, Hite M, Margolin HB, Ramel C, Salamone MF, 1987. Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes. Mutation Research, 189: 103-12.
45. Marques SM, Antunes SC, Pissarra H, Pereira ML, Gonçalves F, Pereira R, 2009. Histopathological changes and erythrocytic nuclear abnormalities in Iberian green frogs (Rana perezi Seoane) from a uranium mine pond. Aquatic Toxicology, 91(2): 187-195.
46. Mateuca R, Lombaert N, Aka PV, Decordier I, Kirsch-Volders M, 2006. Chromosomal changes: Induction, detection, methods and applicability in human biomonitoring. Biochimie, 88: 1515-31.
47. Matson CW, Palatnikov G, Islamzadeh A, Mcdonald TJ, Autenrieth RL, Donnelly KC, Bickham JW, 2005. Chromosomal damage in two species of aquatic turtles (Emys orbicularis and Mauremys caspica) inhabiting contaminated sites in Azerbaijan. Ecotoxicology, 14(5): 513-525.
48. Mavournin HK, Blakey HD, Cimino CM, Salamone FM, Heddle AJ, 1990. The in vivo micronucleus assay in mammalian bone marrow and peripheral blood: A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research, 239: 29-80.
49. Monarca S, Feretti D, Zanardini A, Moretti M, Villarini M, Spiegelhalder B, Zerbini I, Gelatti U, Lebbolo E, 2001. Monitoring airborne genotoxicants in the rubber industry using genotoxicity tests and chemical analyses. Mutation Research, 490: 159-169.
50. Müller W-U, Streffer C, 1994. Micronucleus assays. In: Advances in Mutagenesis Research. Obe, G. (ed.), Vol 5, SpringerVerlag, pp. 1-134.
51. Neumann NJ, Hölzle E, Lehmann P, Rosenbruch M, Klaucic A, Plewig G, 1997. Photo hen’s egg test: A model for phototoxicity. British Journal of Dermatology, 136: 326-330.
52. Nishigori H, Mizuura M and Iwatsuru M, 1992. The hen’s fertile egg screening test (HEST): A comparison between the acute toxicity for chick embryos and rodents of 20 drugs. Cell Biology and Toxicology, 8 (4): 255-265.
53. Öcal A, 2012. İnsan periferik kan lenfositlerinde Hypericum heterophyllum vent. türünün mikronükleus, mitotik indeks ve replikasyon indeksi üzerine etkileri. Bozok Üniversitesi Fen Bilimleri Enstitüsü, Yüksek Lisans Tezi, Yozgat.
54. Özdemir F, Kayaaltı Z, Kaya-Akyüzlü D, 2015. Ksenobiyotiklerin DNA üzerindeki toksik etkileri ve toksikogenetik. Marmara Pharmaceutical Journal, 19: 246-251.
55. Özparlak H, 2006. Yumurtaya verilen organik insektisit fpronilin tavukların embriyonik ve kuluçka sonu erken dönem gelişimi üzerindeki zararlı etkilerinin belirlenmesi. Selçuk Üniversitesi Fen Bilimleri Enstitüsü, Doktora Tezi, Konya.
56. Özparlak H, 2008. Organik insektisit fpronilin genotoksik etkilerinin tavuk yumurtası mikronukleus testi ile belirlenmesi. 19. Ulusal Biyoloji Kongresi Özet Kitabı (23-27 Haziran, Trabzon), 561-562.
57. Özparlak H, 2015. Tavuk embriyolarının embriyotoksisite testlerinde kullanımı. Selçuk Üniversitesi Fen Fakültesi Fen Dergisi, 40: 13-22.
58. Özparlak H, Arslan A, Güler GÖ, 2011. Organik insektisit fpronilin genotoksik etkilerinin civciv mikronukleus test sisteminde belirlenmesi. Selçuk Üniversitesi Fen Fakültesi Fen Dergisi, 37: 1-8.
59. Parlak H, Arslan ÖÇ, Boyacıoğlu M, Karaaslan MA, 2011. Ekotoksikoloji. Ege Üniversitesi Basımevi, İzmir, 339 s.
60. Preston W, Bender AMA, Brewen JG, Carrano AV, Heddle JA, McFee AF, Wolff S, Wassom JS, 1981. Mammalian in vivo and in vitro cytogenetic assays: A report of the U.S. EPA’s Gene-Tox Program. Mutation Research, 87: 143-88.
