Fisheries and Aquatic Sciences

The Korean Society of Fisheries and Aquatic Science (한국수산과학회)
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Cellular Biomarker of Membrane Stability and Hydrolytic Enzyme Activity in the Hemocytes of Benzo(a)pyrene-exposed Pacific oyster, Crassostrea gigas

  • Jo Qtae (Aquaculture Department, National Fisheries Research and Development Institute) ;
  • Choy Eun-Jung (Aquatic Life Medicine Department, Pukyong National University) ;
  • Park Doo Won (Biotechnology Research Center, National Fisheries Research and Development Institute) ;
  • Jee Young-Ju (Aquaculture Department, National Fisheries Research and Development Institute) ;
  • Kim Sung Yeon (Aquaculture Department, National Fisheries Research and Development Institute) ;
  • Kim Yoon (Aquaculture Department, National Fisheries Research and Development Institute)
  • Published : 2002.12.01
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Abstract

The Pacific oysters, Crassostrea gigas, were stressed with different concentrations of benzo(a) pyrene and depurated to determine the hemocyte lysosomal membrane stability and hydrolytic enzymatic activity as a biomarker candidate to the chemical, using NRR (neutral red retention) and API ZYM System, respectively. The membrane damage measured as NRR decrease was significant with the increase of chemical concentration and exposure time (P<0.05), providing a possible tool for biomarker. Interestingly, the control showed intrinsic stress probably due to captive life in the laboratory, and a recovering trend was also found during the depuration. The benzo(a)pyrene-exposed oysters showed increased enzyme activities in alkaline phosphatase, esterase (C4), acid phosphatase, naphthol-AS-BI-phospho­hydrolase, $\beta$-galactosidase, $\beta$-glucuronidase, and N-acetyl- $\beta$-glucosaminidase. Of them, only two enzymes, acid phosphatase and alkaline phosphatase, showed some potential available for the generation of enzymatic biomarker in the oyster. The results are suggestive of the potential availability of the cellular and enzymatic properties as a biomarker. However, considering that a robust biomarker should be insensitive to natural stress coming from normal physiological variation, but sensitive to pollutants, a concept of intrinsic stress the animal possesses should be taken into consideration. This reflects the necessity of further research on the intrinsic stress affecting the cellular and enzymatic properties of the chemical­stressed oysters prior to using the data as a biomarker.

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References

  1. Anderson, R.S. 1996. Interaction of Perkinsus marin us with humoral factors and hemocytes of Crassostrea virginica. J. Shellfish Res., 5, 127-134
  2. Baccino, F.M. 1978. Selected patterns of lysosomal response in hepatocytic injury. In Biochemical Mechanisms of Liver Injury, T.F. Slater, ed. Academic Press, New York, pp. 518-557
  3. Bachere, E. 1995. Knowledge and research prospects in marine mollusk and crustacean immunology. Aquaculture, 132, 17-32 https://doi.org/10.1016/0044-8486(94)00389-6
  4. Bachere, E., D. Chagot and H. Grizel. 1988. Separation of Ctsssostree gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation. Dev. Compo lnmunol., 14, 261-168
  5. Bard, S.M. 2000. Multixenobiotic resistance as a cellular defence mechanism in aquatic organisms. Aquat. Toxicol., 48, 357-389 https://doi.org/10.1016/S0166-445X(00)00088-6
  6. Beckmann, N., M.P. Morse and C.M. Moore. 1992. Comparative study of phagocytosis in nomal and diseased hemocytes of bivalve molluscs Mya arenaria. J. Invertebr. Pathol., 59, 124-132 https://doi.org/10.1016/0022-2011(92)90022-V
