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Lab Report: Lab Rotations (Spring 2007) in the laboratory of Prof. Dr. Angela Koehlerunder the supervision of Dr. Katja Broeg and Sonja Einsporn, PhD;,

Alfred Wegener Institute for Polar and Marine Research (AWI)Am Handelshafen 12, 27570 Bremerhaven, Germany

IMPACT OF METAL POLLUTION IN LIVER TISSUES OFCORKWING WRASSE FISH (SYMPHODUS MELOPS L.)AT

CELLULAR LEVEL

ByKedar Ghimire

Jacobs University Bremen


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Lab rotations III, IV in AWI [email protected] 2

Structural differences in organelles and its consequences in the livertissues of Corkwing Wrasse fish (Symphodus melops L.) sampledfrom differently polluted coastal sites of Norway

Kedar Ghimire,

Jacobs University Bremen, School of Engineering and Science, Campus Ring 1, 28759 Bremen,Germany

Wrasse (Symphodus melops L.) is an important marine species for monitoring theenvironmental and health effects of contamination in North Sea. Due to the toxicsubstances like PAH (polycyclic aromatic hydrocarbons), biocide(C-Treat 6), TBT etcreleased by aluminium smelters; metal contamination of coastal water due to coppermines; the habitats of this fish have been negatively effected. Many of these fishes havebeen found to be effected with various diseases that directly affects the vital metabolicorgans of the body like the liver hinting to the fact that the situation of life forms in theseareas are in peril. Through this study, we have attempted to explore the liver tissues(hepatocytes) from various wrasse samples living in metal (copper) contaminated sitesand reference sites and make a comparable analysis of the structural and functional

changes observed in the cell organelles at electron microscope level. We conclude that Cucontamination is harmful and it affects the cell organelles in liver tissues of Wrasse indifferent ways.

Keywords: Hepatocytes, lipid, copper, metallic crystals, metabolism, glycogen, electronmicroscopyAbbreviations: TBT: TributyltinPAH: Polycyclic aromatic hydrocarbonEM: Electron microscopy

Introduction:Wrasse is an interesting fish species

whose gender changes from female to maleduring the life time (a protogyn) (Broeg et al,2007). It has a flat body structure. Specificchemical impacts are expected to changemorphology and consequently, the function ofits organs. Increasing frequencies oftoxipathic lesions and liver tumors have beenreported in other fish from areas with chemicalimpact of pollution (Gardner et al., 1991;Koehler et al., 1992; Johnson et al., 1993;Stein et al., 1990; Stentiford et al., 2003). Wefear Wrasse can be another such victim.

Fish are poikilothermic vertebrates sothey change their metabolism according to thetemperature variations throughout the yearand all those changes are reflected in the liver.Fish are highly susceptible to environmentalvariations and respond sensitively to pollutantsthan other various mammals (Munsi andDutta, fish morphology, 1996). The liver of thewrasse has many digestive and storage

functions. Liver cells secrete bile which

emulsifies fat and helps change the acidic pHof stomach into neutral pH of the intestine. Bilecollects in the bile capillaries, which then unite,forming bile ducts. The bile canaliculus is astructure formed by grooves on the contactsurface of adjacent liver cells, i.e. the dilatedintercellular space between adjacenthepatocytes. Bile forms in these canaliculi andthen flows into small ducts, and finally intolarger hepatic ducts.

Figs. 1.2 and 1.1 in the next pagesshow a liver tissue with a normal nucleus,plenty of glycogen granules, lot of vesicles,lysosome and plenty of mitochondria. It shouldbe noted that the liver is the major site for Cuexcretion (in the bile) in vertebrates. Whilecopper is an endocrine disrupter in the aquaticanimals and has a number of neuro-endocrineeffects in vertebrates (Handy, 2003).

