Responses Of Plasma Transaminase Activity In Cyprinus .
Journal of the Indian Fisheries Association29, 2002, 7-137RESPONSES OF PLASMA TRANSAMINASE ACTIVITY IN CYPRINUSCARPIO VAR. COMMUNIS TO MERCURY TOXICITYHannah Sampath, M. Ramesh and ManavalaramanujamUnit of Pollution Biology, Department of Zoology, Bharathiar University,Coimbatore - 641 046ABSTRACTThe present study reports the behavioural and enzymological responsesin a freshwater teleost fish, Cyprinus carpio var. communis, exposed to acuteand sublethal toxicities of mercuric chloride. During acute treatement,significant behavioural changes like erratic swimming, excess mucus secretion,increased opercular movements, etc. were noticed. During acute and sublethaltreatments, both aspartate amino transferase and alanine amino transferaseactivity increased throughout the study period. Comparing the treatments,the changes in enzyme activities were found high in acute treatement and allthe values were significant at 5% level. The above findings can be used asnon-specific biomarkers of environmental pollutants.Keywords: Mercuric chloride, AST, ALT, Cyprinus carpioINTRODUCTIONIndustrial effluents contaminatingaquatic bodies contain a number of toxicchemicals, which in turn, exercise theireffects on fish and pose threat to aquaticlife. Sankar Narayan and Madhyastha(1985) reported that heavy metals arecontinually released into the aquaticenvironment from natural processes suchas volcanic activity, weathering of rocksand industrial processes. The three heavymetals, which are toxic to most organismsat the least concentrations and probablynever beneficial to living organisms arecadmium, mercury and lead. According toDimou et al. (1989), Hg is a ubiquitous,highly toxic heavy metal, which isbioconcentrated through food chain. In Indiaalone, about 180 t ofHg are introduced intothe environment every year, whichultimately reach the aquatic systems, Hgaccumulates in various organs ofmammalian system and adversely affectstheir functions. In fishes, Hgis accumulatedin the form of methyl mercury (WHO,1986).It is established that any kind of stress;not resulting in gross changes and mortalityproduces certain changes in the fish bloodcharacteristics (Christensen et al., 1977).Methods of clinical diagnosis have beenintroduced in fish biology to assess theeffects of pollutants (Lockhart and Metner,1984). Mayer et al. (1992) reported thatserum enzyme activities have been usedextensively to provide simple accuratemeasures of organ dysfunction in mammalsand have received greater attention fromaquatic toxicologists.
8HANNAH SAMPATH, M. RAMESH AND MANAVALARAMANUJAMAspartate amino tranferase (AST) andalanine amino transferase (ALT) not onlyfunction as link enzymes between theprotein and carbohydrate metabilism, butalso serve as indicators of alteredphysiological or stress condition. AST andALT enzyme activities have been used todemonstrate tissue damage in fishCKristoffersson et al., 197 4; Asztalos andNemcsok, 1985). Hence, in the presentstudy, the degree of enzyme responses (ASTand ALT) of an economically importantfish, Cyprinus carpio, to Hg toxicity wasinvestigated.MATERIAL AND METHODSSpecimens ofC. carpiovar. communis,a freshwater teleost fish, were collectedfrom Tamil N adu Fisheries DevelopmentCorporation Limited, Aliyar Fish Farm,Aliyar (India), and accilimatized tolaboratory conditions for 20 days. Duringthis period, the fish were fed ad libitumwith rice bran and groundnut oil cake inthe ratio of 3:1. Water was changed dailyand aerated in order to reduce anyaccumulation of excretory products and toensure sufficient oxygen supply. The tapwater was analysed for physico-chemicalfeatures following APHA (1981) and thevalues were: pH 7 .4 0.2; temperature27 1.0 C; dissolved oxygen 6.4 0.02 mgll;salinity 0.5 0.02%o and total hardness6.0 1.0 mg/l.Healthy fish with an average weight of5 g and length of 8-9 em were selected forthe experimental study. The LC 50 value ofHgCl 2 for 24 hours is 0.34 ppm (Finney,1978). One-tenth of the acute value (0.034ppm) was taken for sublethal studiesaccording to Sprague (1971). Fish wereexposed to acute concentration for 24 hoursand sublethal concentration for 25 days.During sublethal exposure, fish were fedad libitum and the toxicant was reneweddaily. The