  7. Borenfreund, E. and J.A. Puerner. 1984. Simple quantitive procedure using monolayer cultures for cytotoxicity assays. J Tissue Cult. Meth., 9, 7-9 https://doi.org/10.1007/BF01666038
  8. Camus, L., B.E. Grosvic, J.F. Borseth, M.B. Jones and M.H. Depledge. 2000. Stability of lysosomal and cell membranes in haemocytes of the common mussel (Mytilus edulis) : effect of low temperatures. Mar. Environ. Res., 50, 325-329 https://doi.org/10.1016/S0141-1136(00)00056-8
  9. Carballal, M, C. Lopez, C. Azevedo and A. Villalba. 1997. Enzymes involved in defence functions of hemocytes of mussel Mytilus galloprovincialis. J. Invertebr. Pathol., 70, 96-105 https://doi.org/10.1006/jipa.1997.4670
  10. Cheng, T.C. 1988. In Vitro effects of heavy metals on cellular defence mechanisms of Crassostrea virginica: Phagocytic and endocytic indices. J. Invertebr. Pathol., 51, 215-220 https://doi.org/10.1016/0022-2011(88)90028-6
  11. Cheng, T.C. 1990. Effects of in vitro exposure of Crassostrea virginica to heavy metals of haemocyte viability and activity levels of lysosomal enzymes. In Pathology in Marine Science, F.O. Perkins and T.C. Cheng, eds. Academic Press, San Diego, USA, pp. 513-524
  12. Chu, F.-L.E., A.K Volety, R.C. Hale and Y. Huang. 2002. Cellular responses and disease expression in oysters (Crassostrea vitginice) exposed to suspended field - contaminated sediments. Mar. Environ. Res., 53, 17-35 https://doi.org/10.1016/S0141-1136(01)00104-0
  13. Domouhtsidou, G.P. and V.K. Dimitriadis. 2001. Lysosomal and lipid alterations in the digestive gland of mussels, Mytilus galloprovincialis (L.) as biomarkers of environmental stress. Environ. Pollut., 115, 123-137 https://doi.org/10.1016/S0269-7491(00)00233-5
  14. Dyrynda, E.A., R.K. Pipe and N.A. Ratcliffe. 1995. Host defence mechanisms in marine invertebrate larvae. Fish Shellfish Immunol., 5, 569-580 https://doi.org/10.1016/S1050-4648(95)80042-5
  15. Gallager, S.M. and R. Mann. 1986. Growth and Survival of larvae of Mercenaria mercenaria (L.) and Crassostrea virginica (Gmelin) relative to broodstock conditioning and lipid content of eggs. Aquaculture, 56, 105-121 https://doi.org/10.1016/0044-8486(86)90021-9
  16. Grundy. M.M., N.A. Ratcliffe and M.N. Moore. 1996. Immune inhibition in marine mussels by polyclclic aromatic hydrocarbons. Mar. Environ. Res., 42, 187-190 https://doi.org/10.1016/0141-1136(95)00033-X
  17. Guillard, R.R.L. and J.H. Ryther. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana and Detonula confervaceae Hustedt (Cleve). Can. J. Microbiol., 8, 229-239 https://doi.org/10.1139/m62-029
  18. Holland, D.L. 1978. Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. In Biochemical and Biophysical Perspectives in Marine Biology, P. L. Malins and J.R. Sargent, eds. Academic Press, London and New York, pp. 85-123
  19. Holm, N.P. and J. Shapiro. 1984. An examination of lipid reserves and the nutritional status of Daphnia pulex fed Aphanizomenon flos-aquae. Limnol. Oceanogra., 29, 1137-1140 https://doi.org/10.4319/lo.1984.29.5.1137
  20. Kang, C.-K., M.S. Park, P.-Y. Lee, W-.J. Choi and W-.C. Lee. 2000. Seasonal variations in condition, reproductive activity, and biochemical composition of the Pacific oyster, Crassostrea gigas (THUNBERG), in suspended culture in two coastal bays of Korea. J. Shellfish Res., 19, 771-778
  21. Kenchington, E.L.R. 1994. Spacial and temporal variation in adductor muscle RNA/DNA ratio in sea scallops (Placo-pecten megellsnicus) in the Bay of Fundy, Canada. J. Shellfish Res., 13, 19-24
