Studies were conducted to evaluate the molecular changes induced by aflatoxin,

the most frequently encountered dietary carcinogen in Oreochromis

mossambicus. Three sub-lethal test doses of aflatoxin B1 incorporated in feed

(1.5, 5.0 and 15.0mg/kg feed) were given to the fishes for a period of 30 days and

the changes in serum protein profile were resolved through SDS PAGE at regular

intervals. Similarly, the changes in immune responses were also studied. The

study revealed significant changes in both protein profile and immune response

in the experimental fishes compared to the control, indicating that the toxin

exerts its effect even at sublethal levels

Paulton, M P

Thomas, P C

Zacharia, Sherly

CMFRI Digital Repository

421Changes in serum proteins and immune responsein oreochromis mossambicus induced by aflatoxin B1SHERLY ZACHARIA, P. C. THOMAS AND M. P. PAULTONCentral Marine Fisheries Research Institute, Cochin - 682 014, IndiaABSTRACTStudies were conducted to evaluate the molecular changes induced by aflatoxin,the most frequently encountered dietary carcinogen in Oreochromismossambicus. Three sub-lethal test doses of aflatoxin B1 incorporated in feed(1.5, 5.0 and 15.0mg/kg feed) were given to the fishes for a period of 30 days andthe changes in serum protein profile were resolved through SDS PAGE at regularintervals. Similarly, the changes in immune responses were also studied. Thestudy revealed significant changes in both protein profile and immune responsein the experimental fishes compared to the control, indicating that the toxinexerts its effect even at sublethal levels.IntroductionAflatoxicosis is an important andfrequently encountered problemseriously affecting fish production intropical countries. It is mostly producedby the fungal strains of Aspergillusflavus. Aflatoxins are the only naturallyoccurring dietary carcinogens currentlyrecognized by the International Agencyfor Cancer Research (IARC, 1993) ascarcinogenic to humans. It functions asan electrophilic reactant, which afterbiotransformation induces carcinogeniceffects. The toxin is best known for theirhepatotoxic, nephrotoxic, carcinogenic,mutagenic and immunosuppressiveproperties in animals.Initial changes due to aflatoxins areexpected to be manifested in thetransporting medium, viz., blood. Thisaspect has received ample attentionespecially in fish immunological studies.A prolonged time for blood clotting wasreported by Woerr et al. (1987) indicatingsevere coagulopathy. The defect wasmainly due to the failure of hepaticsynthesis of factors V, VI, VII andfibrinogen (Baker and Green, 1987).Anticoagulant activity has beenattributed to aflatoxin mainly due to itschemical relation to caumarin, which canbind to serum albumin. Severe anaemiatogether with the changes in haematocrit,haemogloblin concentration anderythrocyte and leukocyte counts wereobserved as prominent signs of acuteaflatoxin B1 (AFB1) toxicity in channelcatfish (Jantrarotori et al., 1990).Determination of the serum proteins infish contributes to an appraisal of thehealth of fish. The activities of serumconstituents were found to be differentin many farm animals on aflatoxicosis.Indian J. Fish., 50(4) : 421-426, Oct.-Dec., 2003422The total protein levels were found to bedecreased in aflatoxin-fed animals, butthe protein expressions were found to beelevated. Expressions of serumcholesterol, albumin and globulin weredecreased in AFB1 fed cockerels, whilethe serum globulins were elevated(Jakab, 1994).  Reductions in the totalserum protein and serum calcium andphosphorus levels were noted in AFB1treated animals (Dafalla et al., 1987;Singh et al., 1987; Jakab, 1994).The more advanced teleosts arereported to possess an immune systemapproaching the complexity andefficiency of higher vertebrates. Intereston the effect of xenobiotics, particularlyon the immune system, has increased inthe recent past making it a sensitivebiological parameter to monitor theimmunotoxicity of chemicals. Mycotoxinshave been reported to suppress theimmune function and reduce resistanceto infection on chronic exposure to lowlevels of toxin in a number of species(Jakab, 1994). Although a number ofstudies have been made on the effects ofxenobiotics on the immune response offish, studies involving aflatoxin in fish