Author Institution: Department of Biological Sciences, Kent State UniversityThere were qualitative and quantitative changes in the serum proteins of bluegills, Lepomis macrochirus, exposed to 8.728 X 10~4 ppb (W/V) of methyl mercury (MeHg) for 24, 48, and 72 hr. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of serum proteins revealed significant changes in the qualitative and quantitative profiles at 24 and 48 hr. However, at 72 hr a trend to return to control levels was noted. The data suggest that at the dose tested, MeHg produced repairable lesions in certain tissues or organs of bluegills

Dutta, Hiran M.

Haghighi, Ali Z.

Lall, S. B.

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OhioJ. Sci. DEVONIAN POLYCHAETE BORINGS 119Copyright © 1983 Ohio Acad. Sci. OO3O-O95O/83/OOO3-O119 $2.00/0METHYL MERCURY INDUCED CHANGES IN THE SERUM PROTEINSOF BLUEGILLS — LEPOMIS MACROCHIRUS (TELEOSTEI)1HIRAN M. DUTTA, S. B. LALL2 and ALI Z. HAGHIGHI3, Department of Biological Sciences, KentState University, Kent, OH 44242ABSTRACT. There were qualitative and quantitative changes in the serum proteins ofbluegills, Lepomis macrochirus, exposed to 8.728 X 10~4 ppb (W/V) of methyl mercury(MeHg) for 24, 48, and 72 hr. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) of serum proteins revealed significant changes in the qualitative and quanti-tative profiles at 24 and 48 hr. However, at 72 hr a trend to return to control levels wasnoted. The data suggest that at the dose tested, MeHg produced repairable lesions in cer-tain tissues or organs of bluegills.OHIOJ . SCI. 83 (3): 119-122, 1983INTRODUCTIONMethyl mercury (MeHg) can cause a va-riety of abnormalities in the gills, gut,liver, kidney, or brain of fishes. The in-tensity of these changes depends on thespecies, its age, physiological status, con-manuscript received 23 April 1982 and in re-vised form 12 July 1982 (#82-9).2Present Address: Associate Professor, Depart-ment of Zoology, University of Udaipur, Udaipur,India.3Present Address: Ph. D. Student, Department ofChemistry, Kent State University, Kent, OH44242.centration of MeHg and exposure time(Choi et al. 1978, Mehra and Choi 1981).Data are available in the literature on themovement of Hg in aquatic system(Hildebrand et al. 1980), haematologicalalterations in the blood of fishes exposed tovarious forms of Hg (Hilmy et al. 1980),mucus secretion (Lock and Van Overbeeke1981), changes in cellular elements(Harper 1968), developmental anamolies(Weis and Weis 1977, Sharp et al. 1980)and the bioaccumulation and biomagni-fication of Hg in fish tissues (Waldichuk1974, Yamazaki 1978). However, there islittle information on changes in serum pro-120 DUTTA ET AL. Vol. 83teins of fishes exposed to MeHg (Lagler etal. 1977, Hi lmyetal . 1980).Estimation of qualitative and quantita-tive changes in serum proteins as measuredby sodium dodecyl sulphate-polyarcyl-amide gel electrophoresis (SDS-PAGE) canbe correlated with toxicological effects ofchemicals. The purpose of this study wasto measure any changes in serum proteinsof bluegills {Lepomis macrochirus) exposedto MeHg.METHODS AND MATERIALSAdult bluegills (5-8 cm in total length) were col-lected locally. The bluegills were acclimated toaquaria water (pH 7.25—7.35, temperature 25—27 C) for 3 days, and food was available ad libitum.Concentrations of MeHg ranging from 1.255 X10"5, 1.255 x 10~4, 1.255 X 10"3, 1.255 X10"2 and 8.728 X 1(T4 ppb (W/V) were tested todetermine a sub-lethal dose.Fish (N = 4-5) were exposed to 8.728 X 1(T4ppb (W/V) MeHg in all glass aquaria. Controlswere treated under identical conditions, minus theMeHg. Samples of blood were taken from unan-esthetized control and treated fishes at 24, 