다이아지논에 의해 야기된 송사리의 이상행동 연관 분자생물지표의 선발

Selection of Molecular Biomarkers Relevant to Abnormal Behaviors of Medaka Fish (Oryzias latipes) Caused by Diazinon

  • 고성철 (한국해양대학교 건설환경공학부) ;
  • 신성우 (한국해양대학교 건설환경공학부) ;
  • 조현덕 (부산대학교 생명과학부) ;
  • 전태수 (부산대학교 생명과학부) ;
  • 김정상 (경북대학교 동물공학과) ;
  • 이성규 (안전성평가연구소)
  • Koh, Sung-Cheol (Division of Civil and Environmental Engineering, Korea Maritime University) ;
  • Shin, Sung-Woo (Division of Civil and Environmental Engineering, Korea Maritime University) ;
  • Cho, Hyun-Duk (Department of Biological Sciences, Pusan Naitonal University) ;
  • Chon, Tae-Soo (Department of Biological Sciences, Pusan Naitonal University) ;
  • Kim, Jong-Sang (Department of Animal Biotechnology, Kyungpook Naitonal University) ;
  • Lee, Sung-Kyu (Korea Institute of Toxicology)
  • 발행 : 2009.12.31

초록

본 연구의 목적은 다이아지논(Diazinon; O, O-diethyl O-[6-methyl-2 (1-methylethyl)-4-pyrimidinyl] phosphorothioate)에 노출된 모델 생물체(송사리)의 행동변화와 관련된 분자생물학적 기전 규명을 통하여 비정상적 행동의 모니터링을 위한 생물지표(biomarker)를 개발하는데 있다. 이를 위해 우선 suppression subtractive hybridization (SSH) 및 DNA microarray 기법을 활용하여 다양한 유전자를 스크리닝하였다. 다이아지논에 노출시킨 송사리에서 발현의 차이가 나는 상향 조절된 유전자 97개 (알려지지 않은 유전자 27개 포함)와 하향 조절된 유전자 99개 (알려지지 않은 유전자 60개 포함)를 동정 하였고 이들 중 이상행동과 관련되는 것으로 보이는 유전자 10개 (상향조절 5개, 하향조절 5개)를 선발하였다. 이들 중에서 primer 제작이 잘된 beta-1, Orla C3-1, parvalbumin 및 apolipoprotein E을 선발하여 그 유전자 발현을 real-time PCR 기법을 사용하여 정량적으로 모니터링 하였다. Orla C3-1, parvalbumin 및 apolipoprotein E는 고농도의 다이아지논 처리(1000 ppb; 24 h)에서 그 발현이 억제됨이 관찰되었다. 다이아지논 처리 시 신경질환 (알츠하이머 병 및 다운신드롬)에 관련된 apolipoprotein E와 근육세포의 유연화에 작용하는 parvalbumin 등의 발현억제는 송사리의 인지능력 교란 및 근육세포의 경직 등을 각각 유도하여 송사리의 비정상적 행동을 야기하는 것으로 판단되었다. 따라서 이들 생물지표는 신경독성물질에 의한 송사리 및 기타 어류의 이상행동의 변화의 감지에 활용될 수 있을 것으로 사료된다.