arescanty.Most of the available information onaflatoxicosis in teleost is based on thestudies conducted on coldwater fishes,especially salmonids and cyprinidis. Withsuch information on tropical fisheslacking, an attempt was made in thepresent study to determine the changesin serum protein profile and antibodyresponse in tilapia, Oreochromismossambicus, fed a diet fortified withAFB1. Of the several blood componentsin fish, determination of changes in theserum protein profile, serum proteinconcentration and immune response canprovide to be a reliable appraisal of thecondition of fish.Material and methodsTilapia (50 ± 10 g) collected frombrackishwater ponds were used for thestudy. They were acclimatized tofreshwater conditions in the laboratoryand transferred to experimental units ofcircular polypropylene tanks of 40 lcapacity. The animals were fed initiallyfor a week with a semi-purified dietcontaining 35% protein as suggested byJauncey and Ross (1982) for freshwatertilapia during the period ofacclimatisation.Experimental dietThree experimental diets, having thesame composition as that of the control(35% protein) was prepared byincorporating different concentrations ofAFB1 from a stock solution (100 µg AFB1/ml of chloroform) prior to final mixing ofthe diets.  Since studies had indicatedthat warmwater fishes are generally lesssensitive to AFB1 than coldwater fishes(Jantrarotai et al., 1990) higher doses ofAFB1 were included in the present study.Diets with 1.5, 5.0 and 15.0 mg AFB1/ kgfeed were prepared by adding adequatealiquots of the stock solution beforeblending and chloroform was allowed toevaporate. The ingredients were thenmixed with water, extruded and dried.Experimental designThree experimental groups eachconsisting of nine animals were exposedto three dietary levels of AFB1- 1.5, 5.0,15.0mg/kg diet (test groups I, II and III),while the fourth group, fed on semi-purified diet devoid of AFB1 formed thecontrol. The fishes, starved for two daysprior to feeding the experimental diet,were fed at the rate of 2% body weightper day to ensure that they consumed thefeeds offered. Each group was routinelyexamined for physical and behaviouralabnormalities. Thirty days observationSherly Zacharia et al.423was used in this study.Serum protein profileBlood samples were obtained bycardiac puncture on the 7th, 14th, 21st and30th days of the treatment. Samples wereallowed to clot at room temperature.Serum was separated immediately andstored at -200C until they were analyzedelectrophoretically. The changes in theserum protein profile were analysed bySodium Dodecyl Sulphate Polyacry-lamide Gel (11.5%),  Electrophoresis(SDS PAGE) (Table 1) using 1.5 M Tris-HCl buffer (pH 8.8) along with a stackinggel of 6% at a constant voltage of 140 V(Laemmli, 1970). The molecular weightsof the subunits were determined usingmolecular weight standards run alongwith the samples. The volumes ofsamples were adjusted to a concentrationof 120 µg proteins per well.On completion of electrophoresis, thegels were stained for proteins with 0.1%Coomassie brilliant blue R250 accordingto conventional methods and destainedin a solution of 50% methanol containing10% acetic acid.  Relative mobilities (Rf)of the various protein fractions estimatedand the molecular weights weredetermined in comparison with markers.Evaluation of immune responseIn order to study the changesbrought about by AFB1 in the immuneresponse of tilapia, haemagglutinationtest was conducted (Ambrosius andSchaker, 1964). Rabbit erythrocytessuspended in normal saline were used asantigen in the study.Immunisation of the fishes: Treatedand control fishes were immunised byintra-muscular injection of the antigen(rabbit RBC suspension containing 104-106 cells / kl) emulsified with completeFreund’s adjuvant.  Booster doses weregiven on the 10th day after primaryinjection without the adjuvant.Antibody detection: The antiserumfrom the blood of immunized fishes wereseparated on the 30th day of exposure. Thesamples were analyzed for antibodyproduction at different serial dilutions byhaemagglutination test.ResultsChanges in serum profileElectrophoretic analyses of theserum protein profile of the test groupsSerum protein changes in O. mossambicusTABLE 1. Composition of 11. 