The fish were sampled from fivedifferent fjord sites in Norway. Site 1 was


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Salvy, considered to be an outer referencesite on the west side of Karmoy. Site 2 wasVisnes- a highly copper and zinc contaminatedsite on the west side of Karmoy. In this site,both tailings and slag was dumped too. Site 3was Frlandsfjorden- an extremely shelteredfjord representing the inner part of the fjordsystem, with small boat traffic and some smallfarms that drain to the fjord with vast amountof mussels found along the shores of the fjord.Site 4 was Bokn- a reference site in theexposed part of the fjord system. Site 5 wasHgevarde- a site just north of the PAHdischarge from the Alumina smelter inKarmsund. Other discharge there consisted ofbiocide, TBT. Among these sites, our studyfocused on site 2 and 3. Site 2 was influencedby an old copper mine which was inproduction 1865-94 and a new production fora few years until 1965. The area closest to the

old mine had no sign of life. The sea waterwas exposed to the metals mainly copper andzinc from the fillings and the run off from land.Station 3 was our reference site.

We used the methods of microscopicanalysis at light and EM level in our study toobserve structural changes in the liver tissuesand the consequences of these changestowards the physiology and adaptation ofWrasse.

Materials and Methods:Our samples were embedded in the

year September 2001 and were preservedsafely.

For electron microscopy, afterembedding liver tissues into epoxy resin, amicrotome (Model Leica EM UC6) equippedwith a diamond knife was used to cut first verythin sections for examining under lightmicroscope (Zeiss Axioskop). The settings ofmicrotome was speed (mm/s) =1 andfeed/nm=500. For this, three samples (Nr. 2,3, 4) from station 2 and two samples (Nr. 1, 5)from station 1 were chosen. Five slides fromeach sample were prepared. Among them,

two of each sample were stained with TolueneBlue 0.5% in Na2CO3. Toluene blue wasfiltered and the tissue sections were stainedfor 1-2 minutes in it. We had used variationsfor this process. Two slides for each samplewere prepared. One sample was heated(Stuart SB 300 heater) to magnitude 2 andwas stained for 2 minutes. The other samplewas stained for 1 minute with heat magnitude

of 3. The sections were then washed with dist.water and dipped in ethanol for dehydrationand quickly taken out and dried. After lightmicroscopic analysis, it was found thatsamples heated at 2 and stained for 2 minutesproduced better results and weresubsequently used.

After light microscopy, theblock sections were marked after consideringtheir special characteristics to observe underEM. These marked sections were prepared inblock removing other unnecessary areas withblade. Then the microtome (Model Leica EMUC6) was used to prepare ultra sections forEM. The settings of 1 mm/s speed and 60feed/nm was used for this purpose. The slicedsections were placed in small grids carefully.Then staining was performed. First, thesample was stained with Uranyl acetate for 5

minutes, and then washed thoroughly withddH2O. Then the sample was again stained inlead citrate for exactly 1 minute and washedthoroughly well and let to dry for a night. Fewequivalent samples were not stained so thatcomparable analysis could be done betweenstained (contrasted) and unstained(uncontrasted) sections of the same region.

Results:We tried to see and note the

differences in structure and consequently infunction of cellular organelles of Corkwing

wrasse fish from metal sites and referencesite. The differences between normal andpollution-effected tissues will be discussed infull detail in the discussion. Our results couldbe explained through various EM images ofthe liver tissue of Corkwing Wrasse fish.

Overview

Fig 1. Transmission EM Overview of the tissuefrom station 2 (polluted site) at 3000 magnification


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Fig 1.1 Transmission EM Overview of the liver

tissue from station 3 (reference site) at 12000 magnification

Observations on mitochondria andlysosome

Fig 2. Normal mitochondria seen in liver tissuesfrom station 3 at 12000 magnification

Fig3.Transmission EM of a mitochondrial overviewin section of liver tissues from station 3 at 20000magnification

Figs. 4, 4.1, 5, 6, 7, 8, 9, 10 under the sameheading as fig. 2 could be found in appendix 1.

Observations on nucleus andEndoplasmic Reticulum

Fig 11. EM of Rough endoplasmic reticulum fromstation 2 at 20000 magnification


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Observations on metal deposits intissues and bile canaliculus

Fig 12. Assumed metal deposits inside the cellsfrom station 2 at 7000 magnification

The black deposits in figure 12 are assumedto be pieces of metallic elements. It was seenat random places and not uniformly.

