{"id":496,"date":"2015-01-22T07:05:24","date_gmt":"2015-01-22T07:05:24","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=496"},"modified":"2020-04-25T06:24:42","modified_gmt":"2020-04-25T06:24:42","slug":"cytotoxic-effect-of-coelomic-fluid-of-earthworm-eudrilus-eugeniae","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol1no2\/cytotoxic-effect-of-coelomic-fluid-of-earthworm-eudrilus-eugeniae\/","title":{"rendered":"Cytotoxic Effect of Coelomic Fluid of Earthworm Eudrilus Eugeniae"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Coelomic fluid (CF) of earthworms contain cytolytic, antibacterial and\/or agglutinating components1-5 <\/sup>(, Roch et al., 1989; Valembois et al., 1982; Lassegues et al., 1989 Mohrig et al., 1996).\u00a0 The primary function of this cytolytic system may be to destroy membranes of foreign cells which causes cell death by cytosol release.\u00a0 Cytotoxicity of CF of Eisenia fetida<\/em> is studied by a number of workers6-9<\/sup> (Suzuki and Cooper, 1995; Cossarizza et al., 1996; Quaglino et al., 1996).. Lange et al., 1999,)\u00a0 Many peptide molecules from the CF of Eisenia fetida<\/em> were identified, purified and characterized10-11<\/sup> (Yamaji-Hasegawa\u00a0 et al., 2003:Konig et al., 2004.). This is a preliminary report on the cytotoxic effect of coelomic fluid of Eudrilus eugeniae.<\/em>\u00a0 The cytoxicity was checked using BHK21<\/sub> cells.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Earthworms Eudrilus eugeniae<\/em> were obtained from GKVK (Gandhi Krishi Vignyan Kendra) Bangalore, and were cultured. BHK21<\/sub> cells were procured from NCCS, courtesy IAH & VB, Bangalore. TBP, NBCS & cell culture media were from Sigma \u2013 Aldrich fine chemicals.\u00a0 All other chemicals were of analytical grade purchased from S.D. fine chemicals, Mumbai, India.<\/p>\n

Culturing of Earthworms:<\/strong><\/p>\n

Earthworms Eudrilus eugeniae <\/em>were cultured on suitable bedding in plastic trays.\u00a0 Under ideal conditions, they were fed with organic substances from plant and animal origin. The feed stock chosen were dairy and beef manures which is considered as best natural food for the Earthworms12<\/sup>. 75%- 80% moisture content were maintained so that the average worm weight and the reproduction rate increased11<\/sup>. As the worms are aerobic, sufficient aeration was provided.<\/p>\n

Extraction of coelomic fluid<\/strong><\/p>\n

Extraction of Coelomic fluid from chosen earthworm species was performed according to the method of Kale R., (1991)13<\/sup>. The earthworms were washed with cold water 3-4 times at room temperature and their body surface were dried on filter paper.\u00a0 Then they were subjected to cold shock. Cold shock was performed using an icepack at a temperature of 0-2\u00b0C.\u00a0 The icepack was slowly rubbed against the body of the earthworms. The procedure is repeated for 3- 4 times. The coelomic fluid obtained from the body of the earthworms was then collected and stored in vials.<\/p>\n

Estimation of protein<\/strong><\/p>\n

Protein was measured by the method of Lowry et al., (1951)14<\/sup> using bovine serum albumin\u00a0 standard (0-75\u00b5g).<\/p>\n

Culture of BHK21<\/sub> cells<\/strong><\/p>\n

BHK21<\/sub> cells were first grown MD bottles using Sigma media supplemented with 7% New Born Calf Serum (NBCS) and 10% Tryptose Phosphate Broth (TBP).\u00a0 The cultures were incubated in CO2<\/sub> incubator for 48hr at 37\u00b0C.\u00a0 The monolayer formed was further trypsinized and cultures were transferred to different plates.<\/p>\n

Assay of cytotoxic activity<\/strong><\/p>\n

445\u00b5l of the medium containing BHK21<\/sub> cells were taken in two different microtitre plates and 5\u00b5l containing 10\u00b5g protein of sample (CF of Eudrilus eugeniae<\/em>) was added and serially diluted.\u00a0 The plates were observed after three days under invert microscope at 5x magnification.\u00a0 Control was maintained without adding the sample.\u00a0\u00a0 The experiments were performed in triplicates.<\/p>\n

Results and Discussions<\/strong><\/p>\n

The CF treated BHK21<\/sub> cells shows cytopathic effect on the 3rd<\/sup> day, upto a concentration of 0.2\u00b5g\/ml (Fig.1).\u00a0 The effect was dependent on concentration as evidenced in the figure. The monolayer appeared from well-5 containing protein concentration 0.02\u00b5g\/ml. \u00a0These experimental results were reproducible.<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 1:<\/strong><\/p>\n

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Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