5% GelSeparating GelAcrylamide and bisacrylamide -  11.50 mlSeparating gel buffer - 6.00 ml10% SDS - 300.00 lTEMED - 30.00 lAmmonium persulphate - 100.00 lDouble distilled water - 12.07 mlStacking GelAcrylamide and bisacrylamide - 2.00 mlSeparating gel buffer - 2.50 ml10% SDS - 100.00 lTEMED - 10.00 lAmmonium persulphate - 40.00 lDouble distilled water - 5.40 ml424showed some variations from that of thecontrol. Of the 35 proteins resolved bySDS PAGE in the control, only a fewshowed marked changes in theirexpression on exposure to AFB1 on the7th day in the experimental groups. Thegeneral expression of all the serumproteins of molecular weights above 98kDa was weak in all the test groupscompared to the control.  The lowmolecular weight proteins exhibitingvariation in the test groups were 81, 72,70, 54, 24, 23 and 16.5 kDa  (Fig.1).control group was 1:512, whereas in thefishes exposed to 1.5, 5.0 and 15.0 mg ofAFB1/kg diet, the maximum titre atwhich agglutination took place was 1:64,1:32 and 1:16, respectively (Fig. 2).Sherly Zacharia et al.Fig. 2. Immune response of tilapia exposedto different concentrations of   afla-toxin B1 on the 30th dayFig 1. SDS PAGE analysis of serum proteins on 7thdayLane 1 - Molecular weight markerLane 2 - ControlLane 3 - 1.5 mg AFB1Lane 4 - 5.0 mg AFB1Lane 5 - 15.0 mg AFB1The moderate expression of theprotein bands in the test groups I and IIwas maintained till the 21st day, while intest group III  the protein expressionbecame very strong by the 14th day andthis level was maintained till the 21st day.On the 30th day of exposure, theexpression of all the fractions became tooweak to be detected in all the test groups.Immune responseThe sera collected from both controland treated fishes which were immunizedagainst rabbit erythrocytes were testedfor antibodies by haemagglutination testusing different serial dilutions of serumand rabbit RBC. The antibody titre of theDiscussionBlood together with lymph is ahomeostatically controlled internalenvironment and changes induced bycarcinogens are expected to be initiallymanifested in this transporting medium.In the present study, the treated fishesexhibited dose-dependent changes in thenormal serum protein profile on exposureto AFB1 indicating their sensitivity toAFB1. The serum protein profiles of thetest animals were similar to those of thecontrols except for the enhancement ofexpression. The proteins had an initialincrease in their expression on the 7thday, which got further intensified by the14th day and continued till the 21st dayand thereafter declining by the 30th dayof observation.  Scarpelli (1969) reportedelevated levels of serum proteins inelectrophoretic analysis in rainbow troutwith hepatocarcinoma where thecomposition of the serum wasqualitatively similar to that of normalanimals.  In some cases, a three-foldincrease over normal serum value wasnoted, which reflected in an increase ofall the serum protein components.Snieszko (1961) made the first425observation that trout bearing livertumours showed marked elevations oftheir serum proteins.The changes in serum proteinexpression in the treated fishes may bedue to DNA damage, which in turn, leadsto changes in the expression of itsproducts, viz., proteins. Experimentalresults have demonstrated the covalentbinding of aflatoxin to DNA, resulting ininhibitions of DNA replication and alsoinhibition of RNA synthesis thus,resulting in altered expression of thegene. Thus, the initial enhancement inthe protein expression in the treatedgroups in comparison to the control maybe due to the activation of the genescoding for these proteins under theinfluence of AFB1, while the depressionin protein expression by the 30th day mayprobably be due to the genetic alterationprogressing to the point of collapse of themechanism of transcription of DNA andthe subsequent translation into proteins.Studies on the immunologicalaspects of AFB1 have done much toexploit the special susceptibility of thevery young and newborn members incontrast to the adults and aged membersof the same species. In the present study,adult fishes exposed to aflatoxin whenimmunised with rabbit erythrocytesthrough intramuscular routes showed adecrease in agglutination titre ascompared to control, indicating theimmunosuppressive action of AFB1. Theimmunosuppressive effect of AFB1 