The lysosomal system in bivalves is sensitive to changes in the intra and extra cellular environment. The activity pattern of hemolymph acid phosphatase, a marker enzyme of the lysosomal system, was studied in the green mussel (Perna viridis) exposed to sub lethal concentration of 25 μg/l of copper and mercury at 24 hr, 72 hr, 7th day and 14th day. The results of the study revealed that at the sub lethal level, copper can induce remarkably higher acid phosphatase activity (hypersynthesis) than mercury. Hypersynthesis of the enzyme occurs much earlier in copper treated mussels than observed in mercury exposed mussels, but on continued exposure, the acid phosphatase activity shows a considerable decline

Sharma, S R Krupesha

Thomas, Sujitha

CMFRI Digital Repository

226Journal of the Marine Biological Association of India (2007)S. R. Krupesha Sharma and Sujitha ThomasJ. Mar. Biol. Ass. India, 49 (2) : 226 - 229, July - December 2007Because of their coastal and estuarinedistribution, commercially exploited mussel bedsare often subjected to sewage and industrialpollution and these bivalves accumulate andconcentrate most of the potentially toxic heavymetals in their tissues to concentrationssignificantly above ambient level in theenvironment (Roberts, 1976).  Many physiologicalprocesses including activity of many lysosomalhydrolytic enzymes are inhibited by heavy metals(Suresh and Mohandas, 1989) even though thesemetals may also activate certain enzymes (Have,1969). The lysosomal system is very sensitive tochanges in the intra and extra cellular environmentdirectly or indirectly in controlling manyphysiological and pathological processes (Chandyand Patel, 1985).Cheng (1983a) suggested that quantitativeassessment of enzymes is a reliable indicator ofstress imposed on the organism by environmentalpollutants like heavy metals. Acid phosphataseactivity pattern in the hemolymph of the copperstressed clam was investigated by Suresh andMohandas (1989).  However, no information isavailable on hemolymph acid phosphatase activityresponse in Perna viridis exposed to copper andmercury. Considering the wide spread distributionof the green mussel along the coastal areas, a studywas carried out to determine the influence of sublethal levels of copper and mercury on the activityof acid phosphatase in the hemolymph.Materials and methodsGreen mussels (65 to 85 mm shell length) werecollected from a natural mussel bed (salinity: 35‰)off Calicut (740 40’ N lat. 110 30’E long.) andimmediately brought to the laboratory.  After twodays of acclimatization, the mussels were dividedinto three groups of 25 mussels with three replicatesfor each group and maintained in 40 litre plasticbasins containing 30 litres of sand-filtered sea water(salinity: 35‰ ±0.5‰; pH: 7.5±0.1; temperature:28±0.5 OC; dissolved oxygen: >85% saturation).The first group was exposed to copper as coppersulphate [(CuSO4).5H2O, E. Merck, Mumbai] atconcentration of 25 µg/l and the second group wasexposed to mercury as mercuric chloride [(HgCl2),E. Merck, Mumbai] at 25 µg/l. The aboveexperimental concentration was selected based onthe acute toxicity (96 hr LC50 for copper, 86µg/l;mercury, 155µg/l) levels (Krishnakumar et al.,Hemolymph acid phosphatase activity in the green mussel (Perna viridis,Linnaeus, 1758) exposed to copper and mercuryS. R. Krupesha Sharma* and 1Sujitha ThomasCalicut Research Centre of Central Marine Fisheries Research InstituteWest Hill Post, Calicut-673 005, Kerala, India. *E-mail: krupeshsharma@gmail.com,1Mangalore Research Centre of CMFRI, Bolar, Mangalore -575 001, Karnataka, India AbstractThe lysosomal system in bivalves is sensitive to changes in the intra and extra cellular environment. Theactivity pattern of hemolymph acid phosphatase, a marker enzyme of the lysosomal system, was studied inthe green mussel (Perna viridis) exposed to sub lethal concentration of 25 µg/l of copper and mercury at 24hr, 72 hr, 7th day and 14th day. The results of the study revealed that at the sub lethal level, copper can induceremarkably higher acid phosphatase activity (hypersynthesis) than mercury. Hypersynthesis of the enzymeoccurs much