The Korean Journal of Malacology (한국패류학회지)

The Malacological Society of Korea (한국패류학회)
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Cellular Immune Responses of the Manila Clam, Ruditapes philippinarum, Exposed to Cadmium Chloride

카드뮴에 노출된 바지락 (Ruditapes philippinarum) 의 세포성 면역 반응

  • Park, Kyung-Il (Department of Aquatic Life Medicine, College of Ocean Science and Technology, Kunsan National University) ;
  • Choi, Min-Soon (Department of Aquatic Life Medicine, College of Ocean Science and Technology, Kunsan National University) ;
  • Park, Sung-Woo (Department of Aquatic Life Medicine, College of Ocean Science and Technology, Kunsan National University) ;
  • Park, Kwan-Ha (Department of Aquatic Life Medicine, College of Ocean Science and Technology, Kunsan National University) ;
  • Choi, Sang-Hoon (Department of Aquatic Life Medicine, College of Ocean Science and Technology, Kunsan National University) ;
  • Yoon, Jong-Man (Department of Aquatic Life Medicine, College of Ocean Science and Technology, Kunsan National University)
  • 박경일 (군산대학교 해양과학대학 수산생명의학과) ;
  • 최민순 (군산대학교 해양과학대학 수산생명의학과) ;
  • 박성우 (군산대학교 해양과학대학 수산생명의학과) ;
  • 박관하 (군산대학교 해양과학대학 수산생명의학과) ;
  • 최상훈 (군산대학교 해양과학대학 수산생명의학과) ;
  • 윤종만 (군산대학교 해양과학대학 수산생명의학과)
  • Received : 2009.06.12
  • Accepted : 2009.07.20
  • Published : 2009.08.31
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Abstract

The Manila clam, Ruditapes philippinarum, has been considered as a sentinel species due to dominant distribution along the coast of Korea and well developed regulatory system. In order to develop and understand immune responses of the Manila clams, clams were exposed to $50\;{\mu}g/L$ of cadmium chloride (Cd) for 8 days and monitored the cellular immune parameters of the hemocytes including blast cell composition, DNA damage, necrosis, apoptosis and hemocyte mortality using a flow cytometer. The results showed that all immune parameters analyzed in the present study increased remarkably compared to the controls and the increases were statistically significant. Apoptosis rate was higher than necrosis rate in the clams exposed to Cd suggesting that apoptosis was preferably induced by the concentration of Cd used in the present study. Our study indicates that the measurement of cellular immune responses of the Manila clam using flow cytometer will be a useful technique for assessment of heavy metal contamination in marine environment.

바지락 (Ruditapes philippinarum) 은 우리나라 조간대에 널리 분포하며 잘 발달된 생체조절 능력을 가지고 있어 환경모니터링 종으로서 가치가 높은 생물이다. 본 연구는 산업활동으로 해양환경에서 그 농도가 높아지고 있으며 생물체에 강한 독성을 가진 카드뮴이 바지락의 세포성 면역에 미치는 영향을 연구함으로써 바지락의 면역 기작을 이해하고 환경 모니터링 기법으로의 적용 가능성을 조사하기 위하여 실시되었다. 실험을 위하여 바지락은 $50\;{\mu}g/l$의 카드뮴에 8일간 노출되었으며, 유세포 분석기를 이용하여 혈구의 미분화 세포의 구성비율, DNA 손상도, 혈구괴사율, 혈구 apoptosis 비율 및 혈구사망율을 측정하였다. 조사 결과 카드뮴에 노출된 바지락은 대조구와 비교하여 미분화세포, DNA 손상도, 세포괴사율, 세 apoptosis 비율 및 세포 사망율이 통계적으로 유의하게 증가하였다. 특히 apoptosis 비율은 세포괴사율보다 높게 증가함에 따라 바지락 혈구의 경우 본 연구에서 사용된 카드뮴의 농도는 세포괴사보다는 apoptosis를 유도하는데 더 효과적인 농도인 것으로 판단된다. 본 연구를 통하여 중금속 오염에 대한 바지락의 세포성 면역반응은 유세포 분석기를 이용하여 정량이 가능하였으며, 세포성 면역반응을 이용한 환경모니터링에 유용하게 사용될 수 있을 것으로 기대된다.