Fig 14. Bile canaliculi with black deposits in thesurrounding along intracellular pathways at 4400 magnification from the station 2 metal site.

Fig 18. Black deposits in intracellular space at20000 from station 2.

Figs. 13, 15, 16, 16.1, 17 and 21 under thesame heading as fig. 12 could be found inappendix 1.

Fig19. Assumed metal deposits in Bile canaliculuswith lipids alongside at 3000 magnification,(station 2- nC)


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Fig 20. Black deposits in the intercellular space

near bile canaliculi at 3000 magnification- non-contrasted image (station 3)

Observations on lipid and glycogenpresent in tissues

Fig 22. Glycogen penetrating lipid and

mitochondria attached to lipid from livertissues at station 2 at 30000 magnification

Fig 23, 24, 25 and 26 under the same headingas fig. 22 could be found on the appendix 1.

Discussion:Aqueous Cu has been reported to

accumulate in several tissues like gill, kidneyand liver during chronic exposure and there islesser accumulation in muscle (Handy, 2003).The metal site at Visnes is a chronic exposure

to fish since there is an abandoned mine. Insuch type of exposure, it had been found fishhave more time to down regulate Cu uptakethrough the gills and distribute newly acquiredcopper to the liver for excretion to minimizethe toxological effects of copper (Grosell et al.,1996, 1997, 1998). It is also known that fish tryto adjust to the metal exposure by initiatingcomplex physiological adjustments likeincreased oxygen consumption, increasingneutrophils, altered immunity, increasing ionicregulation and altered cellularity (Handy,2003). Copper demonstrates a high affinity forthiol groups and is therefore capable ofseverely disrupting many metabolic functionsin the cell (Hultberg et al., 1998).

Fig 1.2 (see Appendix 1) is atransmission electron micrograph which showsa clear overview of the wrasse liver tissue at3000 magnification. The section was fromstation 3 which consisted of our reference site.Lysosome engulfing a lipid molecule could beseen. A normal nucleus with general lipiddroplets was seen. No abnormalities wereseen in the cells from the liver tissues ofWrasse from reference site as expected. Fig1.1 shows another section from the station 3at higher magnification of 12000. Anoticeable observation was that lots ofvesicles could be seen, implicates that the cellwas quite active with all types of intra-cellularactivities going on and consequently, shouldbe a very healthy cell. Mitochondria seemed tobe coupled with the lipid droplet.

Compared to cells from reference site,fig 1 shows EM of the liver tissue from site 2-metal contaminated sites. No. of glycogengranules was much higher than those seen in

the reference site. It can be inferred that thereis at least some disruption in gluconeogenesisdue to which glycogen couldnt convertsufficiently into glucose. Carattino et al. (2004)had shown that Cu significantly inhibitsglucose-6-phosphate dehydrogenase activityin-vitro in their study on effects of Cu onmetabolism through pentose phosphatepathway in toad ovaries. Cu has also been


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found to interfere with the glycolytic pathway(Strydom et al., 2006). Glycogen granuleswere seen attached to the periphery of thelipid molecules. The reason for this closeassociation could be that glycogen as apolymer of glucose can form covalent bondswith the fatty acid chains in lipid. Fatty acidsare made by repeatedly joining together thetwo-carbon fragments found in acetyl-CoA andthen reducing the (-CO-) part of the moleculeto (-CH2-). In this way, the hydrocarbon chaincan become the hydrophobic, energy storingpart of the fatty acid.

The number of lipid molecules isconsiderably high in comparison to cells fromreference site. This clearly hints towards somenegative effects on the reactions catalyzinglipid break down. In reverse, there is increasedimportance of the catabolism of lipids in suchcells so that they wouldnt accumulate to

harmful level. Lipid peroxidation in response tocopper exposure has been reported infreshwater crab (Vosloo et al, 2002). In ourimages, lysosome was seen attached to thelipid and could be in the process of degradinglipids. Lipid peroxidation is considered as ameasure of oxidative stress and general stressthus is an indicator of fish health as a whole(Marcogliese et al, 2005). A suspectedtransport of black deposits (possibly metalcrystals) was seen at 3000 M denoted in thefigure as SP. An increased stimulation of ROSproduction by metals may lead to an

imbalanced oxidative stress condition in fishthat may result in physiological alterations(Sies, 1993, Paris-Palacios et al., 2000 andVaranka et al., 2001). Huge lipids were seenin sections from polluted sites.

