Yumurtaya Verilen Siklofosfamid ve C Vitamini’nin Tavuk Embriyoları Üzerindeki Bazı Etkileri

Year 2018, Volume: 44 Issue: 2, 157 - 173, 11.10.2018

Haluk Özparlak , Bülent Çelik Döndü Balta

Abstract

Literatürdeki
in vivo ve in vitro ortamda gerçekleştirilen pek çok
genotoksisite-antigenotoksisite çalışmasında genotoksik bir madde olarak
Siklofosfamid (CP) ve antigenotoksik bir madde olarak C vitamini (Askorbik
Asit, AA) yaygın olarak kullanılmaktadır. Bunlar alternatif bir mikronükleus
testi olan tavuk yumurtası mikronükleus testinde (Hen’s Egg Test for Analysis
of Micronucleus Induction, HET-MN) de 50 µg/yumurta (50 µg/y) dozunda
kullanılmaktadır. Bu çalışmayla 50 µg/y dozunda CP ve AA’nın tavuk embriyoları
üzerindeki bazı etkilerinin ilk kez tespit edilmesi amaçlandı. Çalışma için
döllü tavuk yumurtalarından oluşan dört grup oluşturuldu. CP, AA, CP+AA
birlikte ve steril bidistile su (kontrol olarak) gruplardaki yumurtaların hava
kamarasına kuluçkanın 8. gününde enjekte edildi. Kuluçkanın 11. gününde her bir
grubun ölü ve anormal embriyo oranları, malformasyon tipleri, canlı ve rölatif
embriyo oranları tespit edildi. Ayrıca kemik gelişiminin belirlenebilmesi için
embriyoların bir kısmı total olarak Alizarin Red-S yöntemiyle boyandı. Çalışma
iki tekrarlı olarak gerçekleştirildi. İki denemenin sonucunun birbiriyle uyumlu
olduğu tespit edildi.  Sonuç olarak, test
edilen dozda CP, AA ve CP+AA’nın önemli bir embriyotoksik veya teratojenik etki
göstermediği ve embriyoların kemik gelişimini makroskobik düzeyde etkilemediği
belirlendi. 

Keywords

Alizarin Red-S, Askorbik asit, Siklofosfamid, Tavuk Embriyosu

Some Effects of Cyclophosphamide and Vitamin C Given in ovo on Chicken Embryos

Abstract

In the
literature, Cyclophosphamide (CP) as a genotoxic substance and vitamin C
(Ascorbic Acid, AA) as an antigenotoxic substance have been used extensively in
many genotoxicity-antigenotoxicity studies conducted in vivo and in vitro, and
these substances at doses of 50 µg/egg are also used in Hen’s Egg Test for
Analysis of Micronucleus Induction (HET-MN) which is an alternative
micronucleus test. The aim of this study was to determine the some effects of
CP and AA at 50 µg/egg dose on the development of chicken embryos for the first
time. Four groups were formed from the fertilized hen's eggs. CP, AA and CP+AA
together, and sterile bidistilled water (as a control) were injected into the
air sac of eggs in groups at 8th day of incubation. Following parameters of
each group were examined on 11th day of the incubation: rates of dead and
abnormal embryo, malformation types, live and relative embryo weights. In
addition, some of the embryos were totally stained with Alizarin Red-S method
for bone development. Two trials were conducted in the study. The results of
the two trials were compatible with each other. As a result, CP, AA and CP+AA
together at the examined doses did not present significant embryotoxic and
teratogenic effects on chick embryos. They also did not affect the bone
development of chicken embryos at the macroscopic level.

