생태와환경 (Korean Journal of Ecology and Environment)

한국수생태학회 (The Korean Society of Limnology)
권호 표지

Proteomics를 이용한 내분비계장애물질인 nonylphenol에 노출된 무당개구리의 단백질 발현 비교 연구

Proteomics of Liver Tissues of Bombina orientalis Following Exposure to Nonylphenol

  • 김호승 (한양대학교 자연과학대학 생명과학과) ;
  • 계명찬 (한양대학교 자연과학대학 생명과학과)
  • Kim, Ho-Seung (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • Gye, Myung-Chan (Department of Life Science, College of Natural Sciences, Hanyang University)
  • 발행 : 2003.09.30
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초록

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.

키워드

참고문헌

  1. 김호승, 계명찬. 2003. 프로테오믹스를 이용한 내분비계교란물질 환경독성 연구. 환경생물학회지 21: 87-100.
  2. 최영주, 윤춘식, 박주홍, 진정효, 정선우. 2002. 한국산 도롱뇽(Hynobius leechii)의 농경지에서의 배 발생 이상과 살균제 Benomyl의 독성 효과. 한국육수학회지 35: 198-212.
  3. Bevan, C.L., D.M. Porter, A. Prasad, M.J. Howard and L.P.Henderson. 2003. Environmental estrogens alter earlydevelopment in Xenopus laevis. Environ. Health Perspect.111: 88-96.
  4. Blackstock, W.P. and M.P. Weir. 1999. Proteomics: quantitativeand physical mapping of cellular proteins. Trends.Biotechnol. 17: 121-127.
  5. Blom, A., W. Harder and A. Matin. 1992. Unique and overlappingpollutant stress proteins of Escherichia coli.Appl. Environ. Microbiol. 58: 331-4.
  6. Blaustein, A.R. and D.B. Wake. 1995. The puzzle of decliningamphibian populations. Sci. Am. 272: 52-57.
  7. Bradley, B.P., D.C. Brown, T.N. Lamonte, S.M. Boyd andM.C. O’Neill. 1996. Protein patterns and toxicity identification.pp.79-94. In: Aquatic Toxicology and RiskAssessment (Henschel, D. and D. Bengston, eds.)ASTM, Philadelphia.
  8. Carey, C. and C.J. Bryant. 1995. Possible interrelationsamong environmental toxicants, amphibian development, and decline of amphibian populations. Environ.Health Perspect. 103 Suppl. 4: 13-17.
  9. Chapin, R.E., J. Delaney, Y. Wang, L. Lanning, B. Davis,B. Collins, N. Mintz and G. Wolfe. 1999. The effects of4-nonylphenol in rats: a multigeneration reproductionstudy. The effects of 4-nonylphenol in rats: a multigenerationreproduction study. Toxicol. Sci. 52: 80-91.
  10. Cutler, P., D.J. Bell, H.C. Birrell, J.C. Connelly, S.C.Connor, E. Holmes, B.C. Mitchell, S.Y. Monte, B.A.Neville, R. Pickford, S Polley, K. Schneider and J.M.Skehel. 1999. An integrated proteomic approach tostudying glomerular nephrotoxicity. Electrophoresis 20:3647-3658.
  11. Cutler, P., H. Birrell, M. Haran, W. Man, B. Neville, S.Rosier, M. Skehel and I. White. 1999. Proteomics inpharmaceutical research and developmen. Biochem.Soc. Trans. 27: 555-559.
  12. Gilbert, S.F. 2001. Ecological developmental biology: developmentalbiology meets the real world. Dev. Biol. 233:1-12.
  13. Gonzalez, C.R.M. and B.P. Bradley. 1994. Salinity stressproteins in Eurytemora affinis. Hydrobiologia 292/293:462-468.
  14. Jellum, E., A.K. Thorsrud and F.W. Karasek. 1983. Twodimensionalelectrophoresis for determining toxicity ofenvironmental substances. Anal. Chem. 55: 2340-2344.
  15. Kloas, W., I. Lutz and R. Einspanier. 1999. Amphibians asa model to study endocrine disruptors: II. Estrogenicactivity of environmental chemicals in vitro and invivo. Sci. Total Environ. 225: 59-68.
  16. Lahr, J. 1997. Ecotoxicology of organisms adapted to life intemporary freshwater ponds in arid and semi-aridregions. Arch. Environ. Contam. Toxicol. 32: 50-57.
  17. Lee, P.C. and W. Lee. 1996. In vivo estrogenic action ofnonylphenol in immature female rats. Bull. Environ.Contam. Toxicol. 57: 341-348.
  18. Lutz, I. and W. Kloas. 1999. Amphibians as a model tostudy endocrine disruptors: I. Environmental pollutionand estrogen receptor binding. Sci. Total Environ. 225:49-57.
  19. Mann, RM. and J.R. Bidwell. 2000. Application of theFETAX protocol to assess the developmental toxicity ofnonylphenol ethoxylate to Xenopus laevis and two Australian frogs. Aquat. Toxicol. 51: 19-29.
  20. Mann, R.M. and J.R. Bidwell. 2001. The acute toxicity ofagricultural surfactants to the tadpoles of four Australianand two exotic frogs. Environ. Pollut. 114: 195-205.
  21. Mosconi, G., O. Carnevali, M.F. Franzoni, E. Cottone, I.Lutz, W. Kloas, K. Yamamoto, S. Kikuyama and A.M.Polzonetti-Magni. 2002. Environmental estrogens andreproductive biology in amphibians. Gen. Comp. Endocrinol.126: 125-129.
  22. O’Farrell, P.H. 1975. High resolution two-dimensionalelectrophoresis of proteins. J. Biol. Chem. 250: 4007-4021.
  23. Plotner, J. and R. Gunther. 1987. Toxicity of an anionicdetergent to the spawn and larvae of anurans (Amphibia).Int. Rev. Ges. Hydrobiol. 72: 759-771.
  24. Presutti, C., C. Vismara, M. Camatini and G. Bernardini.1994. Ecotoxicological effects of a nonionic detergent(Triton DF-16) assayed by ModFETAX. Bull. Environ.Contam. Toxicol. 53: 405-411.
  25. Shepard, J.L., B. Olsson, M. Tedengren and B.P. Bradley.2000. Protein expression signatures identified in Mytilusedulis exposed to PCBs, copper and salinity stress.Mar. Environ. Res. 50: 337-340.
  26. van Wyk, J.H., E.J. Pool and A.J. Leslie. 2003. The effectsof anti-androgenic and estrogenic disrupting contaminantson breeding gland (nuptial pad) morphology,plasma testosterone levels, and plasma vitellogeninlevels in male Xenopus laevis (African clawed frog).Arch. Environ. Contam. Toxicol. 44: 247-256.
  27. William, K.L. and D.F. Hochsatrasser. 1997. Introductionto the proteome. pp. 1-30. In: Proteome Reserch: NewFrontiers in Functional Genomics (Wilkins, M.R., K.L.Williams, R.D. Appel and D.F. Hochstrasser, eds.)Springer-Verlag, Berlin.
  28. Yamauchi, K., A. Ishihara, H. Fukazawa and Y. Terao.2003. Competitive interactions of chlorinated phenolcompounds with 3, 3′, 5-triiodothyronine binding totransthyretin: detection of possible thyroid-disruptingchemicals in environmental waste water. Toxicol. Appl.Pharmacol. 187: 110-117.