48 and72 hr. Blood was removed by cardiac puncture with1.0 ml sterile plastic syringes. The samples were al-lowed to stand at room temperature for one hr andthen placed in a refrigerator for overnight storage.The following day, the samples were centrifuged at700 g for 10 min and the serum was removed andfrozen for subsequent biochemical and electropho-retic assays.Sodium dodecyl sulphate-polyacrylamide gel(SDS-PAGE) was performed essentially according tothe method of O'Farrel (1975). Serum samples(30—40/U,l) were thawed and applied to slab wells.Albumin, oval albumin, /3-lactoglobulin, pepsin,trypsinogen and lysosyme were run simultaneouslyas marker proteins. The gels were stained withCoomassie brilliant blue G-250 for 1—1.15 hr andthen destained in 7% acetic acid until the poly-peptide bands displayed maximal staining effect andno background staining. Gels were scanned witha densitometer to determine the quantitative profileof peptides. Schematic maps were prepared todisplay the site and number of bands. Total serumproteins were estimated according to the method ofLowry et al. (1951). All protein samples and SDS-PAGE were run in duplicate.RESULTS AND DISCUSSIONMeHg at 8.728 X 1CT4 ppb (W/V)caused the fish to swim erratically and in-creased opercular movements. The levelsof total serum proteins in controls were2.65 mg/ml compared to 1.17, 1.778 and1.40 mg/ml in fish treated with MeHgfor 24, 48 and 72 hr respectively (table 1).SDS-PAGE of serum protein hydrolysatesshowed changes in serum proteins ofMeHg treated fish (fig. 1). A total of 28major plus minor (weakly staining) bandswere observed in the serum of controls asopposed to 22, 51, 27, from fish exposedto MeHg for 24, 48, 72 hr. A varietyof peptides, showing differential loci,staining intensity, mobility, and molecularweight were seen at 48 hr compared to 24hr. However, 72-hr samples showed a re-duction in polypeptide bands approximat-ing a profile similar to controls. Althoughthe number of bands at 72 hr and controlare nearly the same, they differ in their lociand staining intensity indicating quan-titatively and qualitatively different pep-tides. Additional peptides could be addedto the serum of MeHg treated fish as a re-sult of breakdown of intracellular haemo-globin, red blood cells or other cellularcomponents. The total protein as measuredby Lowry's method decreased in MeHgtreated fish, even though there was an in-crease in the total number of bands. Thiscould have resulted from rapidly hydro-lysed low molecular weight proteins whichyielded fast-moving peptides that stainedweakly and occupied different loci onthe gel.Giblin and Massaro (1974) stated thebinding of MeHg to intracellular haemo-globin of rainbow trout can be reversed byextracellular sulfahydryl groups. Nakaoet al. (1973) showed intact red blood cellscan be penetrated by various mercurialcompounds which induce inhibition ofboth N a + - K + ATPase and N a + - K +TABLE 1Total serum protein in control and MeHg treated bluegills.TreatmentTotal serum protein(mg/ml)ControlMeHg (24 hr)(48 hr)(72 hr)2.651.171.781.40OhioJ. Sci. MEHG CHANGES IN FISH SERUM PROTEINS 121ProteinStandardsControl Day 1 Day 2 Day 3FIGURE 1. Diagrammatic representation of serumpolypeptides as resolved by SDS-PAGE from controland MeHg treated bluegills.insensitive ATPase. Dawson (1975) andSaad et al. (1973) emphasized blood altera-tions or damage to the haemopoietic organsalso may be associated with pathologicalconditions related to waterborne pollut-ants. Passow et al. (1961) suggested bind-ing of Hg may be in either "sensitive" or"non-sensitive" areas of subcellular com-partments and may influence the outcomeof toxicity.Regardless of the mechanism(s) of cellu-lar damage, our data show that MeHgcaused considerable changes in the serumproteins of the bluegills. With further re-finements in methodology, serum proteinscould be useful indices of contaminant-induced toxicity in fish populations.LITERATURE CITEDChoi, B. H., L. W. Lapham, L. Amin-Zaki and T.Saleem 1978 Abnormal neuronal migration,deranged cerebral cortical organization and dif-fused white matter astrocytosis of human fetalbrain: A major effect of methyl mercury poisoningin utero. J. Neuropath. Exp. Neurol. 