Keywords

Alizarin Red-S, Ascorbic Acid, Cyclophosphamide, Chick Embryo

References

• Ahmad MS, Afzal M (2004). Amelioration of genotoxic damage by certain phytoproducts in human lymphocyte cultures. Chemico-Biological Interactions 149: 107–115.
• Akpolat M, Tarladaçalışır YT, Kanter M (2008). İyonizan radyasyonun neden olduğu ince bağırsak hasarına karşı curcumin ve C vitamininin koruyucu etkilerinin incelenmesi. Tıp Araştırmaları Dergisi 6: 77–85.
• Ataya KM, Valeriote FA, Ramahi-Ataya AJ (1989). Effect of cyclophosphamide on the immature rat ovary. Cancer Research 49: 1660–1664.
• Balta D (2017). Kültür Ganoderma lucidum (Curtis) P. Karst’ın genotoksik-antigenotoksik etkilerinin tavuk yumurtası mikronükleus testiyle belirlenmesi. Selçuk Üniversitesi Fen Bilimleri Enstitüsü Yüksek Lisans Tezi, Konya.
• Blom J, Tamarkin L, Shiber J, Nelson R (1995). Learned immunosuppression is associated with an increased risk of chemically-induced tumors. Neuroimmunomodulation 2: 92–99.
• Bramwell VH, Mouridsen H, Santoro A, Blackledge G, Somers R, Verwey J, Dombernowsky P, Onsrud M, Thomas D, Sylvester R (1987). Cyclophosphamide versus ifosfamide: final report of a randomized phase II trial in adult soft tissue sarcomas. European Journal of Cancer and Clinical Oncology 23: 311–321.
• Brown LP, Flint OP, Orton TC, Gibson GG (1986). Chemical teratogenesis: testing methods and the role of metabolism. Drug Metabolism Reviews 17: 221–260.
• Budai P, Fejes S, Várnagy L, Somlyay I, Takács I (2001). Teratogenicity test of dimethoate containing insecticide formulation and heavy elements (Cu, Cd) in chicken embryos after administration as single compounds or in combination. Mededelingen (Rijksuniversiteit te Gent. Fakulteit van de Landbouwkundige en Toegepaste Biologische Wetenschappen) 66: 885–889.
• Budai P, Fejes S, Várnagy L, Szabo R, Keserü M (2002). Embryonic toxicity of a dimethoate containing insecticide formulation and Cu-sulphate in chicken after individual or combined administration. Mededelingen (Rijksuniversiteit te Gent. Fakulteit van de Landbouwkundige en Toegepaste Biologische Wetenschappen) 67: 99–103.
• Campana MA, Panzeri AM, Moreno VcJ, Dulout FN (1999). Genotoxic evaluation of the pyrethroid lambda-cyhalothrin using the micronucleus test in erythrocytes of the fish Cheirodon interruptus interruptus. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 438: 155–161.
• 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 Research/Genetic Toxicology and Environmental Mutagenesis 538: 81–91.
• Çavaş T, Ergene-Gözükara S (2005). Induction of micronuclei and nuclear abnormalities in Oreochromis niloticus following exposure to petroleum refinery and chromium processing plant effluents. Aquatic Toxicology 74: 264–271.
• Çavaş T, Könen S (2007). 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: 263–268.
• Çelik B (2016). Türkiye’deki yabani Ganoderma lucidum (Curtis) P. Karst’ın genotoksik-antigenotoksik etkilerinin tavuk yumurtası mikronükleus testiyle belirlenmesi. Selçuk Üniversitesi Fen Bilimleri Enstitüsü Yüksek Lisans Tezi, Konya.
• Çelik I, Oguz H, Demet O, Boydak M, Donmez H, Sur E, Nizamlioglu F (2000). Embryotoxicity assay of aflatoxin produced by Aspergillus parasiticus NRRL 2999. British Poultry Science 41: 401–409.
• Ehrenfried JA, Ko TC, Thompson EA, Evers BM (1997). Cell cycle-mediated regulation of hepatic regeneration. Surgery 122: 927–935.
• Fairchild W, Spence C, Solomon H, Gangai M (1979). The incidence of bladder cancer after cyclophosphamide therapy. The Journal of Urology 122: 163.
• Fejes S, Budai P, Varnagy L, Molnar T, Szabo R, Fancsi T (2002). Toxicity of a mancozeb containing fungicide formulation and Cu-sulphate to chicken embryos after administration as single compounds or in combination. Mededelingen (Rijksuniversiteit te Gent. Fakulteit van de Landbouwkundige en Toegepaste Biologische Wetenschappen) 67: 105–109.
