Dietilnitrozamin (DEN) Uygulanan Gökkuşağı Alabalıklarında Karaciğer Değişikliklerinin Histopatolojik Değerlendirilmesi

Yıl 2023, , 283 - 290, 30.09.2023

Banu Yardımcı , Ertan Emek Onuk , Gökmen Zafer Pekmezci

https://doi.org/10.35229/jaes.1294687

Öz

Bu çalışmanın amacı, endüstriyel ve zirai faaliyetlerde kullanılan birçok ürünün içerisinde ve atıklarında bulunan, canlılarda karsinojenik özellikteki, dietilnitrozamin (DEN) in tatlı sularda en çok yetiştiriciliği yapılan ve tüketilen balık olan gökkuşağı alabalığındaki hepatokarsinojen etkisini ortaya koymaktı. Çalışmada, 50-70 gr ağırlığındaki 120 adet balık kullanıldı. Rastgele seçilen balıklar her grupta 10 balık olacak şekilde ve her doz için 3’erli gruplara ayrıldı. 50 mg/kg, 100 mg/kg ve 150 mg/kg olacak şekilde DEN (dietilnitrozamin) 0,1 ml DMSO (dimetilsülfoksit) da çözdürülerek intraperitoneal (ip) yolla verildi. 15, 30 ve 45. günlerde, her bir grupta bulunan balıklara sedasyon uygulanarak ötenazileri gerçekleştirilip nekropsileri yapıldı. Histopatolojik ve immunohistokimyasal inceleme için örnekler alınarak rutin doku takibi prosedürü uygulandı, ışık mikroskobunda incelendi. Histolojik olarak, karaciğerde hiperemi, parankimal dejenerasyon, mononükleer hücre infiltrasyonu, nekroz, safra kanallarında hiperplazi, kolangiofibrozis ve yağlı dejenerasyon bulguları gözlendi.. İmmunohistokimyasal boyamada, CYP1A yönünden C2, A3, B3 ve C3 gruplarında safra kanalı epitellerinde hafif pozitif, diğer gruplarda ise negatif boyanma; Cytokeratin 18 yönünden tüm gruplar negatif boyanma ve GST yönünden A3, B3 ve C3 gruplarında safra kanalı epitellerinde hafif pozitif, gruplarda ise negatif boyanma tespit edildi.Sonuç olarak, dietilnitrozaminin balıklarda kısa süreli uygulamalarda bile karaciğer dokusunda belirgin hasara neden olduğu ve uzun dönemde karaciğer hücrelerinde neoplastik değişikliklere neden olabileceği kanısına varıldı.

Anahtar Kelimeler

Dietilnitrozamin, gökkuşağı alabalığı (Oncorhynchusmykiss), hepatokarsinojen, histopatoloji, immunohistokimya.

Destekleyen Kurum

Ondokuz Mayıs Üniversitesi

Proje Numarası

PYO.VET.1901.15.001

Histopathological Evaluation Of Liver Changes In Rainbow Trout After Diethlynitrosamine (DEN) Exposure

Öz

The aim of this study was to determine the hepatocarcinogenic effect of diethylnitrosamine in rainbow trout (Oncorhynchus mykiss), the most cultivated and consumed fresh water fish. In this study, 120 fish weighing 50-70 g each were used. Randomly selected fish were divided into 3 dosage groups and a control group that contained 30 fish per group. Diethylnitrosaminewas dissolved in 0.1 ml of dimethylsulfoxideat 50 mg/kg, 100 mg/kg and 150 mg/kg, and respectively administered to each of the dosage groups intraperitoneally. On the 15th, 30th and 45th days of the study, 10 fish from each study group were euthanised while under sedation, and necropsies were performed. For histopathological and immunohistochemical examinations, tissues were routinely processed and sections were stained with haematoxylin-eosin and examined by light microscopy. Histology revealed hyperaemia, parenchymal degeneration, mononuclear cell infusion, necrosis, bile ducts hyperplasia, cholangiofibrosis and fatty degeneration of the liver. In immunohistochemical staining, CYP1A was slightly positive in the bile duct epithelium in the C2, A3, B3 and C3 groups, and negative in the other groups. Cytokeratin 18 showed negative staining in all groups. Mild positive staining in the bile duct epithelium, A3, B3 and C3 and negative staining in other groups for glutathione-S-transferase. It was concluded that diethylnitrosamine causes significant damage to liver tissuein fish, even in short-term applications, and may cause neoplastic changes in liver cells over the long term.