Figure 2 and figure 3 shows an imageof the tissue at 12000 M from station 3,showing normal mitochondria (1-5 m) withparallel cristae. There was no sign of anyprecipitates in these mitochondria. Lysosomeseemed to be in pearl structure and ER wasdilated. Again, a lot of vesicular activity was

seen around. In contrast to these, EM of livertissues from polluted sites (fig 4) showedelongated mitochondrias, which seem to bedamaged and totally irregular in shape. Aconsiderable amount of precipitate was seeninside the mitochondria. The parallel cristaestructure wasnt seen but cristae seemed to bedamaged at various places in the mitochondriaas it was seen at random positions within themitochondria.

Figure 4.1 (reference site) shows thepresence of lipid alongside mitochondria. Thelipids also were seen to be degraded bylysosome. Figure 5 shows a non contrastedimage of a cluster of lipids attached tomitochondria, this combination of mitochondriaand lipid structure hints towards a possibleinteraction between mitochondria and lipid fordegradation of lipids. Eugene P. Kennedy andAlbert Lehninger had already demonstrated in1948 that enzymes of fatty acid oxidation inanimal cells are located in the mitochondrialmatrix. It is known that A-Methylacyl-CoAracemase, found in both mitochondria andperoxisomes, is required for the metabolism ofisoprenoid compounds, e.g. cholesterol to bileacids, and other methyl branched lipids. So,this could well be happening in case of fish aswell. Mitochondria could be well affected dueto metal deposits and thus might not be

functioning properly to produce enzymes forlipid oxidation. Also in the same image, faultsand cuts on lipids could be seen where metalcrystals had aggregated. This could be seenas the harsh physical effects of metal crystalson the cellular organelles. Definitely, metalprecipitates seem to affect the cell organelleschemically as well as physically.

Figure 6 shows glycogen filledlysosome and high amount of glycogen allaround. It shows that there has beenobstruction in the pathway of conversion of

glycogen. The cell seems to be very active asa lot of lysosome and vesicles were seen.Again, an elongated and irregularly shapedmitochondria could be seen in the figure. Infigure 7, lysosome degrading lipid and a lot ofblack metal crystals were seen. However, infig. 6, lysosome seemed to have weakermembrane structure since the membranelining looked very irregular compared tonormal lysosome. It had been already shownthat the lysosomal membrane stability ofwrasse was impaired at the sites of PAHs andorganic contamination (Einsporn and Koehler,

2007) so it seems metallic pollution results insame, but could be in lesser extent as PAHand organic contamination are harsh andmore effective than simple metals like copper.The interior of the lysosomes (pH 4.8) is moreacidic than the cytosol (pH 7). The lysosomesingle membrane stabilizes the low pH bypumping in H

+from the cytosol, and also

protects the cytosol and the rest of the cell,


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from the degradative enzymes within thelysosome.

Fig. 8 clearly shows glycogen andribosome, ribosome attached to endoplasmicreticulum. Glycogen was seen to be of twotypes. Both bigger than ribosome in size, andwere stained differently. One was darklystained electron dense granules while theother was lightly stained. Glycogen appearedas electron-dense rosettes, termed alphaparticles. However, glycogen-rich andglycogen-poor liver is differentiated by no. ofalpha particles rather than in their size.

It is noticeable that some particles ofglycogen seem to have combination of bothlight and dark areas. This could either be dueto complex formation of glycogen withribosome or it is also known that alpha

glycogen contains beta particles within itself.And the alpha particles additionally containvarious enzymatic proteins involved in thesynthesis of glycogen and hence they arecalled glycosomes (Rybicka, 1996). Theshape of black particles in this reference siteimage seemed to be different from anuncontrasted image from metal site (fig. 26). Itis hard to distinguish between ribosomes andglycosomes because both types of organellesappear in U/Pb stained sections, as 20-30 nmparticles. Also both could attach to ERmembranes and cytoskeletal components

(Hesketh and Pryme, 1991).