physico,-chemical.parameters ofthe water were monitored regularly tomaintain the same level. A common controlwas maintained. Fish from acute and fromsublethal concentrations were removed atintervals of 24 hours and 5 clays respectivelywith respective conhols. Blood was drawnfrom the heart by puncturing it using anice-cold microsyringe. The sample wascentrifuged at 9000 rev/min for 5 minutesleaving a clear yellow fluid called plasma,which was used for the estimation of ASTandALT activities. AST (AST, EC 2.6.1.1)and ALT (ALT, EC 2.6.1.2) activities wereestimated by 2, 4 DNPH method (Reitmanand Frankel, 1957). The significancebetween the sample mean of control andexperimental fish was tested usingStudents' 't' test.RESULTBehavioural changes constitute yetanother index to measure toxicity. In thepresent study, during acute treatment ofmercuric chloride, the fish exhibited thefollowing behavioural changes: fast jerky. movements, turning upside down,restlessness, erratic movement, mucussecretion from the body and increasedopercular movements. However, fishes fromthe control did not show such behaviourchanges.Tables 1 and 2 show the changes inAST andALT activity in the plasma offishexposed to acute and sublethal
Table 1 :Changes in aspartate aminotransferase activity and alanine aminotransferase activity in the plasmaof Cyprinus carpio var. communis exposed to acute concentration (0.34 ppm) ofHgCl 2Aspartate aminotransferase activity(U/ml)Alanine aminotransferase activity (U/ml)Exposure periodAcute treatment24 h (0.34 ppm)ControlExperimentalControlExperimental66.067 .0.1566226.186 0.1146*( 242.35)35.7040 0.1295126.492 .0.1194*( 254.00) trj 0c -3Table 2 :Changes in aspartate aminotransferase activity and alanine aminotransferase activity in the plasmaof Cyprinus carpio var. communis exposed to sublethal concentration (0.034 ppm) HgCl2Aspartate aminotransferase activity (U/ml)Exposure period(d)Alanine aminotransferase activity (U/ml)§0 - - z :rj - ControlExperimentalControlExperimental561.074 0.460854.170 0.30951042.215 .0.24391563.083 0.06912084.473 0.28572562.443 0.381976.586 0.2729*( 25.39)56.477 .0.1453*( 33.78)97.686 0.1851*(54.85)142.398 0 2793*( 68.57)151.362 0.1085*( 142.40)59.592 Q.1938*( 10.00)56.028 0.2960*( 23.91)73.708 0.1573*( 28.23)104.257 0.2901*( 81.72)164.592 0.1440*( 81.72)45.215 0.243957.477 0.145272.343 0.285690.574 0.1958-L-- -- -- - Values are mean SE of five individual observations. Values in parentheses are per cent changes over control.Degree of freedom at 8t 0.05 2.306. *Values are significant at 5% level.- - ----w.p::(.0
10HANNAH SAMPATH, M. RAMESH AND MANAVALARAMANUJAMconcentrations ofHgC1 2 . During the abovetreatment period, the enzyme activityincreased both in acute and sublethaltoxicities. In acute toxicity, maximum percent increases of242.35 ofAST and 254.00of ALT were noticed. During sublethaltreatment also the AST and ALT activitiesincreased and were directly proportional tothe exposure period showing minimum percent increases of25.39 and 10.00 at the endof the fifth day and maximum per centincreases of142.40 and 81.72 at the end ofthe 25th day, respectively. All the valueswere significant at 5% level.DISCUSSIONQuantification of enzyme activity inplants and animals can serve as a valuablebiomarker of pollutant exposure and effect.Toxicants can inhibit enzymes at veryspecific sites (e.g., esteratic site ofacetylcholinesterase) or effects can beevoked by less specific interactions withvarious moieties like sulfhydryl groups(Mayer et al., 1992). Serum transaminases,specifically aspartate aminotransferase(ASAT) and alanine aminotransferase(ALAT), have been widely utilized inmammalian toxicology as a biomarker ofspecific organ dysfunction (Wroblewski andLaDue, 1956). ASAT is a non-specificcytosolic and mitochondrial enzyme foundin a variety of tissues including liver,skeletal muscle, cardiac muscle and kidney(Verma et al., 1981). ALAT is also acytosolic enzyme, but is more tissue-specificand is normally associated with liver.Recently, there has been increasing interestin using changes in enzyme activity inaquatic animals as an index reflecting metaltoxicity. Both the alanine and aspartatetransaminases are important in thediagnosis ofliver damage caused due to theexposure to industrial