Anahtar Kelimeler

Diethylnitrosamine, hepatocarcinogen, histopathology, immunohistochemistry, Rainbow trout (Oncorhynchus mykiss).

Proje Numarası

PYO.VET.1901.15.001

Kaynakça

• Aydinlik, H., Nguyen, T.D., Moennikes, O., Buchmann, A.& Schwarz, M.(2001). Selective pressure during tumor promotion by phenobarbital leads to clonalout growth of beta-catenin-mutated mouse liver tumors. Oncogene, 20(53),7812-7816. DOI: 10.1038/sj.onc.1204982
• Bailey, G.S.,Williams, D.E. & Hendricks, J.D. (1996). Fish models for environmental carcinogenesis: the rainbow trout. Environmental Health Perspectives, 104, Supp. 1, 5-21. DOI: 10.1289/ehp.96104s15
• Black, J.J. & Baumann, P .C. (1991). Carcinogens and cancers in fresh water fishes. Environmental Health Perspectives, 90, 27-33. DOI: 10.1289/ehp.90-1519473
• Boorman, A.G., Botts, S., Bunton, T.E., Fournie, J.W., Harshbarger, J.C., Hawkins, W.E., Hinton, D.E., Jokinen, P.M., Okihiro, M.S. & Wolfe, M.J. (1997). Diagnostic criteria for degenerative, inflammatory, proliferative nonneoplastic and neoplastic liver lesions in Medaka (Oryziaslatipes): Consensus of a National Toxicology Program Pathology Working Group. Toxicologic Pathology, 25(2), 202-210. DOI: 10.1177/019262339702500210
• Braunbeck, T.A., Teh, S.J., Lester, S.M. & Hinto, D.E. (1992). Ultrastructural alterations in liver of Medaka (Oryzias latipes) exposed to diethylnitrosamine . Toxicologic Pathology, 20, 141-311. DOI: 10.1177/019262339202000205
• Bunton, T.E. (1996). Review Article: Experimental Chemical Carcinogenesis in Fish. Toxicologic Pathology, 24, 603-618. DOI: 10.1177/019262339602400511
• Chen, Y.Y., Zhu, J.Y. & Chan, K.M. (2014). Effects of cadmium on cell proliferation, apoptosis, and proto-oncogene expression in zebrafish liver cells. Aquatic Toxicology, 157, 196-206. DOI: 10.1016/j.aquatox.2014.10.018
• Chiarello, P.G., Iglesias, A.C., Zucoloto, S., Moreno, F., Jordao, A.A. &Vannucchi, H. (1998). Effect of a necrogenicdose of diethylnitrosamine on vitamin E-deficientand vitamin E- supplementedrats. Food and Chemical Toxicology, 36, 929-935. DOI: /10.1016/s0278- 6915(98)00043-x
• Couch, J.A. & Harshbarger, J.C. (1985). Effects of carcinogenic agents on aquatic animals: an environmental and experimental overview. Journal of Environmental Science and Health. Part C: Environmental Carcinogenesis Reviews, 3, 63-105. DOI: 10.1080/10590508509373329 Daoust, R. & Morais, R. (1986). Degenerative changes, DNA synthesis and mitotic activity in rat liver following single exposure to diethylnitrosamine. Chemico-Biological Interactions. 57(1), 55-64. DOI: 10.1016/0009-2797(86)90048-7
• Dias Guerreiro I. C. (2019). Xenoestrogenic and androgenic impacts on carcinogenesis onset – A mechanistically look at a controversial issue recurring to diethylnitrosamine induced carcinogenesis in zebrafish larvae. Dissertation for the degree of Master in Environmental Contamination and Toxicology, submitted to the Institute of Biomedical Sciences Abel Salazar of the University of Porto (ICBAS – UP).
• Dogan, A.L. & Guc, D. (2004). Sinyal iletim mekanizmaları ve kanser. Hacettepe Medical Journal, 35, 34-42. https://actamedica.org/index.php/actamedica/arti cle/view/180
• Dunn, B.P., Black, J.J. & Maccubbin, A.E. (1987). 32 P- post1abling analysis of aromatic DNA adducts in fish from polluted areas. Cancer Research, 47, 6543-6548. Published December 1987.