Fig. 9 shows an uncontrasted imageof lysosome degrading lipid at 12000. Fig. 10(12000) shows bulged mitochondria incontrast to the normal mitochondria in livertissues of reference site (Fig 2, 12000). Ahuge difference in size could be seen betweenmitochondrias from polluted and unpollutedsites. In polluted site, mitochondria wereconsiderably swelled with cristae at randompositions. Again, we saw certain amount ofgranules like precipitates in the mitochondria.

We propose the swelling of mitochondria isdirectly related to the effect of precipitates onthe mitochondria.

In figure 11, the mitochondria seemedto be damaged with almost no cristae. Themitochondria are greatly enlarged and arefilled with concentric cristae, which isabnormal. Also, the mitochondrial matrixappeared to be inexistent. It is well known that

mitochondria are sensitive to cellular stressand have a pivotal role in the initiation ofprogrammed cell death. It could be proposedthat the structural change in the mitochondriabegins with the degradation of cristae due tometal pollution. In fig. 12 and fig. 14, densemetal deposits could be seen near bilecanaliculi. The tissue sections were fromcopper polluted site. It is of specificobservation that the density of the blackdeposits is around bile canaliculi and metalcrystals could be seen attached to the groovesof bile canaliculus. This could well be theexport and elimination pathway for theseprecipitates from the fish through bilecanaliculi. There is less possibility for thesedeposits to be background staining or dirtparticles because it can be seen clearly that itis not uniform throughout the tissue andlocalized in certain specific areas.

If it had been the remains of stainingprocedure, it should well have been seen allaround the tissue. Also, the sections weredead so there is no reason that the dirt, if itwas, should be attached to the grooves of bilecanaliculus instead of any other parts oftissue. But if these were metal deposits, itmakes perfect sense they were beingeliminated while the samples were taken andprepared for EM in 2001 and remained therefrozen. Fig. 15 (see appendix I) shows thesame at 12000. Our evidence is strongly

supported by Fig. 16 (see appendix I),16.1(see appendix I) and 20 and 21(seeappendix I). These were not stained beforeelectron microscopy and were noncontrastedimages but they still showed black particles onthe canaliculus and intercellular space. Alsoevident was the fact that the cells in directcontact with bile canaliculus showed moreparticles than the secondary cells after themand the particles were polarized at one side ofbile canaliculus, (see figure 12, 14, 15). In fig.13 (see Appendix I); unlike in the middle of thecell, the lining of the tissue was filled with

black deposits. It could be assumed that thesewere metal crystals but it couldnt beconfirmed whether it is copper or the residuesof uranyl citrate and lead acetate, since thiswas a contrasted image stained with these twocompounds. Numerous lipid droplets wereseen and this section was from polluted site.

Fig. 17 (see appendix I) and 18 showsblack deposits passing through an intercellular


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space presumably. When closely observed,the black deposits are minute rectangularshaped crystalline structures with sharpedges. These prove that they are metalliccrystals. Presence of abnormally accumulatedmetal crystals show detoxification function ofliver has been impaired.

Fig. 22 shows us the glycogenenclosed with lipid. Both dark and lightgranules of glycogen can be seen. It wasinteresting to see mitochondria blocking theway for glycogen to come out of the lipiddroplet. It might well be that this is one of theways through which glycogen make their wayinside lipid and are later trapped. Similar casewas seen in fig 24. Mitochondria squeezedbetween lipids, limiting the movement ofglycogen. The occurrence of glycogeninclusions in the liver hints that the metal

deposits could be causing biochemical stressin the fish from polluted areas. Theaccumulation of glycogen in hepatocytes couldresult in type IV glycogen storage disease(amylopectinosis) (Sherlock and Dooley, 1997;Peplow and Edmunds, 2005).

Fig. 23 is a wonderful case showingthe two types of glycogen, dark and lighter incolor complexion. On liver, glycogen appearsas electron dense rosettes, which are alphaparticles so we assumed the darker particlesto be alpha glycogen. The structural backbone

of ER could be seen clearly with ribosomes. Itcould be inferred that glycogen particles couldhave also formed a stable complex withribosomes.