37:719-753.Dawson, A. B. 1975 The Haemopoitic responsein the catfish. Ameiurus nebulosus to chronic leadpoisoning. Biol. Bull. 68: 335-346.Giblin, F.J.B. and E.G. Massaro 1974 Themechanism of methyl mercury transport andtransfer to the tissues of the rainbow trout (Salmogairdnerii). In: D. P. Hemphill (ed.), Trace sub-stances in environmental health. Univ. ofMissouri, Columbia, VIII: 349-355.Harper, T. A. 1968 The Peripheral blood film.Butterworth, London. 134 p.Hildebrand, S.G., R. H. Strand and I. W.Huckabee 1980 Mercury accumulation in fishand invertebrates of the North Fork Holston RiverVirginia and Tennessee. J. Environ. Quality9: 393-400.Hilmy, A.M., M. B. Shabana and M. M. Said1980 Haematological responses to mercury tox-icity in the marine teleost Aphanius dispar (Rupp).Comp. Biochem. Physiol. 67c: 147-158.Lagler, K. F., J .E. Bardach, R. R. Miller andD. R. M. Passino 1977 Ichthyol. 2nd Ed.John Wiley & Sons, NY. p. 211.Lock, R. A. C. and P. Van Overbeeke 1981Effects of mercuric chloride and methyl mercuricchloride on mucus secretion in rainbow troutSalmo gairdnerii Richardson. Comp. Biochem.Physiol. 69c: 67-73.Lowry, O.H., N. C. Rosebrough, A. L. Farr andR.J. Randall 1951 Protein measurementswith folin-phenol reagent. J. Biol. Chem. 193:265-275.Mehra, M. and B. H. Choi 1981 Distribution ofmercury in subcellular fractions of brain, liverand kidney after repeated oral administration ofHg-labelled methyl mercuric chloride in mice.Exptl. Molec. Pathol. 35: 435-447.Nakao, M., T Nakao, M. Ohta, F. Nagai, K.Kawai, Y. Fujiharaand K. Nagano 1973 TheNa+ —K+, ATPase molecule In: M. Nakao andG. Packer (eds.), Organization of energy-trans-ducing membranes. Tokyo Univ. Press, Tokyo, p.23-34.O'Farrel, P. M. 1975 High resolution two-dimensional electrophoresis of proteins. J. Biol.Chem. 250: 4007-4021.Passow, H. , A. Rothstein and T. W.Clarkson 1961 The general pharmacology ofthe heavy metals. Pharmacol. Rev. 13: 185-224.Saad, M.A. , A. A. Mezzat and M.B.Shabana 1973 Effect of pollution on the bloodcharacteristics oiTilapia zillii. Water, air, soil pol-lution 2: 171-179.Sharp, J. R., K. W Fucik and J. M. Neff 1980Physiological basis of differential sensitivity offish embryonic stages to oil pollution. In: W. B.Vernberg, A. Calabrese, F. P. Thurbert and F. J.Vernberg (eds.), Marine pollution: Functional re-sponses. Academic Press, NY. p. 85—107.122 DUTTA ET AL. Vol. 83Waldichuk, M. 1974 Some,biological concerns metals on development of the killiifish —in heavy metal pollution. In: F J. Vernberg and Fundulus heteroclitus. J. Fish. Biol. 11: 49—54.W. B. Vernberg (eds.), Pollution and physiology Yamazaki, S., Y. Dokiya, T. Watanabe and K.of marine organisms. Academic Press, NY. p. Fuwa 1978 Problems in mercury determina-1—57. tion in environmental samples. Ecotoxical. Envi-Weis, J . S . and P. Weis 1977 Effects of heavy ron. Safety 2: 1-8.

Methyl Mercury Induced Changes in the Serum Proteins of Bluegills – Lepomis Macrochirus (Teleostei)

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Methyl Mercury Induced Changes in the Serum Proteins of Bluegills – Lepomis Macrochirus (Teleostei)

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