The mechanism of action of cytolyic proteins from CF of other genus of earthworm Eisenia fetida<\/em> are worked out in other laboratories. The cytolytic protein \u2018Eiseniapore\u2019, a 38 kDa protein from coelomic fluid of the earthworm Eisenia fetida<\/em> induces cytolysis by forming pores, which is confirmed by electron microscopy of erythrocyte membranes treated with the protein9 <\/sup>(Lange et al., 1999). \u2018Lysenin\u2019, from the coelomic fluid of the earthworm Eisenia foetida<\/em>, binds to the plasma membrane of target cells via sphingomyelin10<\/sup> (Yamaji-Hasegawa\u00a0 et al., 2003). When earthworm coelomocytes from Eisenia fetida<\/em> are incubated with the tumor target cells – K562,\u00a0 damage to target membranes has been observed by scanning electron microscopy and transmission electron microscopy6-8 <\/sup>(Suzuki and Cooper, 1995; Quaglino et al., 1996:Cossarizza et al., 1996;). Hemolytic proteins both from coelomocytes (CL39,41<\/sub>) and coelomic fluid (H1\u22123<\/sub>) of wildtype E. fetida <\/em>have been isolated and assigned to fetidin and lysenin using mass spectrometry and bioinformatic tools11<\/sup> (Konig et al., 2004).\u00a0 Efforts are being made to purify the molecule\/s responsible for the cytotoxicity from CF of Eudrilus eugeniae<\/em>. Further studies on \u00a0their mechanism of action may throw light on drug designing.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Valembois P., Roch P., Lassegues M and Cassand P, 1982. Antibacterial activity of the hemolytic system from the earthworm Eisenia fetida Andrei<\/em>, J. Invertebr. Pathol.\u00a0<\/em>40<\/em>, 21-27.<\/li>\n
  2. Roch P., Davant N and Lassegues M, 1984. Isolation of agglutinins from lysins in the earthworm coelomic fluid by gel filtration followed chromatofocusing, J.Chromatogr.<\/em>290<\/em>, 231-235.<\/li>\n
  3. \u00a0Roch P., Canicatti C and Valembois P, 1989. Interaction between earthworm\u00a0 hemolysins and sheep red blood cell membranes, Biochim. Biophys. Acta 2<\/em>, 193-198<\/li>\n
  4. Lassegues M., Roch P and Valembois P, 1989. Antibacterial activity of Eisenia fetida <\/em>\u00a0Andrei<\/em> coelomic fluid: Evidence, induction and animal protection, J. Invertebr.<\/em>\u00a0Pathol.\u00a0 <\/em>1-6.<\/li>\n
  5. Mohrig W., Eue I., Kauschke E and Hennicke F, 1996. Cross reactivity of hemolytic\u00a0and hemeagglutinating proteins in the coelomic fluid of lumbricidae (annelida),Comp. Biochem. Physiol. 115A<\/em>, 19-30.<\/li>\n
  6. \u00a0Suzuki M.M and Cooper E.L, 1995, 1995. spontaneous cytotoxic earthworm leukocytes kill K562 tumor cells. Zool. Sci. 12<\/em>, 443-451.<\/li>\n
  7. Cossarizza A., Cooper E.L., Suzuki M.M., Salvioloi S., Capri M., Gri G., Quaglino \u00a0and Franceschi C, 1996. Earthworm leukocytes that are not phagocytic and cross react with several human epitopes can kill human tumor cell lines, Exp. Cell Res. 224<\/em>,174-182.<\/li>\n
  8. Quaglino D., Cooper E.L., Salvioloi S., Capri M., Suzuki M.M., Ronchetti I,P,Franceschi C and Cossarizza A, 1996. Earthworm coelomocytes in vitro: cellular\u00a0features and granuloma formatin during cytotoxic activity against the mammalian\u00a0tumor cell target \u00a0K562., Eur. J. Cell Biol. 70<\/em>, 278-288.<\/li>\n
  9. Lange S., Kauschke E., Mohrig W and Cooper E. L , 1999. Biochemical\u00a0characteristics of Eiseniapore, a pore forming protein in the coelomic fluid of earthworms, Eur. J. Biochem. 262<\/em>, 547-556.<\/li>\n
  10. Yamaji-Hasegawa A., Makino A., Baba T., Senoh Y., Kimura-Suda H., Sato S.\u00a0B.,\u00a0Terada N., Ohno S., Kiyokawa E., Umeda M. and Kobayashi\u00a0T,<\/strong> 2003.<\/li>\n
  11. \u00a0Oligomerization and Pore Formation of a Sphingomyelin-specific Toxin, Lysenin, J. Biol. Chem. 278, 22762\u201322770.<\/li>\n
  12. \u00a0Konig S., Wagner F., Kauschke E and Eue I, 2004, Sequence analysis of earthworm hemolysins. Spectroscopy 18<\/em>, 347-353.<\/li>\n
  13. George, 2004, Feasibility of developing the organic and transitional form market \u00a0processing municipal and form organic wastes using large scale vermin composting.\u00a0Good earth organic resources group, http\/\/www.alternativeorganic.com. Kale R., D., 1991. Vermiculture: scope for new Biotechnology. In: Earthworms \u00a0<\/em>resources and vermiculture<\/em>. (Ed. Director, ZSI, Calcutta, India). 105-108.<\/li>\n
  14. \u00a0Lowry O.H., Rosebrough N.,J, Farral and Randall R.,J, 1951. Protein measuremen with the folin-phenol reagent. J. Biol. Chem. 193<\/em>, 265-275.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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