Keywords

References

  1. Allam, B., Ashton-Alcox, K.A. and Ford, S.E. (2002) Flow cytometric comparison of haemocytes from three species of bivalve molluscs. Fish & Shellfish Immunology, 13: 141-158. https://doi.org/10.1006/fsim.2001.0389
  2. Auffret, M. and Oubella, R. (1994) Cytometric parameters of bivalve molluscs: effect of environmental factors. In: Modulators of Fish Immune Responses, Vol. 1 (ed. by Stolen, J.S. and Fletcher, T.C.) pp. 23-32. SOS Publication, Fair Haven, USA.
  3. Auffret, M. and Oubella, R. (1995) Cytology and cytometric analysis of bivalve mollusc haemocytes. In: Techniques in Fish Immunology and Pathology of Aquatic Invertebrates Vol. 4 (ed. by Stolen, J.S.) pp. 55-64. SOS Publications, Fair Haven, USA.
  4. Auffret, M. and Oubella, R. (1997) Hemocyte aggregation in the oyster Crassostrea gigas: in vitro measurement and experimental modulation by xenobiotics. Comparative Biochemistry and Physiology Part A, 118: 705-712. https://doi.org/10.1016/S0300-9629(97)00017-0
  5. Blasco, J., Puppo, J. (1999) Effect of heavy metals (Cu, Cd and Pb) on aspartate and alanine aminotransferase in Ruditapes philippinarum (Mollusca: Bivalvia). Comparative Biochemistry and Physiology Part C, 122: 253-263. https://doi.org/10.1016/S0305-0491(99)00006-1
  6. Brousseau, P., Pellerin, J. Morin, Y., Cyr, D., Blakley, B., Boermans, H. and Fournier, M. (1999) Flow cytometry as a tool to demonstrate the disturbance of phagocytosis in the clam Mya arenaria following in vitro exposure to heavy metals. Toxicology, 142: 145-156. https://doi.org/10.1016/S0300-483X(99)00165-1
  7. Cajaraville, M., Maria, P., Bebianno, J., Blasco, J., Porte, C., Sarasquete, C. and Viarengo, A. (2002) The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach. The Science of The Total Environment, 247: 295-311.
  8. Cheng, T. (1988) In vivo effects of heavy metals on cellular defense mechanisms of Crassostrea virginica: Total and differential cell counts. Journal of Invertebrate Pathology, 51: 207-214. https://doi.org/10.1016/0022-2011(88)90027-4
  9. Coughlan, B.M., Hartl, M.G.J., O'Reilly, S.J., Sheehan, D., Morthersill, C., van Pelt, F.N.A.M., O'Halloran, J. and O'Brien, N.M. (2002) Detecting genotoxicity using the Comet assay following chronic exposure of Manila clam Tapes semidecussatus to polluted estuarine sediments. Marine Pollution Bulletin, 44: 1359-1365. https://doi.org/10.1016/S0025-326X(02)00254-0
  10. Dailianis, S. (2009) Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: A possible involvement of $Na^+/H^+$ exchanger in the induction of cadmium toxic effects. Fish & Shellfish Immunology, 27: 446-453. https://doi.org/10.1016/j.fsi.2009.06.016
  11. Davey, H.M. and Kell, D.B. (1996) Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiology Review, 60: 641-696.
  12. Fisher, W.S. (1988) Environmental influence on host response; environmental influence on bivalve hemocyte function. American Fisheries Society Special Publication, 18: 225-237.
  13. Galan, A., Garcia-Bermejo, L., Troyano, A., Vilaboa, N.E., Fernandez, C., de Blas, E. and Aller, P. (2001) The role of intracellular oxidation in death induction (apoptosis and necrosis) in human promonocytic cells treated with stress inducers (cadmium, heat, X-rays). European Journal of Cell Biology, 80: 312-320. https://doi.org/10.1078/0171-9335-00159
  14. Gauthier-Clerc, S., Pellerin, J. and Amiard, J.C. (2006) Estradiol-17 beta and testosterone concentrations in male and female (Mya aenaria) (Molusca Bivalvia) during the reproductive cycle. General and Comparative Endocrinololgy, 145: 133–139.
