Etlik piliçlerde çörek otu tohumu ve timokinonun karaciğerde Aflatoksin biyotransformasyonu üzerine etkilerinin immunohistokimyasal olarak araştırılması
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Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada, broyler civcivlerde çörek otu tohumu (ÇÖT) ve bunun farmasötik olarak etkin
maddesi kabul edilen uçucu yağ asiti timokinonun (TQ), aflatoksinin (AF) biyotransformasyon
mekanizmaları ve detoksifikasyonu üzerine olan etkinlikleri, histopatolojik ve immunohistokimyasal
(İHK) olarak moleküler düzeyde araştırıldı. Çalışmada, her bir grupta 20’şer adet 1 günlük broyler
civciv bulunan 6 grup oluşturuldu; kontrol grubu (normal yem), AF grubu (2mg/kg aflatoksin), TQ
grubu (300mg/kg timokinon), ÇÖT grubu (%5 çörek otu tohumu), AF+TQ grubu (2mg/kg
aflatoksin+300mg/kg timokinon) ve AF+ÇÖT grubu (2mg/kg aflatoksin+%5 çörek otu tohumu) içeren
yemlerle 28 gün süreyle beslendi. Deneme süresi sonunda her bir gruptan rastgele 10’ar adet broyler
civciv seçilerek, AST, ALT, ALP, GGT, bilirubin, total protein ve total kolesterol ölçümleri için kan
alındı. Çalışmada kullanılan hayvanların canlı ağırlıkları belirlendikten sonra sistemik nekropsileri
yapılarak karaciğerin ağırlığı ve makroskobik bulgular kaydedildi. Histopatolojik ve
immunohistokimyasal (AFB1-DNA adüktü, PXR, AhR, CAR, CYP1A1, CYP2A6, glutatyon, GSTA3)
incelemeler için örnekler alınarak %10’luk tamponlu formaldehit solüsyonunda tespiti ve rutin doku
takibi yapıldı. Aflatoksinin AST, ALT, ALP ve GGT miktarlarında neden olduğu artış ile total protein
ve kolesterol düzeylerinde neden olduğu azalmanın, rasyona çörek otu tohumu veya timokinon ilave
edilmesiyle normale yaklaştırıldığı belirlendi (p<0,05). Aflatoksinin karaciğerde neden olduğu relatif
organ ağırlığı artışının yanı sıra histopatolojik olarak görülen hidropik dejenerasyon ve yağlanma ile
periportal mononükleer hücre -heterofil infiltrasyonları, asiner dizilim ve safra kanalı proliferasyonunun
ÇÖT veya TQ tarafından önemli oranda azaltıldığı saptandı (p<0,05). İHK boyamalarda AF’nin,
biyotransformasyonunda görevli nükleer reseptörler olan PXR, AhR ve CAR ile yine
metabolizmasından sorumlu CYP1A1 ve CYP2A6 enzim aktivitesinde artışa, glutatyon ve GSTA3
seviyelerinde ise azalmaya neden olduğu, AFB1-DNA adükt oluşumunu güçlü bir şekilde uyardığı
gözlendi. Çalışmada rasyona yapılan TQ veya ÇÖT ilavesinin AF’nin bu olumsuz etkilerini önemli
oranda (p<0,05) düzelttiği moleküler düzeyde ilk kez ortaya konuldu. Ayrıca, TQ ve ÇÖT’ün incelenen
parametreler yönünden kontrol grubuna kıyasla önemli bir değişikliğe neden olmadıkları ve GSH ve
GSTA3 seviyelerini artırarak antioksidan aktivite sergiledikleri saptandı. AF’nin detoksifikasyonunda
TQ’nun ÇÖT’e kıyasla daha etkili olduğu ve etkisini hem AF’nin metabolizmasını inhibe ederek hem
de antioksidan sistem enzimlerini stimüle ederek gösterdiği sonucuna varıldı.
