Çocuklarda Sevoflurane ve Desflurane'ın periferik lenfositler üzerine genotoksik etkilerinin "Comet assay" yöntemiyle araştırılması
Research of Sevofurane and Desflurane genotoxic effects on children peripheral lymphocytes by comet assay method
Yazar: YEŞİM IŞIKÇI
Danışman: PROF. DR. GUNER KAYA
İstanbul Üniversitesi / Cerrahpaşa Tıp Fakültesi / Anesteziyoloji ve Reanimasyon Ana Bilim Dalı
Derece: Tıpta Uzmanlık, 2007
Konu: Anestezi ve Reanimasyon = Anesthesiology and Reanimation ; Genetik = Genetics ; Çocuk Cerrahisi = Pediatric Surgery
Dizin: Anestezi-inhalasyon = Anesthesia-inhalation ; Bilimsel araştırmalar = Scientific researchs ; Comet tekniği = Comet assay ; Genotoksisite = Genotoxicity ; Lökosit kültür testi-mikst = Leukocyte culture test-mixed ; Çocuklar = Children

Özet:
Anestezik gazlarin kromozomal degisikliklere neden oldugunu gosteren pek cok klinik ve sitogenetik calisma yapilmistir ancak cocuk hasta grubunda yapilanlar sinirlidir. Biz calismamizda, sik kullanilan inhalasyon anesteziklerinden olan Sevofluran ve Desfluranin cocuklarda olasi genotoksik etkilerini arastirmayi hedefledik.Arastirma icin cabuk, basit ve ucuz bir gorsel teknik olan Comet Assay yontenimini kullanarak periferik kandan elde edilen lenfositleri inceledik. Bu deneyde agaroz jelin icerisine gomulen hucreler deterjan ve tuz solusyonlarinda parcalanirlar. Hasarli DNA molekulu elektroforez ortaminda yurutulur. Kirik iceren DNA molekulleri super sarmal yapidan kurtularak anoda dogru hareket eder.Fluoresan boyalarla boyanan hucreler mikroskopta incelendiklerinde kuyruklu yildiza benzer bir goruntu olustururlar. Kuruk kismindaki DNA yogunlugu olusan DNA hasarini gosterir.Calisma kapsamina, premedikasyon uygulanmayan, anestezi suresi en az 120 dakika olan pediatrik cerrahi girisim uygulanacak 1-10 yas arasi 26 cocuk alindi. Aneztezi oncesi Heparinli enjektore alinan 3 mL venoz kan ornekleri kontrol olarak kabul edildi. Satndard induksiyon sonrasi olgular rastgele iki gruba ayrilarak Oksijen\Hava icinde Desfluran (Grup D) uygulanan grup ve Sevofluran (Grup S) uygulanan grup olarak incelendi. Anestezi baslangicindan 60 dakika, 120 dakika, 1. gun, ve 5 gun sonra alinan kan orneklerinden periferik lenfositler izole edilerek Comet Assay yontemiyle incelendi. Lenfositler fluoresan mikroskopta degerlendirildi ve gorsel siniflama yapilarak % kuyruk faktorleri hesaplandi.Her iki grupta kontrol degerleri farkli degildi. Hem Desfluran hem de Sevofluran grubunda anestezi baslangicindan 60 dakika, 120 dakika, 24. saat, ve 5. gunlerde alinan kan orneklerinde bazal degerlere yakin veya ayni sonuclar elde edildi. Desfluran ile Sevofluran arasinda tum zamanlar kan orneklerinde istatiksel olarak anlamli fark bulunmadigi goruldu.Sonuc olarak, standard bir anestezi suresi boyunca Sevofluran ve Desfluran anestezisi uygulanan cocuk hasta gruplarinin lenfositlerinde cekirdek yapinin etkilenmeyerek hasarlanmadigi gosterildi.

Abstract:
The potential mutagenicity of inhalational anestetic gases was investigated by a lot of clinical and cytogenetic studies but those concerning children were very limited. In the presen paper, we report data on the possible genotoxic properties of two inhalational anaesthetics ; Sevoflurane and Desflurane in peripheral blood lymphocytes of children before, during and after anaesthesia. Both anaesthetics were evaluated for genotoxic activity using the comet assay.The Comet assay is a single cell based technique that allows to detect and quantitate DNA damages. It is simple, rapid and easy to perform. In this method, cells are suspended in molten low melting point agarose , then, they are immersed in an electrophoresis solution. An electric field is applied, the slides are then neutralised and stained with DNA specific fluorescent stain and analysed using a microscope. The human eye readily discriminates comets representing different levels of damage, a scheme was developped for visual scoring based on 5 recognasible classes of comet, from class 0 ( undamaged , no discernable tail) to class 4 (almost all DNA in tail, insignificant head) and tail length % of total fluorescence in head and tail was calculated.In our study, the exposed group consisted on 26 unpremedicated children (aged 1-10 years, anesthetized minimum 120 minutes) . After standard induction, anesthesia was maintained with inhalation of Sevoflurane 1,5-2% ( n=13) or Desflurane 3% ( n=13) in oxygen-air mixture.Venous blood samples were obtained before the induction of anesthesia ( control), at 60 and 120 min of anesthesia and on the first and fifth days. Blood samples were evaluated by comet assay method.The mean comet response was not different between two groups before anesthesia. However, similar results were observed in the mean comet response in blood sampled from patients at 60 or 120 min and on the first and fifth days in S and D treated groups respectively.We demonstrated that Sevoflurane and Desflurane did not induce any increase in DNA migration in the studied clinical conditions.