  15. Giamberini, L. and Cajaraville, M.P. (2005) Lysosomal responses in the digestive gland of the freshwater mussel, Dreissena polymorpha, experimentally exposed to cadmium. Environmental Research, 98: 210-214. https://doi.org/10.1016/j.envres.2004.11.003
  16. Gielazyn, M.L., Ringwood, A.H. Piegorsch, W.W. and Stancyk, S.E. (2003) Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg). Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 542: 15-22. https://doi.org/10.1016/j.mrgentox.2003.07.010
  17. Goedken, M. and De Guise, S. (2004) Flow cytometry as a tool to quantify oyster defence mechanisms. Fish & Shellfish Immunology, 16: 539-552. https://doi.org/10.1016/j.fsi.2003.09.009
  18. Goldberg, E.D. (1986) The mussel watch concept. Environmental Monitoring and Assessment, 7: 91-103. https://doi.org/10.1007/BF00398031
  19. Goulletquer, P. (1997) A Biography of the Manila Clam Tapes philippinarum. IFREMER, RIDRV-97.02/RA, LaTremblade, France.
  20. Hegaret, H., da Silva, P.M., Wikfors, G.H., Lambert, C., De Bettignies, T., Shumway, S.E. and Soudant, P. (2007) Hemocyte responses of Manila clams, Ruditapes philippinarum, with varying parasite, Perkinsus olseni, severity to toxic-algal exposures. Aquatic Toxicology, 84: 469-479. https://doi.org/10.1016/j.aquatox.2007.07.007
  21. Hegaret, H., Wikfors, G.H. and Soudant, P. (2003a) Flow-cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation: I. Haemocyte types and morphology. Journal of Experimental Marine Biology and Ecology, 293: 237-248. https://doi.org/10.1016/S0022-0981(03)00236-3
  22. Hegaret, H., Wikfors, G.H. and Soudant, P. (2003b) Flow cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation: II. Haemocyte functions: aggregation, viability, phagocytosis, and respiratory burst. Journal of Experimental Marine Biology and Ecology, 293: 249-265. https://doi.org/10.1016/S0022-0981(03)00235-1
  23. Ji, J., Choi, H.J. and Ahn, I.Y. (2006) Evaluation of Manila clam Ruditapes philippinarum as a sentinel species for metal pollution monitoring in estuarine tidal flats of Korea: Effects of size, sex, and spawning on baseline accumulation. Marine Pollution Bulletin, 52: 447-453. https://doi.org/10.1016/j.marpolbul.2005.12.012
  24. Kang, S.G., Park, S.W. and Kim, Y.G. (2000) The morphology of the hemocytes of the clam, Ruditapes philippinarum (Mollusca: Bivalvia). Journal of Fish Pathology, 13: 129-136. [in Korean]
  25. Ketata, I., Smaoui-Damak, W., Guermazi, F., Rebai, T. and Hamza-Chaffai, A. (2007) In situ endocrine disrupting effects of cadmium on the reproduction of Ruditapes decussates. Comparative Biochemistry and Physiology Part C, 146: 415-430. https://doi.org/10.1016/j.cbpa.2006.11.018
  26. Kim, H.Y., Kim, J.C., Kim, S.Y., Lee, J.H., Jang, Y.M., Lee, M.S., Park, J.S. and Lee, K.H. (2007) Monitoring of heavy metals in fishes in Korea -As, Cd, Cu, Pb, Mn, Zn, total Hg. Korean Journal of Food Science and Technology, 39: 353-359. [in Korean]
  27. Kim, J. and Soh, J. (2009) Cadmium-induced apoptosis is mediated by the translocation of AIF to the nucleus in rat testes. Toxicology Letters, 188: 45-51. https://doi.org/10.1016/j.toxlet.2009.03.006
  28. Kim, J.H., Im, C.W., Kim, P.J. and Park, J.H. (2003) Heavy metals in shellfishes around the South Coast of Korea. Journal of Food Hygiene and. Safety, 18: 125-132. [in Korean]
  29. Lambert, C., Soudant, P., Choquet, G., Paillard, C. (2003) Measurement of Crassostrea gigas hemocyte oxidative metabolism by flow cytometry and the inhibiting capacity of pathogenic vibrios. Fish & Shellfish Immunology, 15: 225-240. https://doi.org/10.1016/S1050-4648(02)00160-2