  22. Livingstone, D.R., J.K. Chipman, M.D. Lowe, C. Minier, C.L. Mitchelmore, M.N. Moore, L.D. Peters and R.K. Pipe. 2000. Development of biomarkers to detect the effects of organic pollution on aquatic invertebrates: revent molecular, genotixic, cellular and immunological studies on the common mussel (Mytilus edulis L.) and other mytilids. Int. J. Environ. Pollut., 13, 56-91 https://doi.org/10.1504/IJEP.2000.002311
  23. Lopez, C., M.J. Carballal, C. Azevedo and A. Villalba. 1997. Differential phagocytic ability of the circulating haemocyte types of the carpet shell clam Ruditapes decussates (Mollusca: Bivalvia). Dis. Aquat. Org., 30, 209-215 https://doi.org/10.3354/dao030209
  24. Lowe, D.M. 1988. Alterations in cellular structure of Mytilus edulis resulting from exposure to environmental contaminants under field and experimental conditions. Mar. Ecol. Prog. Ser., 46, 91-100 https://doi.org/10.3354/meps046091
  25. Lowe, D.M. and V.U. Fossato. 2000. The influence of environmental contaminants on lysosomal activity in the digestive cells of mussels (Mytilus gelloptovincielis) from the Venice Lagoon. Aquat. Toxicol., 48, 75-85 https://doi.org/10.1016/S0166-445X(99)00054-5
  26. Lowe, D.M., M.N. Moore and B.M. Evans. 1992. Contanminant impact on interations of molecular probes with lysosomes in lyving hepatocytes from dab Limanda limanda. Mar. Ecol. Prog. Ser., 91, 135-140 https://doi.org/10.3354/meps091135
  27. Lowe, D.M., C. Soverchia and M.N. Moore. 1995a. Lysosomal membrane responses in the blood and digestive cells of mussels experimentally exposed to flouranthene. Aquatic Toxicol., 33, 105-112 https://doi.org/10.1016/0166-445X(95)00015-V
  28. Lowe, D.M., U. Valentono and M.H. Depledge. 1995b. Contaminant- induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: an in vitro study. Mar. Ecol. Prog. Ser., 129, 189-196 https://doi.org/10.3354/meps129189
  29. Moore, M.N. 1976. Cytochemical demonstration of latency of lysosomal hydrolases of digestive cells of the common mussel, Mytilus edulis, and changes induced by thermal stress. Cell Tissue Res., 175, 279-287
  30. Moore, M.N. 1985. Cellular responses to pollutants. Mar. Pollut. Bull., 16, 134-139 https://doi.org/10.1016/0025-326X(85)90003-7
  31. Moore, C.A. and S.R. Gelder. 1985. Demonstration of lysosomal enzymes in hemocytes of Mercenaria mercenaria (Mollusca: Bivalvia). Trans. Am. Microsc. Soc., 104, 242-249 https://doi.org/10.2307/3226436
  32. Moore, M.N. and D.M. Lowe. 1985. Cytological and cytochemical measurements. In The effects of Stress and Pollution on marine Animals, B.L. Bayne, ed. Praeger Scientific, New York, pp. 46-74
  33. Moore, M.N., R.K. Pipe, S.V. Farrar, S. Thomson and P. Donkin. 1987. Lysosomal and microsomal responses to oil-derived hydrocarbons in Littorina littorea. In Oceanic Processes in Marine Pollution, J.R. Capuzzo and J.R. Kester, eds. ER Kriegger, Malabar, FL, USA, pp. 89-96
  34. NFRDI Report. 1997. Studies on the bad seed collection in oyster growing area and investigation of the new oyster seed collection area. National Fisheries Research and Development Institute, Pusan, Korea, pp. 12-26 (in Korean)
  35. Nicholson, S. 1999. Cytological and physiological biomarker responses from green mussels, Perna viridis (L.) transplanted to contanminated sites in Hong Kong coastal waters. Mar. Pollut. Bull., 39, 261-268 https://doi.org/10.1016/S0025-326X(98)90189-8
  36. Nicholson, S. 2001. Ecocytological and toxicological responses to copper in Perna viridis (L.) (Bivalbia: Mytilidae) haemocyte lysosomal membranes. Chemosphere, 45, 399-407 https://doi.org/10.1016/S0045-6535(01)00039-X
