내분비계장애물질 (환경호르몬)의 일종인 노닐페놀(nonylphenol, NP)에 의한 수서 환경 내 생태독성 평가의 일환으로 한국에 서식하는 무당개구리 (Bombina orientalis) 수컷에서 NP에 의한 간조직 내 발현 단백질의 변화를 추적하였다. 체중 10${pm}$0.1g의 수컷 무당개구리에 NP을 10mg/kg 농도로 복강 주사한 후 48 및 96시간 후에 간을 절취 한 뒤 마쇄하여 2차원 전기영동을 수행하였다. Coomassie brilliant blue로 염색한 gel 상에서 전체적으로 50${sim}$60개 정도의 protein spots을 확인할 수 있었으며 단백질 spots의 변화를 비교 분석한 결과 NP처리 48시간 후 8개의 spots이 증가한 반면 12개의 spots이 감소하였다. 96시간 후에는 30개의 spots이 증가되었고 8개의 spots이 감소하였다. 전체적으로는 약20%정도의 단백질의 변화가 있었다. 단백질 발현의 동태는 투여 후 2일 까지는 단백질 생산이 일시적으로 감소하지만 다시 새로운 단백질을 생성하는 것으로 사료된다. NP 노출에 따른 무당개구리 간조직 내 단백질 발현의 변화는 한국의 수서환경에서 내분비계 장애물질의위해성 평가에 요구되는 단백질 biomaker의 개발에 이용 할 수 있을 것이다.
Nonylphenol (NP), an well known aquatic contaminant, has been known to induce abnormalities in various aquatic animals. In an effort to develop proteome in the study of aquatic contamination of NP and its impact on the amphibia, protein changes in liver tissues of Korean red bellied frog, Bombina orientalis was investigated following the NP exposure. NP was administered intraperitoneally to male B. orientalis at 10 mg/kg body weight. At 48 and 96h after the treatment, the frog livers were sampled, and the protein fraction was separated using two dimensional gel electrophoresis (2D/E) and visualized with Coomassie brilluant blue staining. The 2D/E Images of the tissue from the animals treated with NP showed marked changes of protein spots (about 20% of total protein spots). Analysis of the 50-60 separated spots allowed identification of the major protein changes in the overall pattern for the stressor (NP) by time (0,48 and 96 h). At 48h after treatment, 8 spots were increased and 12 spots were reduced. Then, at 96h after treatment, 10 spots were increased and 8 spots were reduced. In total, approximately 29% of liver proteins showed the altered expression following the NP treatment. It is suggested that protein expression was repressed by blocking of certain metabolisms at 48 hand induced by the synthesis of new proteins for adaptation at 96 h following NP exposure. This application for 2D/E analysis may show promise in searching biomarkers for environmental proteomics in amphibians.