iscorroborated by the serum proteinchanges also. Aflatoxin toxicity wasreported to impair the systemic innateand acquired defence by suppressingperitoneal macrophage phagocytosis andspleenic antibody production resulting inincreased susceptibility to infection bythe domesticated animals (Jakab, 1994).Similarly, references to AFB1 initiatedreduction in blood parameters(haematocrit, haemoglobulins, RBC andWBC) and reduction in leukocytes andmacrophages in spleenic corpuscles werereported in channel catfish (Jantrarotaiet al., 1990).The molecular weight of tetramers,H chain and L chain of theimmunoglobulin IgM4 reported in teleostare 610-720 kDa and 24-26 kDarespectively (Roberts, 1978). The serumprotein bands exhibiting variations in thepresent study, viz., 81, 72 and 70 kDa areregarded as 1, 2 and  protein fractionsof the H chain and 24, 25 and 16.5 kDaare their corresponding L chains. Thereduced expression of the serum proteinfraction as well as the general reductionin the total serum proteins in the treatedfishes suggest a decline inimmunoglobulins leading to dose-dependent reduction in the antibody titreon exposure to AFB1.The present study demonstrate thattilapia exposed to AFB1 exhibit variationin the protein expression suggesting analteration in the genes which encodethem. Thus, it could be used as a rapidbiological test for detecting carcinogenicpotential of chemical compound. Thisapproach has the advantage that fishfrom the same treatment group could bemaintained to determine the long-termeffects. However, the exact mechanismunderlying the increase in expressionremains to be established. The presentstudy confirms the extreme sensitivity oftilapia to sublethal levels of dietary AFB1.AcknowledgementsWe are thankful to the Director,Central Marine Fisheries ResearchInstitute, Kochi, for providing facilities.One of the authors wishes to expressthanks to the Indian Council ofSerum protein changes in O. mossambicus426Agricultural Research, New Delhi, for theaward of a fellowship.ReferencesAmbrosius, H. and W. Schaker 1964.Contribution to the immunobiology ofpoikilothermic vertebrates. I.Immunological investigations on thecommon carp, Cyprinus carpio. Zool. J.Physiol., 71: 74-88.Baker, D. C. and R. A. Green 1987.Coagulation defects of aflatoxinintoxicated rabbits. Vet. Pathol., 24 (1):62- 70.Dafalla R., A. Yagi and A. Adam 1987.Experimental aflatoxicosis in hybridtype chicks, sequential changes ingrowth and serum constituents andhistopathological changes. Vet. Hu.Toxicol., 29(3): 222-226.IARC, 1993. Monographs on the Evaluationof the Carcinogenic Risk of Chemicals toHumans: Aflatoxins, Vol.56International Agency for CancerResearch, Lyon, p. 245 - 396.Jakab, G. T. 1994. Respiratory aflatoxicosis:suppression of pulmonary and systemichost defences in rats and mice. Toxicol.Appl. Pharmacol., 125 (2): 198- 205.Jantrarotai, W., R.T. Lovell and J.M. Grizzle1990. Acute toxicity of AFB1 to channelcatfish.  J. Aquat. Anim. Health, 2: 237-247.Jauncey, K. And B. Ross 1982. A Guide toTilapia Feeds and Feeding. ScotlandInstitute of Aquaculture, University ofStirling, 111 pp.Laemmli, U.K. 1970. Cleavage of structuralprotein during the assembling of thehead of bacteriophage T4.  Nature, 227:680-685.Roberts, R. J. 1978.  Fish Pathology. BailliereTindall, London.Scarpelli, D. J.  1969. Comparative aspectsof neoplasia in fish and other laboratoryanimals. In: Fish in Research, O. W.Neuhaus and J. E. Halver (Eds.).Academic  Press, New York, p 45- 49.Singh, J., R. P. Tiwari, G. Singh, S. Singh andD. V. Vadhera  1987. Biochemical andimmunological effects of aflatoxins inrabbits. Toxicol., 35 (2-3): 225- 230.Snieszko,  S. F. 1961.  Hepatoma and visceralgranuloma in trouts. N. Y. Fish and   Game J.,  8(2): 145-147Woerr, J. A., W. E. Huff and P. B. Hamilton1987. Increased sensitivity tostaphylococcal betahaemolysin oferythrocytes from chicken duringaflatoxicosis. Poult. Sci., 66(12): 1929-1933.Sherly Zacharia et al.

Changes in serum proteins and immune response

in oreochromis mossambicus induced by aflatoxin B

http://eprints.cmfri.org.in/120/1/IJF-4_Oct-Dec_-_1.pdf

Changes in serum proteins and immune response in oreochromis mossambicus induced by aflatoxin B

Abstract

Similar works

Full text

Available Versions

CMFRI Digital Repository