• Furuta K, Kakita A, Takahashi T, Tomiya T, Fujiwara K (2000). Experimental study on liver regeneration after simultaneous partial hepatectomy and pancreatectomy. Hepatology Research 17: 223–236.
• Goncharova R, Kuzhir T (1989). A comparative study of the antimitagenic effects of antioxidants on chemical mutagenesis in Drosophila melanogaster. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 214: 257–265.
• 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: 118–134.
• Guha B, Das JK, Khuda-Bukhsh AR (2007). Ameliorative effects of vitamin supplementation on ethyl methane sulphonate-induced genotoxicity in a fish, Anabas testudineus. Ecotoxicology and Environmental Safety 68: 63–70.
• Guha B, Khuda-Bukhsh A (2002). Efficacy of vitamin-C (L-ascorbic acid) in reducing genotoxicity in fish (Oreochromis mossambicus) induced by ethyl methane sulphonate. Chemosphere 47: 49–56.
• Hamburger V, Hamilton HL (1951). A series of normal stages in the development of the chick embryo. Journal of Morphology 88: 49–92.
• Hamilton JW, Denison MS, Bloom SE (1983). Development of basal and induced aryl hydrocarbon (benzo [a] pyrene) hydroxylase activity in the chicken embryo in ovo. Proceedings of the National Academy of Sciences 80: 3372–3376.
• Hartman PE, Shankel DM (1990). Antimutagens and anticarcinogens: a survey of putative interceptor molecules. Environmental and Molecular Mutagenesis 15: 145–182.
• Jelinek R (1977). The chick embryotoxicity screening test (CHEST). Methods in Prenatal Toxicology 381–386.
• Jelinek R, Peterka M, Rychter Z (1985). Chick embryotoxicity screening test-130 substances tested. Indian Journal of Experimental Biology 23: 588.
• Jiraungkoorskul W, Sahaphong S, Kosai P, MyungHuk K (2010). Micronucleus test: the effect of ascorbic acid on cadmium exposure in fish (Puntius altus). Research Journal of Environmental Toxicology 4: 103–112.
• Kaya B (2003). Anti-genotoxic effect of ascorbic acid on mutagenic dose of three alkylating agents. Turkish Journal of Biology 27: 241–246.
• Kaya B, Creus A, Velázquez A, Yanikoğlu A, Marcos R (2002). Genotoxicity is modulated by ascorbic acid: studies using the wing spot test in Drosophila. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 520: 93–101.
• Kemper F, Luepke N (1986). Toxicity testing by the hen's egg test (HET). Food and Chemical Toxicology 24: 647–648. Kinlen L, Peto J, Doll R, Sheil A (1981). Cancer in patients treated with immunosuppressive drugs. British Medical Journal (Clinical research ed.) 282: 474.
• Könen S (2007). Trifluralin ve askorbik asit kombinasyonlarının Oreochromis niloticus üzerindeki genotoksik ve antigenotoksik etkilerinin mikronükleus testi kullanılarak araştırılması. Yüksek Lisans Tezi, Mersin Üniversitesi Fen Bilimleri Enstitüsü Biyoloji Ana Bilim Dalı, Mersin.
• Kumar K, Kuttan R (2005). Chemoprotective activity of an extract of Phyllanthus amarus against cyclophosphamide induced toxicity in mice. Phytomedicine 12: 494–500.
• Landino LM, Koumas MT, Mason CE, Alston JA (2006). Ascorbic acid reduction of microtubule protein disulfides and its relevance to protein S-nitrosylation assays. Biochemical and Biophysical Research Communications 340: 347–352.
• Mauldin JM (1993). Quality control procedures for the hatchery. Poultry Science 93: 1-24.
• Moore F, Urda G, Krishna G, Theiss J (1995). An in vivo/in vitro method for assessing micronucleus and chromosome aberration induction in rat bone marrow and spleen 1. Studies with cyclophosphamide. Mutation Research/Environmental Mutagenesis and Related Subjects 335: 191–199.
• Nishigori H, Mizumura M, 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: 255–265.
• Özcan M (1992). Hidrokinon’un (HK) gelişim toksisitesinin döllenmiş tavuk embriyosunda analiz ve değerlendirilmesi. Yüksek Lisans Tezi, GÜ Sağlık Bilimleri Enstitüsü, Ankara.
• Öznurlu Y (2003). Yumurtaya verilen Aflatoksin B1’in, etçi piliçlerin kemik dokularının embriyonik gelişimi üzerindeki etkilerinin belirlenmesi. SÜ Sağlık Bilimleri Enstitüsü, Doktora Tezi, Konya.