chemicals.The toxicity data in the present studyshow that HgC1 2 is toxic to C. carpio var.communis. Heavy metals are highly toxic,water soluble, non-degradable, vigorousoxidizing agents and are strongly bonded tomany chemicals, especially polypeptides andproteins, and also to other materials whichposses electron-rich functional groups suchas sulfhydryl, amino and imidazole (Albert,1965). Metals may bind to macromoleculesinside the cells potentially with enzymes orchange the concentration of cofactors orreactants by altering membranepermeability and indirectly affectingenzyme activity (Tucker and Matte, 1980).Metals have been found to affect theactivities of transaminases in fish. Fishexposed to acutely toxic concentrations ofCd, Hg, or Cu had increased transaminaseactivities (McKim et al., 1970).Alternatively, chronic exposure to Cu hasbeen reported to decrease serum ASATactivity while chronic cadmium exposuredoes not affect activities of thistransaminase in serum (McKim et al.,1970).The degree of increase in the activity ofcellular enzymes in serum dependsprimarily on the magnitude and severity ofcell damage (Kristoffersson et al., 1974;Aszatalos and N emcsok, 1985;Radhakrishnaiah, 1988). Tissue necrosisis one of the most used signs of damagedorganisms (Cheneryetal., 1981; Nemcsok,1993.). In the event of tissue necrosis,transaminases pass into the circulation
MERCURY TOXICITY IN FISHfrom the damaged organ and their increaseactivities in the blood indicate thP degree oftissue damage (Kristoffersson et al., 197 4).The enzymes AST and ALT aregenerally associated with cellular metabolicactivity (Abston and Yarbrough, 1976).During cellular damage or lysis,b·ansminases are released. Elevatedtransaminase could be taken as a measureof compensatory mechanism as aconsequence to impaired carbohydratemetabolism (Reddy and Venugopal. 1991).Radhakrishnaiah (1988) observed anincreased accumulation ofCu in liver andgill of Labeo rohita .and suggested thatelevation or inhibition of enzyme activitymay be due to the accumulation fo heavymetals in these tissues leading to damage.Kiran et al. (1990) reported that theincrease in the levels of serum enzymescan be attributed to the efflux of enzymesfrom damaged liver and other organs intocirculation. Hepatocellular disorder causesplasma AST and ALT levels to go up (Raoet a1., 1992) and Moorthy et al. (1984)reported that the increase of AST activitycould be a result of general tissue damageparticularly liver, musde and heart.Elevated AST and ALT levels werepres1.nnably due to damage of liver, butother organs may also have been damaged(kidney or/and gill). Reichenback-Klinke(1972), and Schreck and Lorz (1978)observed epithelial necrol:lis ofthe gills andglomerular atrophy of kidney when cohosalmon, Oncorh.ynchus kisutch, wasexposed to Cu.According to Mayer et al. (1992),basically, there are four different processesthat may suggeHt the responses of enzyme11to specific or non-specific chemicals stress;they are (1) direct enzyme inhibition, (2)enzyme induction by specific classes ofchemicals, (3) elevation Of::;erum enzymes,viz., tissue damage and (4) alterations inenzyme activity as a result of changes inmetabolic pathways or fluxes. The authorsfurther added that enzyme activity isgenerally regulated such that specificsubstances or entire pathways may behomeostatically adjusted to compensate forendogenous or exogenous changes.In the present study, the significantincrease in AST and ALT levels duringacute and sublethal studies may be due toaccumulation of Hg in tissues leading toth;sue (liver) damage reBulting in release ofthese enzymes into blood or impairedcarbohyrate metabolism. In 'the presentstudy, long-term exposure enhanced theactivities of enzymes suggesting that withincrease in exposure period, the organismtries to mitigate the toxicant induced stressby increased rate ofmetaboHsm.REFERENCESAbston, P. A. and Yarbrough, J. D.,1976. In vivo effect ofmirex on solublehepatic enzymes in the rat. Pestic.Biochem. Physiol., 6: 192-199.Albert, A . , 1965. Selective Toxicity a grded., .John Wiley and Sons, New York,394pp.APHA, 1981. Standard lvlethods for theExamination of Water and Wastewater. 15th ed. American Public HealthAssociation, Washington, DC, 1134pp.Asztalos, B. and Nemcsok, J·., 1985.