• Etchin, J., Kanki, J. & Look, A. (2011). Zebrafish as a model for the study of human cancer. Methods in Cell Biology, 105, 309-337. DOI: 10.1016/B978- 0-12-381320-6.00013-8
• Frey,S., Buchmann, A., Bursch, W., Schulte-Hermann, R. & Schwarz, M. (2000). Suppression of apoptosis in C3H Mouse liver tumors by activated Ha-ras oncogene. Carcinogenesis, 21(2), 161- 166. DOI: 10.1093/carcin/21.2.161
• Gardner, G.R. (1988). Histopathology of feral and experimental flounder exposed to contaminated harbor sediments. Proceedings of the 14th Annual Aquatic Toxicity Workshop, November 2-4, 1987, Toronto, Ontario. Canadian Technical Report of Fisheries anda Aquatic Sciences No. 1607, p. 145.
• Goodsell, D.S. (2004). The molecular perspective: Nicotine and nitrosamines. Oncologist, 9(3), 353- 354. DOI: 10.1634/theoncologist.9-3-353
• Hendricks, J.D., Meyers, T.R. & Shelton, D.W. (1984). Histological progression of hepatic neoplasia in rainbow trout (Salmo gairdneri). National Cancer Institute Monographs, 65, 321-336.
• Hendricks, J.D., Cheng, R., Shelton, D.W., Pereira, C.B. & Bailey, G.S. (1994). Dose-dependent carcinogenicity and frequent K ria-s proto- oncogene activation by dietary N- nitrosodiethylamine in rainbow trout. Fundamental and Applied Toxicology, 23: 53-62. DOI: 10.1006/faat.1994.1078
• Howe, K., Clark, M. D., Torroja, C. F., Torrance, J., Berthelot, C., Muffato, M., Collins, J. E., Humphray, S., McLaren, K., Matthews, L., McLaren, S., Sealy, I., Caccamo, M., Churcher, C., Scott, C., Barrett, J. C., Koch, R., Rauch, G. J., White, S., Chow, W., Kilian, B., Quintais, L. T., Guerra-Assuncao, J. A., Zhou, Y., Gu, Y., Yen, J., Vogel, J. H., Eyre, T., Redmond, S., Banerjee, R., Chi, J., Fu, B., Langley, E., Maguire, S. F., Laird, G. K., Lloyd, D., Kenyon, E., Donaldson, S., Sehra, Harley, J., Holt, K., Panagiotidis, G., Lovell, J., Barker, G., Whitehead, S., Kay, M., Brown, J., R., Andrews, R., Harrison, E., Kimberley, A., Garnett, J., Fosker, N., Hall, R., Garner, P., Kelly, D., Bird, C., Palmer, S., Gehring, I., & Stemple, D. L. (2013). The zebrafish reference genome sequence and its relationship to the human genome. Nature, 496(7446), 498-503. DOI: 10.1038/nature12111
• Kuroda, H., Ohtsuru, A., Futakuchi, M., Kawashita, Y., Nagayama, Y., Fukuda, E., Namba, H., Shirai, T., Kanematsu, T. & Yamashita, S. (2002). Distinctive gene expression of receptor type Tyrosine-kinase families during rat hepatocarcinogenesis. International Journal of Molecular Medicine, 9, 473-480. DOI: 10.3892/ijmm.9.5.473
• Kurutaş, B.E. & Kılınç, M. (2003). Pestisitlerin biyolojik sistemler üzerine etkileri. ARŞİV, 12, 215-228, 2003.
• Lee, B.C., Hendricks, J.D. & Bailey, G.S. (1989). Metaplastic pancreatic cells in liver tumors induced by diethylnitrosamine. Experimental and Molecular Pathology, 50, 104-113. DOI: 10.1016/0014-4800(89)90060-9
• Maccubbin, A.E., Black, J.J. & Dunn, B.P. (1990). 32P- postlabeling detection of DNA adducts in fish from chemically contaminated water ways. Science of the Total Environment, 94, 89-104. DOI: 10.1016/0048-9697(90)90366-3