61. Purchase IFH, Longstaff E, Ashby J, Styles JA, Anderson D, Lefevre PA, Westwood FR, 1978. An evaluation of 6 shortterm tests for detecting organic chemical carcinogens. British Journal of Cancer, 37: 873-959.
62. Rajaguru P, Fairbairn LJ, Ashby J, Willington MA, Turner S, Woolford LA, Chinnasamy N, Rafferty JA, 1999.
63. Genotoxicity studies on the azo dye direct red 2 using the in vivo mouse bone marrow micronucleus test. Mutation Research, 444: 175-180.
64. Rosenbruch M, Holst A, 1990. The chick embryo yolk-sac blood vessel system as an experimental model for irritation and inflammation. Toxicology In Vitro, 4(4/5): 327-331.
65. Savage JRK, 2000. Micronuclei: Pitfalls and problems. Atlas of Genetics and Cytogenetics in Oncology and Haematology.
66. http://www.infobiogen.fr/ services/chromcancer/Deep/ MicronucleiID20016.html (28.05.2012)
67. Shimizu N, Itoh H, Utiyama H, Wahl GM, 1998. Selective entrapment of extrachromosomally amplifed DNA by nuclear budding and micronucleation during S phase. The Journal of Cell Biology, 140: 1307-1320.
68. Stopper H, Müler SO, 1997. Micronuclei as a biological endpoint for genotoxicity: A Minireview. Toxicology In Vitro, 11: 661- 667.
69. USEPA, 1996. In vivo mammalian cytogenetics test: Erythrocyte micronucleus assay. Health effects test guidelines EPA OPPTS 870. 5395. U.S. Environmental Protection Agency, Washington, USA. http://www.epa.gov/opptsfrs/publications/ OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/ Series/870-5395.pdf (28.05.2012)
70. Üstün F, 2007. Albendazol’ün olası genotoksisitesi üzerine askorbik asitin etkisi. İstanbul Üniversitesi Sağlık Bilimleri Enstitüsü, Doktora Tezi, İstanbul.
71. Vanparys P, Vermeiren V, Sysmans M, Temmerman R, 1990. The micronucleus assay as a test for the detection of aneugenic activitiy. Mutation Research, 244: 95-103.
72. Von Ledebur MM, Schmid W, 1973. The micronucleus test: Methodological aspects. Mutation Research, 19: 109-17.
73. Vural N, 2005. Toksikoloji. Ankara Üniversitesi Eczacılık Fakültesi Yayınları, Ankara, 659 s.
74. Waters MD, Stack HF, Brady AL, Lohman PHM, Haroun L, Vainio H, 1988. Use of computerized data listings and activity profles of genetic and related efects in the review of 195 compounds. Mutation Research, 205: 295- 312.
75. Wolf T, Luepke NP, 1997. Formation of micronuclei in incubated hen’s eggs as a measure of genotoxicity. Mutation ResearchGen Tox En, 394: 163-175.
76. Wolf T, Niehaus-Rolf C, Luepke N-P, 2002. Some new methodological aspects of the hen’s egg test for micronucleus induction (HET-MN). Mutation Research-Gen Tox En, 514: 59-76.
77. Wolf T, Niehaus-Rolf C, Luepke N-P, 2003. Investigating genotoxic and hematotoxic effects of N-nitrosodimethylamine, N-nitrosodiethylamine and N-nitrosodiethanolamine in the hen’s egg-micronucleus test (HET-MN). Food and Chemical Toxicology, 41: 561-573.
78. Wolf T, Niehaus-Rolf C, Banduhn N, Eschrich D, Scheel J, Luepke N-P, 2008. The hen’s egg test for micronucleus induction (HET-MN): Novel analyses with a series of well-characterized substances support the further evaluation of the test system. Mutation Research, 650: 150-164.
79. Yırtıcı Ü, 2007. Tartrazinin Cyprinus carpio’daki genotoksik etkisinin mikronükleus yöntemiyle araştırılması. Erciyes Üniversitesi Fen Bilimler Enstitüsü, Yüksek Lisans Tezi, Kayseri.
80. Zeiger E, 2004. History and rationale of genetic toxicology testing: an impersonal, and sometimes personal. Environmental Molecular Mutagenesis, 44: 363-371.