  30. Lau, P.S. and Wong, H.L. (2003) Effect of size, tissue parts and location on six biochemical markers in the green-lipped mussel, Perna viridis. Marine Pollution Bulletin, 46: 1563-1572. https://doi.org/10.1016/S0025-326X(03)00321-7
  31. Lee, Y., and Shacter, E. (1999) Oxidative stress inhibits apoptosis in human lymphoma cells. Journal of Biological Chemistry, 274: 19792-19798. https://doi.org/10.1074/jbc.274.28.19792
  32. Marigomez, I., Sotoa, M., Orbeaa, A., Cancioa, I. and Cajaravillea, M.P. (2004) Biomonitoring of environmental pollution along the Basque coast, using molecular, cellular and tissue-level biomarkers: an integrative approach. Elsevier Oceanography Series, 70: 335-364. https://doi.org/10.1016/S0422-9894(04)80052-7
  33. Micic, M., Bihari, N., Labura, Z., Muller, W.E.G. and Batel, R. (2001) Induction of apoptosis in the blue mussel Mytilus galloprovincialis by tri-n-butyltin chloride. Aquatic Toxicololgy, 55: 61-73. https://doi.org/10.1016/S0166-445X(01)00156-4
  34. O'Connor, T.P. (2002) National distribution of chemical concentration in mussels and oysters in the USA. Marine Environmental Research, 53: 117-143. https://doi.org/10.1016/S0141-1136(01)00116-7
  35. Park, K.I., Park, H.S., Kim, J.M., Park, Y.J., Hong, J.S. and Choi, K.S. (2006) Flow cytometric assesment of immune parameters of the Manila clam (Ruditapes philippinarum). Journal of Korean Fisheries Society, 39: 123-131. [in Korean] https://doi.org/10.5657/kfas.2006.39.spc1.123
  36. Pipe, R.K. and Coles, J.A. (1995) Environmental contaminants influencing immunefunction in marine bivalve molluscs. Fish & Shellfish Immunology, 5: 581-595. https://doi.org/10.1016/S1050-4648(95)80043-3
  37. Rank, J., Jensen, K. and Jespersen, P.H. (2005) Monitoring DNA damage in indigenous blue mussels (Mytilus edulis) sampled from coastal sites in Denmark. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 585: 33-42. https://doi.org/10.1016/j.mrgentox.2005.04.008
  38. Risso-de Faverney, C., Devaux, A., Lafaurie, M., Girard, J.P., Bailly, B. and Rahmani, R. (2001) Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygene species. Aquatic Toxicology, 53: 65-76. https://doi.org/10.1016/S0166-445X(00)00154-5
  39. Rittschof, D. and McClellan-Green, P. (2005) Molluscs as multidisciplinary models in environment toxicology. Marine Pollution Bulletin, 50: 369-373. https://doi.org/10.1016/j.marpolbul.2005.02.008
  40. Tsangaris, G.Th. and Tzortzatou-Stathopoulou, F. (1998) Cadmium induces apoptosis differentially on immune system cell lines. Toxicology, 128: 143-150. https://doi.org/10.1016/S0300-483X(98)00032-8
  41. Waalkes, P.M. (2000) Cadmium carcinogenesis in review. Journal of Inorganic Biochemistry, 79: 241–244.
  42. Waisberg, M., Joseph, P., Hale, B. and Beyersmann, D. (2003) Molecular mechanisms of cadmium carcinogenesis. Toxicology, 192: 95-117. https://doi.org/10.1016/S0300-483X(03)00305-6
  43. Walsh, G.M., Dewson, G., Wardlaw, A.J., Levi-Schaffer, F. and Moqbel, R. (1998) A comparative study of different methods for the assessment of apoptosis and necrosis in human eosinophils. Journal of Immunological Methods, 217: 153-163. https://doi.org/10.1016/S0022-1759(98)00103-3
  44. Watanabe, M., Henmi, K., Ogawa, K. and Suzuki, T. (2003) Cadmium-dependent generation of reactive oxygen species and mitochondrial DNA breaks in photosynthetic and non-photosynthetic strains of Euglena gracilis. Comparative Biochemistry and Physiology Part C, 134: 227-234.