  37. Nott, J.A. and M.N. Moore. 1987. Effects of polycyclic aromatic hydrocarbons on molluscan lysosomes and endoplasmic reticulum. Histochem. J., 19, 357-368 https://doi.org/10.1007/BF01680453
  38. Paon, L.A. and E.L.R. Kenchington. 1995. Changes in somatic and reproductive tissues during artificial conditioning of the sea scallop, Placopecten magellanicus (Gmellin, 1791). J. Shellfish Res., 14, 53-58
  39. Park, M.S., H.J. Lim, Q. Jo, J.S. Yoo and M. Jeon. 1999. Assessment of reproductive health in the wild seed oysters, Crassostrea gigas, from two locations in Korea. J. Shellfish Res., 18, 445-450
  40. Petrovic, S., B. Ozretic, M. Krajnovic-Ozretic and D. Bobinac. 2001. Lysosomal membrane stability and methallothioneins in digestive gland of mussels (Mytilus galloprovincialis Lam.) as biomarkers in a field study. Mar. Pollut. Bull., 42, 1373-1378 https://doi.org/10.1016/S0025-326X(01)00167-9
  41. Pieters, H., J.H. Kluytmans, W. Zurberg and I. Zandee. 1979. The influence of seasonal changes on energy metabolism in Mytilus edulis (L) I. Growth rate and biochemical composition in relation to environmental parameters and spawning. In Cyclic Phenomena in Marine Plants and Animals, E Naylor and R.H. Hartnoll, eds. Pergamon Press, NY, USA. pp. 285-292
  42. Reader, S.J., V. Blackwell, R. O'Hara, R.H. Clothier, G. Griffin and M. Balls. 1989. A vital dye release method for assessing the short term cytotoxic effects of chemicals and formulations. ATLA, 17, 28-37
  43. Renwrantz, L., T. Yoshino, T.C. Cheng and K. Auld. 1979. Size determination of hemocytes from the American oyster, Crassostrea virginica, and the description of a phagocytosis mechanism. Zool. J. Physiol., 83, 1-12
  44. Ringwood, A.H., D.E. Conners and J. Hoguet. 1998. Effects of natural and anthropogenic stressors on lysosomal destabilization in oysters Crassostrea virginica. Mar. Ecol. Prog. Ser., 166, 163-171 https://doi.org/10.3354/meps166163
  45. Ruiz, C., M. Abad, F. Sedano, L.O. Garcia-Martin and J.L. Sanchez Lopes. 1992. Influence of seasonal environmental changes on the gamete production and biochemical composition of Crassostrea gigas (Thunberg) in suspended culture in EL Grove, Galicia, Spain. J. Exp. Mar. BioI. Ecol., 155, 249-262 https://doi.org/10.1016/0022-0981(92)90066-J
  46. Suresh, K. and A Mohandas. 1990. Hemolymph acid phosphatase activity pattern in copper-stressed bivalves. J. Invertebr. Pathol., 55, 118-125 https://doi.org/10.1016/0022-2011(90)90041-4
  47. Sutherland, J.B., F. Rafic, A.A. Khan and C.E. Cerniglia. 1995. Mechanism of Polycyclic aromatic hydrocarbon degradation,. In Microbial Transformation and Degration of Toxic Organic Chemicals, L.Y. Yong and C.E. Cerniglia, ed, pp. 269-300. Wiley-Liss, New York, USA
  48. Torreilles, J., M.C. Guerin and P. Roch. 1997. Perioxidaserelease associated with phagocytosis in Mytilus galloprovincialis haemocytes. Dev. Compo Immunol., 21, 267-275 https://doi.org/10.1016/S0145-305X(96)00034-1
  49. Tripp, M.R. 1992. Phagocytosis by hemocytes of hard clam, Mercenaria mercenaria. J. Invertbr. Pathol., 59, 222-227 https://doi.org/10.1016/0022-2011(92)90125-N
  50. Winstead, J.T. 1995. Digestive tubule atrophy in eastern oysters, Crsssostree virginica (Gmelin, 1791), exposed to salinity and starvation stress. J. Shellfish Res., 14, 105-111
  51. Xue, Q. and T. Renault. 2000. Enzymatic activities in European flat oyster, Ostrea edulis, and Pacific oyster, Crassostrea gigas, hemolymph. J. Invertebr. Pathol., 76, 155-163 https://doi.org/10.1006/jipa.2000.4965