12HANNAH SAMPATH, M. RAMESH AND MANAVALARAMANUJAMEffect of pesticides on the LDH activityand isoenzyme pattern of carp, Cyprinuscarpio (L.), sera. Camp. Biochem.Physiol} 82 (1): 214-219.tool. In: V.W. Cairns, P.V. Hodson,and .J.O. Nriagu, (Eds.) ContaminantEffects on Fisheries} John Wiley andSons, New York, pp. 73-85.Chenery, R., George, M. and Krishna,G., 1981. TheeffectofiodophoreA23187and calcium on carbon tetrachlorideinduced toxicity in cultured rathepatocytes. Toxicol. Appl. Pharmacal.}50:241-252.Mayer, F.L., Versteeg, D.J., Mckee,M.J., Folmar, L.C., Graney, R.L.,McCume, D.C. and Rattner, B.A.,1992. Physiological and non-specificbiomarkers. In: R.J. Huggeth, R.A.Kimerle, P.M. Mehrle, Jr., and H.L.Bergman,(Eds.) Biomarher}Biochemical} Physiological andHistological Marhers ofAnthropogenicStress. Lewis Publishers, Boca Raton,pp. 5-85.Christensen, G., Hunt, E. and Fiant,J., 1977. The effect of methyl mercuricchloride, cadmium chloride and leadnitrate on six biochemical factors ofthe brook trout Salvelinus fontinalis.Toxicol. Appl. Pharmacal.} 42: 523527.Dimou, A., Kilikidis, S. andKamariano, A., 1989. Methyl mercuryconcentration in broiler's meat andhen's meat and egg. Bull. Environ.Contam. Toxicol., 42:728-734.Finney, D.J., 1978. Statistical Methodsin Biological Assay} 3rd ed., GriffinPress, London, 508 pp.Kiran, R., Gera, R. and Sidhu, H., 1990.Effect of aldicarb administration on liverand serum enzymes in female rats.Poll. Res. 10(4): 235-238.Kristoffersson, R., Broberg, S., Oikari,A. and Pekkarinen, M., 197 4. Effectof a sublethal concentration of phenolon some blood plasma enzyme activitiesin the pike, Esox lwdus .(L. )jn brackishwater. Ann. Zool. Feimici} 11: 220223.Lockhart, W.L. and Metner, D.A., 1984.Fish serum chemistry as a pathlogy,McKim, J.M., Christensen, G.M. andHunt, E.P., 1970. Changes in the bloodof brook trout (Salvelinus fontinalis)after short-term and long-term exposureto copper, J. Fish. Res. Bd. Can.} 27:1883-1889.Moorthy, S.K., Reddy, K.B , Swami,L.S. and Chetty, C.S., 1984. Effectsof dichlorvos on tissue metabolism infreshwater mussel. Arch. Toxicol.} 42:1-8.Nemcsok, J., 1993. Research into thebiochemical effects of xenobiotics onfish.ActaBiol. Szeged} 39:113-118.Radhakrishnaiah,K.,1988.Accumulation of copper in the organsof freshwater fish, Labeo rohita(Hamilton), on exposure to lethal andsublethal concentration of copper. J.Environ. Biol.} 9: 319-326.Ra.o, K.V.R. Surendrana.th, P. andKodavanti, P.R S., 1992. Levels oftransminases in tissues of the penaeid
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