earlier in copper treated mussels than observed in mercury exposed mussels, but on continuedexposure, the acid phosphatase activity shows a considerable decline.Note227Journal of the Marine Biological Association of India (2007)Hemolymph acid phosphatase activity in green mussel1987). The third group served as control. Waterwas changed daily to replenish the metals.Hemolymph was collected from five mussels ofeach group at zero hr, 24 hr, 72 hr, on 7th and 14thday from the anterior adductor muscle with thehelp of a three ml syringe fitted to a 23 gauzeneedle after making a notch on the shell using atriangular file (Santarem and Figueras, 1995). Thehemolymph (approximately 0.5 ml) was transferredinto a clean test tube for acid phosphatase activityand total protein assay.Acid phosphatase activity in the hemolymphwas determined following the method describedby Andersch and Szcypinski (1947) with slightmodification. Hemolymph sample of 0.1 ml wastransferred into a test tube containing one ml ofchilled 0.1M citrate buffer (pH: 3.6), kept in awater bath at 37 OC and 100µl of p-nitrophenylphosphate disodium salt (20 mg/ml of citratebuffer) was added as substrate to start the reaction.After incubation for one hour, 2 ml of 0.25N NaOHwas added to stop the reaction. The absorbancewas measured at 410 nm in a Jenway 6300spectrophotometer. Different concentrations of p-nitrophenol (HiMedia, Mumbai) in 0.25N NaOHwere used to construct the standard curve. Thevelocity of the reaction was measured in terms ofµmols of p- nitrophenol liberated/ hour. The enzymeactivity was expressed as µmols of p- nitrophenolliberated/mg protein/hour.The enzyme activity of experimental groupsand that of the control group was analysed byemploying paired sample t test using Systatsoftware (Systat Software Inc, USA, Version:10.2.01).Results and discussionThe acid phosphatase activity pattern of themussels exposed to copper and mercury at zerohour, 24 hr, 72 hr, 7th and 14th  day are shown inFig.1 & 2 respectively.Mussels exposed to copper showed significantly(P<0.01) higher enzyme activity than the controlgroups at 24 hr, 72 hr and 7th day.  The highestactivity was observed at 24 hr of exposure to copperwhich was sustained up to 72 hr after which therewas a decline in the activity till seventh day. On14th day the activity in the copper treated groupswas not significantly different from that of themussels in the control groups.In the case of mercury treated groups, theenzyme activity was significantly (P<0.01) higherthan the control groups on the third and seventhdays. The highest activity was observed on seventhday followed by a sharp decline in the enzymeactivity and on 14th day, the enzyme activity in themercury treated groups was not different from thatof the control groups.The toxicity of metals affects the integrity ofthe lysosomal membrane and causes itsdestabilization followed by release of storedFig. 1. Acid phosphatase activity pattern in the mussel(Perna viridis) exposed  to  copper   ( n copper;control; vertical bars indicate standard deviation;n=15)Fig. 2. Acid phosphatase activity pattern in the mussel(Perna  viridis) exposed  to mercury ( n mercury;u control; vertical bars indicate standard deviation;n=15)t228Journal of the Marine Biological Association of India (2007)S. R. Krupesha Sharma and Sujitha Thomaslysosomal hydrolases into the hemolymphcomponent, thereby increasing the activity of theenzyme in hemolymph (Moore, 1976; Baccino,1978). However, the increased activity in thepresent study may not be due to increase in thenumber of hemocytes as these metals do notstimulate hemopoesis in the early hours of exposureto copper and mercury (Cheng, 1987).The difference in acid phosphatase activitypattern between mussels exposed to   copper andmercury could be attributed to the nature ofpathogenesis of the cellular changes caused by thetwo metals. The onset of destabilization leading torelease of the enzymes into the cytosol could alsobe responsible for the difference in the activitypattern induced by the metals.The higher activitymay be due to the hypersynthesis of acidphosphatase. Hypersynthesised enzymes play