Figures 25 and 26 show lipid dropletsin tissues from metal site at 12000 . Alsoevident are the lipids being degraded bylysosome selectively. Generally, Lysosomeswere seen to degrade lipids with black metaldeposits before others. In Cu-loaded animalsthere is overburden on biliary excretionpathways. It is typified by the sub-cellular

localization of Cu in non-cytosolic fractions,especially lysosomes (Klaverkamp et al.,1991). This is clearly seen in fig 26, which isan uncontrasted image of wrasse liverexposed to metal site.

To summarize, the study wassuccessful to illustrate that various cellorganelles could be affected due to physicaland chemical toxicity of metal pollution

especially copper. Very important organelleslike the mitochondria which are the powerhouse of cells were found to have irregularstructures, excess of glycogen was seen andamount of lipid droplets were by far, in lotmore amount than a normal cell should have.

On the other hand, due to the smallscale and time of this lab study, manyobservations could not be decidedconclusively. In future, Autometallographyshould be performed at the light and electronmicroscope levels to provide information onthe intracellular distribution of metals as wellas evidence of different responses to metalaccumulation. By checking the pH in andaround lysosome, it could be found outwhether they had weak membrane structure inreal since a considerable variation from pH 4.8means the proton pumps and chloride ion

channels on the membrane to maintain the pHis not functioning well, which is related todamages in membrane of lysosome. Theeffects of structural differences in mitochondriashould be studied thoroughly and quantitativeanalysis should be done on the unknowneffects on ATP production due to loss ofcristae seen on affected mitochondria.

AcknowledgementsI would like to thank Ute Marx

(Alfred Wegener Institute) for her technical

assistance in electron microscopy and SonjaEinsporn, PhD. and Dr. Katja Broeg for theirguidance and supervision during theexperiments.

References:

Broeg, K., Kaiser, W., Bahns, S., Koehler, A.(2007)Theliver of wrasse-morphology and function as a mirror ofpoint source chemical impact. Marine environmentalresearch. 14th international symposium pollutantresponsed in marine organisms. May,6-9 2007, Brazil

CARATTINO MD, PERALTA S, PREZ-COLL C, NAABF, BURLN A, KREINER AJ, PRELLER AF andFONOVICH DE SCHROEDER TM (2004) Effects of long-term exposure to Cu2+ and Cd2+ on the pentose phosphatepathway dehydrogenase activities in the ovary of adultBufo arenarum: Possible role as biomarker for Cu2+toxicity. Ecotoxicol. Environ. Saf. 57 311-318

C Strydom, C Robinson, E Pretorius, JM Whitcutt, JMarx, and MS Bornman (2006) The effect of selectedmetals on the central metabolic pathways in biology: Areview. ISSN 0378-4738 = Water SA Vol. 32 No. 4


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Einsporn S. and Koehler A., (2007) Lysosomal changesin Wrasse and in blue mussel from differently pollutednorwegian fjord sites (2007), Ecotoxicology

Grosell, M.H., Botius, I., Hansen, J.M. andRosenkilde, P., 1996. Influence of pre-exposure to sub-lethal levels of copper on

64Cu uptake and distribution

among tissues of the European eel (Anguilla anguilla).Comp. Biochem. Physiol. Part C114, pp. 229235

Grosell, M.H., Hogstrand, C. and Wood, C.M., 1997.Copper uptake and turnover in both Cu acclimated andnon-acclimated rainbow trout (Oncorhynchus mykiss).Aquat. Toxicol.38, pp. 257276.

M.H., Hogstrand, C. and Wood, C.M., 1998. Renal Cuand Na excretion and hepatic Cu metabolism in both Cuacclimated and non acclimated rainbow trout(Oncorhynchus mykiss). Aquat. Toxicol.40, pp. 275291.

Handy D. R. (2003) Chronic effects of copper exposureversus endocrine toxicity: two sides of the sametoxicological process? CBP. Part A 135 25-38.

