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The Greater Susceptibility of North Ronaldsay SheepCompared with Cambridge Sheep to Copper-inducedOxidative Stress, Mitochondrial Damage and Hepatic

Stellate Cell Activation

S. Haywood, D. M. Simpson*, G. Ross and R. J. Beynon*

Departments of Veterinary Pathology and *Veterinary Preclinical Sciences, Faculty of Veterinary Science,

University of Liverpool, Liverpool L69 3BX, UK

Summary

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Sheep of the semi-feral North Ronaldsay (copper-sensitive) and domesticated Cambridge (copper-tolerant) breeds were compared in respect of pathological changes and protein expression in the liver as aresult of excessive dietary copper. Acute mitochondrial damage and hepatic stellate cell (HSC) activationwith collagen synthesis occurred in response to moderate copper overload in North Ronaldsay but not inCambridge sheep. Mitochondrial degradative changes occurred either as ballooning degeneration andrupture with subsequent autophagic degradation or as mitochondrial matrical condensation (pyknosis).In North Ronaldsay sheep prolonged exposure to copper produced mitochondrial hyperplasia andhypertrophy, and nuclear damage with necrosis. Cytosolic isocitrate dehydrogenase (IDH), an enzymeresponsive to oxidative stress, was induced in the liver of Cambridge sheep receiving a Cu-supplementeddiet but was undetectable in the non-supplemented control sheep. Conversely, IDH was detected atsimilar levels in both control and copper-supplemented North Ronaldsay sheep, indicating a lowerthreshold response, and an enhanced susceptibility, to oxidative stress. “Upregulation” of mitochondrialthioredoxin-dependent peroxidase reductase (antioxidant protein-1) in the hepatic cytosol of the NorthRonaldsay (but not Cambridge) sheep affirmed the increased susceptibility of the mitochondria to Cu-induced oxidative stress in this breed. Likewise the upregulation of cathepsin-D indicated increasedlysosomal activity and HSC activation. The findings may be relevant to copper toxicosis in human infants.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: copper; hepatic stellate cells; isocitrate dehydrogenase; mitochondria; North Ronaldsay sheep; oxidative stress

Introduction

Sheep are intolerant of dietary copper excess dueto an impaired ability to excrete copper in bile,which in turn leads to liver copper accumulationand toxicity (Soli, 1980). The pathogenesis of thedisease is well documented in domesticated sheep,in which copper accumulates in the liver inconcentrations of 500–1000 mg/g in the cumulative(prehaemolytic) phase without clinical conse-quences, although sub-lethal pathological changesoccur (Ishmael et al., 1971; Dincer, 1994). At a livercopper concentration of O1000 mg/g, hepatic

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necrosis may culminate in a haemolytic crisis withprofound jaundice which is generally fatal (Ishmaelet al., 1971). Fibrosis, limited to a mild expansion ofportal connective tissue, has been reported in somecopper-poisoned domesticated sheep that recov-ered naturally (Ishmael et al., 1971) or followingcessation of copper-dosing (Gopinath and Howell,1975).

Sheep breeds vary in their susceptibility tocopper. The Merino sheep is probably the mosttolerant, and the North Ronaldsay the mostsusceptible (Wiener et al., 1978). The NorthRonaldsay is a primitive sheep which occupies an

J. Comp. Path. 2005, Vol. 133, 114–127

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Cu-induced Oxidant Stress in Ronaldsay Sheep 115

ecological niche on the copper-impoverishedforeshore of North Ronaldsay island, to which it iswell adapted by its powers of copper-storage(Maclachan and Johnston, 1982). If removed tothe mainland, however, the North Ronaldsay sheepis in danger of succumbing to copper toxicity(Wiener et al., 1977).

Distinctive pathological changes have beenreported in North Ronaldsay sheep exposed tothe copper-replete conditions of the mainland.These changes are characterized by a floridpericellular fibrosis similar to that occurring innon-Wilsonian hepatic copper toxicosis of infancyand childhood (Haywood et al., 2001). Thesimilarities were confirmed in a trial in whichNorth Ronaldsay lambs received different concen-trations of dietary copper, similar to those ingestedby susceptible bottle-fed infants (Haywood et al.,2004).