키워드

참고문헌

  1. Chon TS, Chung N, Kwak IS, Kim JS, Koh SC, Lee SK, Leem JB and Cha EY. Movement behavior of medaka (Oryzias latipes) in response to sublethal treatments of diazinon and cholineesterase activity in semi-natural conditions, Environmental Monitoring and Assessment, 2005; 101(1-3): 1-21
  2. Coker GT, Vinnedge L and O'Malley KL. Characterization of rat and human tyrosine hydroxylase gene: functional expression of both promoters in neuronal and non-neuronal cell types, Biochemistry and Biophysical Research Communications 1988; 157: 1341-1347 https://doi.org/10.1016/S0006-291X(88)81022-2
  3. Depledge MH, Amaral-Mendes JJ, Daniel B, Halbrook RS, Kloepper-Sams P, Moore MN and Peakall DP. The conceptual basis of the biomarker approach. In: D G Peakall and L R Shugart. Editors, Biomarkers? Springer, Berlin 1993; 15-29
  4. Dutta H, Marcelino J and Richmonds C Brain. Acetylcholinesterase activity and optomotor behavior in bluegills, Lepomis macrochirus exposed to different concentrations of diazinon, Arch. Int. Physiol. Biochim. Biophys 1992; 100: 331-334 https://doi.org/10.3109/13813459209000721
  5. Elaine R, Brown GT, Coker GT and Karen LO. Organization and evolution of the rat tyrosine hydroxylase gene, Biochemistry 1987; 26: 5208-5212 https://doi.org/10.1021/bi00390a046
  6. Erwin WR, Keijzers R and Cornelis AM. Acetylcholinesterase inhibition and increased food consumption rate in the zebrafish, Danio rerio, after chronic exposure to parathio, Aquatic Toxicology 2003; 64: 451-460 https://doi.org/10.1016/S0166-445X(03)00100-0
  7. Feng Q, Boone, AD and Vijayan MM. Copper impact on heat shock protein 70 expression and apoptosis in rainbow trout hepatocytes, Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 2003; 135(3): 345-355 https://doi.org/10.1016/S1532-0456(03)00137-6
  8. Huriaux F, Vandewalle P and Focant B. Immunological study of muscle parvalbumin isotopes in three African catfish during development, Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 2002; 132(3): 579-584 https://doi.org/10.1016/S1096-4959(02)00071-4
  9. Jordan BD, Relkin NR, Ravdin LD, Jacobs AR, Bennett A and Gandy S. Apolipoprotein E epsilon4 associated with chronic traumatic brain injury in boxing, J. Am. Med. Association 1997; 278: 136-140 https://doi.org/10.1001/jama.278.2.136
  10. Kim JS, Koh SC, Lee SK and Chon TS. Regulation of acetylcholine esterase and neurotransmitters in Oryzias Latipes by diazinon, J. Environmental toxicology 1999; 14(3): 81-85
  11. Kwak IS, Chon TS, Kang HM, Chung NI, Kim JS, Koh SC, Lee SK and Kim YS. Pattern recognition of the movement tracks of medaka (Oryzias latipes) in response to sublethal treatments of an insecticide by using artificial neural networks. Environ. Pollution 2002; 120: 671-681 https://doi.org/10.1016/S0269-7491(02)00183-5
  12. Lee HK and Lee YH. A study of system development for the detection of toxic materials into the source of water supply, J. Civil. Engineering 1998; 18: 289-297
  13. Lucarelli P, Piciullo A, Verdecchia M, Palmarino M, Arpino C and Curatol P. The role of p-850 tumor necrosis factor- $\alpha$ and apolipoprotein E genetic polymorphism in patients with Down's syndrome-related dementia, Neuroscience Letters 2003; 352(1): 29-32 https://doi.org/10.1016/j.neulet.2003.08.021
  14. Ma Y, Cao L, Kawabata T, Yoshino T, Yang BB and Okada S. Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells, Free Radical Biology and Medicine 1998; 25: 568-575 https://doi.org/10.1016/S0891-5849(98)00088-4
  15. Majachrzak R, Sroczynski J and Chelmeska E. Evaluation of the nervous system in workers in the furnace and coal divisions of the coke-producing plants, Med. Pr. 1990; 2: 108-113
  16. Mandal PK and Kulshrestha AK. Histopathological changes induced by sublethal summit in Clarias batrachus, Linn. Ind. J. Expt. Biol. 1980; 18: 547-558
  17. Mandelbort BB. The fractal geometry of nature, Freeman, San Francisco 1982
  18. McCarron MO, Muir KW, Weir CJ, Dyker AG, Bone I, Nicoll JA and Lees KR. The apolipoprotein E allele and outcome in cerebrovascular disease, Stroke 1998; 29: 1882-1887 https://doi.org/10.1161/01.STR.29.9.1882
  19. Murali DR and Krishna G. Neurobehavioral changes in freshwater fish Channa punctatus exposed to fenitrothion, Bull. Environ. Contam. Toxicology 1991; 47: 455-458 https://doi.org/10.1007/BF01702210
  20. Oehlmann VD, Berger S, Sterner C and Korsching SI. Zebrafish beta tubulin 1 expression is limited to the nervous system throughout development, and in the adult brain is restricted to a subset of proliferative regions. Gene Expression Patterns 2004; 4: 191-198 https://doi.org/10.1016/j.modgep.2003.09.001
  21. Pan G and Dutta HM. The inhibition of brain acetylcholinesterase activity of juvenile largemouth bass Micropterus salmoides by sublethal concentrations of diazinon, Environ. Res. 1988; 79: 133-137
  22. Qui$\tilde{n}$ones A and Rainov NG. Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression: Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2001; 495: 73-85
  23. Rumelhart DE, Hinton GE and Williams RJ. Learning internal representations by error propagation, MIT Press, Cambridge MA 1986. 318-362
  24. Salminen WF, Voellmy (Jr) R and Roberts SM. Induction of hsp 70 in HepG2 cells in response to hepatotoxicants, Toxicology and Applied Pharmacology 1996; 141(1): 117-123 https://doi.org/10.1016/S0041-008X(96)80016-0
  25. Sambrook J and Russell DW. Molecular Cloning 'A Laboratory manual', Third edition, cold spring harbor laboratory press, New York 2001
  26. Schuller E, Gulesserian T and Seidl R. Barin t-complex polypeptide 1 (PCP-1) related to its natural substrate $\beta$1 tubulin is decreased in Alzheimer’s disease, Life Sciences 2001; 69: 263-270 https://doi.org/10.1016/S0024-3205(01)01126-2
  27. Shin SW, Chung NI, Kim JS, Chon TS, Kwon OS, Lee SK and Koh SC. Effect of diazinon on behavior of Japanese Medaka (Oryzias Latipes) and gene expression of tyrosine hydroxylase as a biomarker, J. Environ. Sci. Health 2001; B36(6): 783-795
  28. Stafford JL and Belosevic M. Transferring and the innate immune response of fish: identification of a novel mechanism of macrophage activation, Developmental & Comparative Immunology 2003; 27: 539-554 https://doi.org/10.1016/S0145-305X(02)00138-6
  29. Thomas N, McEachron D, Freedman W and Yang WP. Cold acclimation increases gene transcription of two calcium transport molecules, calcium transporting ATPase and parvalbumin beta, in carassius auratus lateral musculature, Journal of Thermal Biology 2003; 28(3): 227-234 https://doi.org/10.1016/S0306-4565(02)00068-2
  30. Tyler CR, Filby AL, Aerle R, Lange A, Ball J and Santos EM. Fish toxicogenomics: Advances in Experimental Biology, Volume 2 2008; 75-132, 324-325 https://doi.org/10.1016/S1872-2423(08)00003-3
  31. Zar, JH. Biostatistical analysis, Prentice-Hall International, Englewood cliffs. 1984
  32. Zarkadis IK, Sarrias MR, Sfyroera G, Sunyer JO and Lambris JD. Cloning and structure of three rainbow trout C3 molecules: a plausible explanation for their functional diversity, Developmental & Comparative Immunology 2001; 25(1): 11-24 https://doi.org/10.1016/S0145-305X(00)00039-2