In this study, the activity of Nigella sativa L. seed (NS) and its pharmaceutically active substance volatile fatty acid thymoquinone (TQ) on the biotransformation and detoxification mechanisms of aflatoxin (AF) in broiler chicks were investigated by histopathological and immunohistochemical (IHC) technics as molecular level. Six groups of 20 broiler chicks at the age of 1 day were formed for the study; control group (normal feed); AF group (2mg/kg aflatoxin), TQ group (300mg/kg thymoquinone) NS group (%5 Nigella sativa L. seed), AF+TQ group (2mg/kg aflatoxin + 300mg/kg thymoquinone) and AF+NS group (2mg/kg aflatoxin + % 5 Nigella sativa L. seed) containing diets were fed for 28 days. At the end of the trial period, 10 broiler chicks were randomly selected from each group and blood was collected for AST, ALT, ALP, GGT, bilirubin, total protein, and total cholesterol measurements. After determining the live weights of each animal used in the study, systemic necropsies were performed and liver weight and macroscopic findings were recorded. Samples taken for histopathological and immunohistochemical examinations (AFB1-DNA adduct, PXR, AhR, CAR, CYP1A1, CYP2A6, glutathione, GSTA3) were fixed in 10% buffered formaldehyde solution and routine tissue processing was performed. It was determined that the increase in AST, ALT, ALP and GGT amounts and the decrease in total protein and cholesterol levels caused by aflatoxin were normalized by adding NS or TQ to the ration (p<0,05). In addition to the relative organ weight increase, also the histopathological findings such as hydropic degeneration and fatty changes and periportal mononuclear cell-heterophil infiltrations, acinar arrangement and biliary duct proliferation in the livers caused by AF, were significantly reduced by NS or TQ (p<0,05). In IHC staining, AF strongly stimulated the formation of the AFB1-DNA adduct by inducing an increase in the nuclear receptors PXR, AhR and CAR and in the enzyme activity of the CYP1A1 and CYP2A6 which are responsible for its metabolism and reduced the glutathione and GSTA3 amounts in hepatocytes. It was demonstrated for the first time at the molecular level that the addition of TQ or NS to the diet ameliorated these negative effects of AF significantly (p<0,05). In addition, it was found that TQ and NS did not cause a significant change in terms of the parameters examined compared to the control group and showed antioxidant activity by increasing GSH and GSTA3 levels. It was concluded that TQ was more effective in detoxification of AF than NS and showed its effect both by inhibiting the metabolism of AF and by stimulating antioxidant system enzymes.
In this study, the activity of Nigella sativa L. seed (NS) and its pharmaceutically active substance volatile fatty acid thymoquinone (TQ) on the biotransformation and detoxification mechanisms of aflatoxin (AF) in broiler chicks were investigated by histopathological and immunohistochemical (IHC) technics as molecular level. Six groups of 20 broiler chicks at the age of 1 day were formed for the study; control group (normal feed); AF group (2mg/kg aflatoxin), TQ group (300mg/kg thymoquinone) NS group (%5 Nigella sativa L. seed), AF+TQ group (2mg/kg aflatoxin + 300mg/kg thymoquinone) and AF+NS group (2mg/kg aflatoxin + % 5 Nigella sativa L. seed) containing diets were fed for 28 days. At the end of the trial period, 10 broiler chicks were randomly selected from each group and blood was collected for AST, ALT, ALP, GGT, bilirubin, total protein, and total cholesterol measurements. After determining the live weights of each animal used in the study, systemic necropsies were performed and liver weight and macroscopic findings were recorded. Samples taken for histopathological and immunohistochemical examinations (AFB1-DNA adduct, PXR, AhR, CAR, CYP1A1, CYP2A6, glutathione, GSTA3) were fixed in 10% buffered formaldehyde solution and routine tissue processing was performed. It was determined that the increase in AST, ALT, ALP and GGT amounts and the decrease in total protein and cholesterol levels caused by aflatoxin were normalized by adding NS or TQ to the ration (p<0,05). In addition to the relative organ weight increase, also the histopathological findings such as hydropic degeneration and fatty changes and periportal mononuclear cell-heterophil infiltrations, acinar arrangement and biliary duct proliferation in the livers caused by AF, were significantly reduced by NS or TQ (p<0,05). In IHC staining, AF strongly stimulated the formation of the AFB1-DNA adduct by inducing an increase in the nuclear receptors PXR, AhR and CAR and in the enzyme activity of the CYP1A1 and CYP2A6 which are responsible for its metabolism and reduced the glutathione and GSTA3 amounts in hepatocytes. It was demonstrated for the first time at the molecular level that the addition of TQ or NS to the diet ameliorated these negative effects of AF significantly (p<0,05). In addition, it was found that TQ and NS did not cause a significant change in terms of the parameters examined compared to the control group and showed antioxidant activity by increasing GSH and GSTA3 levels. It was concluded that TQ was more effective in detoxification of AF than NS and showed its effect both by inhibiting the metabolism of AF and by stimulating antioxidant system enzymes.
Açıklama
Anahtar Kelimeler
Aflatoksin, Aflatoxin, GSTA3, İmmunohistokimya, Timokinon, Thymoquinone
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Ateş, M.B.(2019). Etlik piliçlerde çörek otu tohumu ve timokinonun karaciğerde Aflatoksin biyotransformasyonu üzerine etkilerinin immunohistokimyasal olarak araştırılmasI.(Doktora Tezi).