aprotective role in the removal of inflammation-provoking agents (Cheng, 1983a). Thehypersynthesis of the enzyme in hemocytesunderline the involvement of the enzyme in defencereaction, so as to destroy the susceptible bioticand abiotic agents (Cheng, 1983ab).The decline in acid phosphatase activity aftera certain period in the case of both the metals maybe either due to destabilization of the lysosomesor inhibition of hypersynthesis by the same metalresponsible for the early enhanced activity. Besides,response of the enzyme activity to copper wasmore conspicuous than mercury in terms of onsetand sustenance. Krishnakumar et al. (1990) in theirstudy on physiological and cellular responses tocopper and mercury in green mussel observed thatcopper was more toxic than mercury in inducinglysosomal changes in the digestive gland.  Coppercauses lysosomal membrane damage by promptinglipid oxidation (Baccino, 1978). This results indestabilization of the lysosomal membrane andrelease of hydrolytic enzymes into the hemolymph.This activity was delayed in the case of exposureto mercury when compared to copper.The peak enzyme activity in the copper treatedgroups was at 24 hr whereas it was on 7th day inmercury treated mussels suggesting that the releaseof enzyme into the hemolymph occurred wellbefore 24 hr in the case of copper exposed mussels,while it occurred after 24 hr in mercury exposedones.Exposure to heavy metals can cause decreasein the total hemocyte count in the later stages(Cheng, 1987) and continued exposure can lead toinhibition of hypersynthesis of the hydrolyticenzymes. Copper inhibited the acid phosphataseactivity in the tropical blood clam Anadaragranosa at sub lethal levels (Anthony and Patel,2000). Based on the findings of the study, it canbe concluded that at sub lethal level, copper caninduce remarkably higher acid phosphatase activitythan mercury and after a period of exposure, bothmetals can cause a decline in the enzyme activityprobably due to inactivation of hypersynthesis.AcknowledgementsThe authors are thankful to the Director,C.M.F.R.I, Cochin for providing facilities tocarryout the work and Head, PNP Division, forcritically reviewing the manuscript.ReferencesAndersch, M. A. and A. J. Szcypinski. 1947. Amer. J. Clin.Pathol., 17: 571-574.Anthony, K. and  B. Patel. 2000. Indian J. Fish., 47: 311-319.Baccino, F. M. 1978. Selected patterns of lysosomal responsein hepatocytic injury. In: Slater, T.F. (Ed.) BiochemicalMechanisms of Liver Injury, Academic Press, NewYork, p. 518-557.Chandy, J. P. and B. Patel. 1985. Dis. Aquat. Org., 1: 39-47.Cheng, T. C. 1983a.  Amer. Zool., 23: 129-144.Cheng, T. C. 1983b. Mar. Tech. Soc. J., 17:18-25.Cheng, T. C.  1987. J. Invertebr. Pathol., 51: 207-214.Have, N. 1969. Acta. Endocrinol.  Suppl., 139: 1-231.Krishnakumar, P. K., R. Damodaran and P. N. K. Nambisan.1987. Indian J. Mar. Sci., 16: 263-264.Krishnakumar, P. K., P. K. Asokan and V. K. Pillai. 1990.Aqua. Toxicol., 18: 163-174.Moore,  M. N. 1976. Cell Tissue Res., 175: 279-287.Roberts, D. 1976. Mussels and Pollution. In: Bayne. B.L.,(Ed.) Marine Mussels: Their Ecology and Physiology,Cambridge University Press, Cambridge, 25: p. 4-39.229Journal of the Marine Biological Association of India (2007)Hemolymph acid phosphatase activity in green musselSantarem, M., and A. Figueras. 1995. Basic studies ondefence mechanisms in mussel. In: Stolen, J. S., T. C.Fletcher, S. A. Smith, J. T. Zelikoff, S. L. Kaattari,R. S. Anderson, K. Soderhall, and B.A. Weeks-Perkins,(Eds.) Immunology and Pathology of Aquaticinvertebrates, Fair Haven: SOS Publications, FairHaven. p. 87-92.Sumner, A. T. 1969. J. Zool. London, 158: 277-291.Suresh, K. and A. Mohandas. 1989. J. Invertebr Pathol.,55: 118-125.Received: 1 July 2006Accepted: 17 March 2008

Hemolymph acid phosphatase activity in the green mussel (Perna viridis, Linnaeus, 1758) exposed to copper and mercury

http://eprints.cmfri.org.in/2143/1/Krupesha_226-229.pdf

Hemolymph acid phosphatase activity in the green mussel (Perna viridis, Linnaeus, 1758) exposed to copper and mercury

Abstract

Similar works

Full text

Available Versions

CMFRI Digital Repository