HULTBERG B, ANDERSSON A and ISAKSSON A(1998) Alterations of thiol metabolism in human cell linesinduced by low amounts of copper, mercury or cadmiumions. Toxicol. 126 203-212.

J E Hesketh and I F Pryme (1991) Interaction betweenmRNA, ribosomes and the cytoskeleton. Biochem. J. 277 (10)

J.F. Klaverkamp, M.D. Dutton, H.S. Majewski, R.V.Hunt and L.J. Wesson, Evaluating the effectiveness of

metal pollution controls in a smelter by usingmetallothionein and other biochemical responses in fish.In: M.C. Newman and A.W. McIntosh, Editors, MetalEcotoxicologyConcepts and Applications, LewisPublishers Ltd., Chelsea, MI, USA (1991), pp. 3364

Marcogliese DJ, Brambilla LG, Gagne F, Gendron AD(2005) Joint effects of parasitism and pollution on

oxidative stress biomarkers in yellow perch Percaflavescens. Diseases of aquatic organisms. Vol 63: 77-84

Munshi J.S., Dutta H.M. (1996)Fish Morphology: Horizon of New Research, CRC press

Peplow D, R Edmonds, 2005. The effects of mine wastecontamination at multiple levels of biological organization.Ecological Engineering, 2005 (Vol. 24) (No. 1/2)

Rybicka KK (1996) Glycosomes- the organelles ofglycogen metabolism. Tissire Cell 28:253

Sherlock S. and Dooley J.S. Diseases of the Liver andBiliary System, 10th ed. Blackwell science, Oxford

Sies H. (1994) Oxidative stress: oxidants and

antioxidants, Experimental Physiology82.2 pp 291-295

Vosloo A., Aardt van W.J., Mienie, L.J. (2002) Sublethaleffects of copper on the freshwater crab Potamonautes.Comp. Biochem and Physiology 695-702


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APPENDIX 1, Lab rotation III and IV

Figures included in the discussion

Fig 1.2 Overview of the liver tissue from station 3(reference site) at 3000 magnification

Fig 13. The bile duct with a black metal liningthroughout edges at 3000 magnification, frommetal site.

Fig 15. Bile canaliculi with black crystal depositsat 12000 magnification from metal site at station2.

Fig 16. Bile canaliculi with black crystal depositsat 20000 magnification from station 2(uncontrasted image)


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Fig 16.1. Metal deposits in the bile canaliculi at12000 magnification from station 2(uncontrasted image)

Fig 17. Black deposits along an intracellularspace at 12000 magnification from station 2

Fig 21. Assumed metal crystals from station 2 at12000 magnification (non contrasted image)

Fig 23. Glycogen around lipid and ER at 12000 magnification (station 2)

L represents lipid while G shows glycogengranules.


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Fig 24. Glycogen around lipid and at 20000 magnification (station 2)

Fig 25. Lipids being degraded by lysosome inliver tissues at 12000 magnification from station

2 (uncontrasted image- metal site)

Fig26. Lipid filled with metal crystals beingdegraded by lysosome at 12000magnification from station 2 (non contrastedimage)

Fig 4. EM of Elongated mitochondria from station2 at 12000.

Fig. 4 shows a section of liver of Wrassewhich was collected from station 2- themetal polluted sites. Unusually long,stretched mitochondria were found. A lot ofglycogen was seen at most of the places.


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Fig 4.1. Lysosome in liver tissues degradinglipids from station 3 at 12000

Fig 5. EM of Mitochondria in an uncontrastedimage, from station 2 at magnification 12000magnification

Mitochondria can be seen squeezedbetween lipid droplets. Faults on lipids couldalso be seen where black deposits hadaccumulated.

Fig 6. Mitochondria and lysosome from station 2at 12000

Fig7. Lysosome filled with black deposits fromstation 2.


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Fig 8. Ribosome, glycogen from liver tissuesfrom station 3 at 20000 magnification

Fig9. Lysosome degrading lipid in uncontrastedTM image from station 3 at 12000 magnification

Fig 10. An uncontrasted image of mitochondriaand lipid in liver tissues from metal site at 12000 magnification

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