• Özparlak H (2006). Yumurtaya verilen organik insektisit fipronil'in tavukların embriyonik ve kuluçka sonu erken dönem gelişimi üzerindeki zararlı etkilerinin belirlenmesi. Doktora Tezi, Selçuk Üniversitesi Fen Bilimleri Enstitüsü, Konya.
• Özparlak H (2015). Tavuk embriyolarının embriyotoksisite testlerinde kullanımı. Selçuk Üniversitesi Fen Fakültesi Fen Dergisi 40: 13–22.
• Özparlak H, Aslan A, Güler GÖ (2011). Organik insektisit fipronil’in genotoksik etkilerinin civciv mikronukleus test sisteminde belirlenmesi. Selçuk Üniversitesi Fen Fakültesi Fen Dergisi 2: 1–8.
• Perini P, Calabrese M, Rinaldi L, Gallo P (2008). Cyclophosphamide-based combination therapies for autoimmunity. Neurological Sciences 29: 233–234.
• Prelusky D, Hamilton R, Foster B, Trenholm H, Thompson B (1987). Optimization of chick embryotoxicity bioassay for testing toxicity potential of fungal metabolites. Journal-Association of Official Analytical Chemists 70: 1049–1055.
• Premkumar K, Bowlus CL (2003). Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 542: 99–103.
• Romanoff A (1997). Life in Twenty-one Days. Extention Bulletin, 205. Senthilkumar S, Yogeeta SK, Subashini R, Devaki T (2006). Attenuation of cyclophosphamide induced toxicity by squalene in experimental rats. Chemico-Biological Interactions 160: 252–260.
• Sur E (2001). Yumurtaya verilen Aflatoksin B1 (AFB1)’in Tavukların Lenfoid Organlarının Embriyonal Gelişimi Üzerindeki Etkilerinin Enzim Histokimyasal Yöntemlerle Araştırılması. Doktora Tezi, SÜ Sağlık Bilimleri Enstitüsü, Konya.
• Surjyo B, Anisur K (2004). Protective action of an anti-oxidant (L-Ascorbic acid) against genotoxicityand cytotoxicity in mice during p-DAB-induced hepatocarcinogenesis. Indian Journal of Cancer 1: 72–80.
• Tamayo Arango LJ, Suárez Avendaño PA, Cano Valderrama AI, Martínez C, Brayian A, Ciro Y, Sergio A, Mejía Giraldo CA, Lenis Sanin YY (2012). Didactic model of the chicken embryo development using modified Dawson's diaphanization and staining technique. Revista Colombiana de Ciencias Pecuarias 25: 620–624.
• Ural M, Özgüner M, Şenal D, Sütçü R, Delibaş N (2005). Siklosporin A'nın sıçanlarda oluşturduğu nefrotoksisiteye Vitamin C ile Vitamin E'nin ve verapamilin etkilerinin ışık mikroskobunda değerlendirilmesi. SDÜ Tıp Fakültesi Dergisi 12: 28–35.
• Varga T, Cravedi J, Fuzesi I, Varnagy L (2002). Residues of fenitrothion in chick embryos following exposure of fertile eggs to this organophosphorus insecticide. Revue De Medecine Veterinaire 153: 275–278.
• Varga T, Hlubik I, Várnagy L, Budai P, Molnár E (1999). Embryonic toxicity of insecticide Sumithion 50 EC and herbicide Fusilade S in pheasants after individual or combined administration. Acta Veterinaria Hungarica 47: 123–128.
• Varnagy L (1999). Degradation of some pesticides in avian embryos. Acta Veterinaria Hungarica 47: 117–122.
• Varnagy L, Budai P, Molnar E, Füzesi I, Fáncsi T (2001). Teratogenicity testing of BI 58 EC (38% dimethoate) in chicken embryos with special respect to degradation of the active ingredient. Acta Veterinaria Hungarica 49: 355–361.
• Watanabe M, Yamaguchi K, Chijiiwa K, Tanaka M (2001). FR167653 improves survival and pulmonary injury after partial hepatectomy under ischemia/reperfusion in rats. Journal of Surgical Research 101: 146–151.
• Wolf T, Luepke N-P (1997). Formation of micronuclei in incubated hen's eggs as a measure of genotoxicity. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 394: 163–175.
• 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/Genetic Toxicology and Environmental Mutagenesis 650: 150–164.
• 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/Genetic Toxicology and Environmental Mutagenesis 514: 59–76.
• 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.
• Wyttenbach CR, Thompson SC (1985). The effects of the organophosphate insecticide malathion on very young chick embryos: malformations detected by histological examination. American Journal of Anatomy 174: 187–202.