• Machado, S. P., Cunhaa, V., Reis-Henriques M. A., Ferreiraa, M. (2014). Histopathological lesions, P-glycoprotein and PCNA expression in zebrafish (Danio rerio) liver after a single exposure to diethylnitrosamine. Environmental Toxicology and Pharmacology, 38(3), 720-732. DOI: 10.1016/j.etap.2014.09.002
• Manalo, J.V.I. & Hemavathy, R.V. (2023). Effects of water pollution on the quality of fish. Journal of Survey in Fisheries Sciences, 10(1S), 6029-6035.
• Metcalfe, C.D., Cairns, V.W. & Fitzsimons, J.C. (1988). Experimental induction of liver tumors in rainbow trout (Salmo gairdneri) by contaminated sediment from Hamilton Harbor, Ontario. Canadian Journal of Fisheries and Aquatic Sciences, 45, 2161-2167. DOI: 10.1139/f88-251
• Mizgireuv, I.V., Majorova, I.G., Gorodinskaya, V.M., Khudoley, V.V. & Revskoy, S.Y. (2004). Carcinogenic effect of N-nitrosodimethylamine on diploid and triploid zebrafish (Danio rerio). Toxicologic Pathology, 32(5), 514-518. DOI: 10.1080/01926230490496311
• Montella, M., D'Arena, G., Crispo, A., Capunzo, M., Nocerino, F., Grimaldi, M., Barbieri, A., D'Ursi, A. M., Tecce, M. F., Amore, A., Galdiero, M., Ciliberto, G. & Giudice, A. (2015). Role of Sex Hormones in the Development and Progression of Hepatitis B Virus-Associated Hepatocellular Carcinoma. International Journal of Endocrinology, 2015, 1- 9. DOI: 10.1155/2015/854530
• Singh, J., Yadav, P., Pal, A.K. & Mishra, V. (2020). Water pollutants: Origin and status. In: Pooja, D., Kumar, P., Singh, P., Patil, S. (eds) Sensors in water pollutants monitoring: Role of material. advanced functional materials and sensors. Springer, Singapore. DOI: 10.1007/978-981-15- 0671-0_2
• Stanton, M. (1965). Diethylnitrosamine-induced hepatic degeneration and neoplasia in the aquarium fish, Brachydanio rerio. JNCI: Journal of the National Cancer Institute, 34, 117-130. DOI: 10.1093/jnci/34.1.117
• Straif, K., Weiland, S.K., Bungers, M., Holthenrich, D., Taeger, D. & Keil, U. (2000). Exposure to high concentrations of nitrosamines and cancer mortality among a cohort of rubber workers. Occupational and Environmental Medicine, 57, 180-187. DOI: 10.1136/oem.57.3.180
• Thirunavukkarasu, C. & Sakthisekaran, D. (2003). Effect of dietary selenite on N- nitrosodiethylamine-induced and phenobarbital promoted multistage hepatocarcinogenesis in rat: reflection in some minerals. Biomedicine & Pharmacotherapy, 57(9), 416-21. DOI: 10.1016/j.biopha.2003.08.023
• Ustuner, C.M. (2006). Karaciğerde tümör oluşmasına neden olan dietilnitrozamin ve 2- asetilaminofloran uygulanmış sıçanlara demetoksiviridin ve 1-alfa-hidroksi demetoksiviridin'in etkileri. Eskişehir Osmangazi University, Graduate School of Health Science, PhD Thesis.
• Varanasi, U., Stein, J.E., Nishimoto, M., Reichart, W.L. & Collier, T.K. (1987). Chemical carcinogenesis in feral fish: uptake, activation, and detoxification of organic xenobiotics. Environmental Health Perspective, 71, 155-170. DOI: 10.1289/ehp.8771155
• Varanasi, U., Reichart, W.L. & Stein, J.E. (1989). 32P- postlabeling analysis of DNA adducts in liver of wild English sole (Parophrysvetulus) an winter flounder (Pseudopleurnectesamencanus). Cancer Research, 49, 1171-1177.
• Yamada, K., Yanamiya, I. & Utsumi, H. (2006). Invivo detection of free radicals induced by diethylnitrosamine in rat liver tissue. Free Radical Biology and Medicine, 40(11), 2040-2046. DOI: 10.1016/j.freeradbiomed.2006.01.031