Genetic factors undoubtedly influence theresponse to copper, in which hepatocytes, hepaticmacrophages, Kupffer cells and hepatic stellatecells (HSCs) all play a role. However, at the cellularlevel it is the gene-products or proteins thatdetermine the nature of the pathological response.

To clarify the pathogenesis of copper toxicosis inNorth Ronaldsay sheep (and by extension child-hood copper toxicosis) the present study wasdesigned to assess protein expression andhistopathological and ultrastructural changes incopper-overloaded sheep of the North Ronaldsay(Cu-sensitive) and Cambridge (copper-tolerant)breeds. It was hoped, by these means, to identifythe sites responsible for the distinctive response ofthe North Ronaldsay sheep to copper exposure.

The pathological changes at the ultrastructurallevel are reported in detail here, substantiated bysome major changes in protein expression.

Materials and Methods

Animals and Experimental Procedure

Fourteen North Ronaldsay ewes aged 10 monthswere used, of which six received a diet of hay (Cu5 mg/kg dry weight) and 250 g/head/day of beet(Cu 5.4 mg/kg dry weight) over a 6-month period.The remaining eight animals were given the samediet except that the beet contained Cu 15 mg/kg, asupplement of CuSO4 having been added. Thecopper-supplemented animals were killed in pairsfor examination at intervals of 1, 2, 4 and 6 monthsafter the commencement of the experiment, toachieve liver copper concentrations similar to thoseproduced in Cambridge sheep by Simpson et al.

(2004). The non-supplemented animals were killedin pairs at 1, 2 and 6 months. Tissues were collectedand treated as described by Simpson et al. (2004).

Nine Cambridge ewes were housed and fed for aperiod as above, except that six animals receivedcopper-supplemented pellets containing Cu155 mg/kg copper. Full details are given in anearlier paper in this series (Simpson et al., 2004).

Metal Analysis

This was carried out as before (Simpson et al.,2004).

Histology

Formalin-fixed tissue was paraffin wax-embeddedand sections (5 mm) were cut and stained withhaematoxylin and eosin, rhodanine for copper andreticulin van Gieson for collagen.

Electron Microscopy (EM)

Samples of liver (1 mm3) were fixed in sodiumcacodylate-buffered glutaraldehyde 2.5% (v/v) for16–24 h, transferred to Zetterquist’s veronal acet-ate-buffered osmium tetroxide fixative for 1 h,dehydrated with acetone and embedded in epoxyresin (Spurr, 1969). Thin sections (5 mm) fromseveral blocks obtained from two hepatic lobes(dorsal and ventral) were stained with toluidineblue and scanned under the light microscope.Subsequently, ultrathin sections from selectedblocks were stained with uranyl acetate and leadcitrate, placed on metal grids, examined under atransmission electron microscope (Hitatchi H600). Whole sections were scanned and cellularchanges recorded with respect to their microana-tomical location.

Proteomics

The soluble protein fractions obtained fromcentrifugation of homogenized liver samples wereseparated on 2D gels on the basis of charge (pH 5–8) and size. The gels were subsequently analysed bygel comparison software and proteins whoseexpression pattern had been modified as a resultof copper challenge excised and subjected to in-geltryptic digestion followed by MALDI-ToF MS(matrix-assisted laser desorption ionization–timeof flight mass spectrometry). The peptide massfingerprint of the protein obtained was searchedagainst data bases for sufficient sequence homologyto identify the protein. If this proved unsuccessful,partial sequence tags were obtained by tandemmass spectrometry, which greatly increased the

S. Haywood et al.116

chances of identification. Full details of thesetechniques were given by Simpson et al. (2004).

Results

The appearance and behaviour of all the sheepremained normal throughout.

Cambridge Sheep

Liver copper concentrations. The results of copperanalysis have already been given (Simpson et al.,2004).

Liver pathology. The results are given in Table 1.

Light microscopy. After 3 months of copper sup-plementation (liver copper concentration 400–500 mg/g) a fine deposit of rhodanine-positivegranules (at the limit of light microscope resol-ution) was identified in perivenular hepatocytes(zone 3), which by 4 months (liver copperconcentration O1000 mg/g) had extended to alllobular zones. Histopathological changes wereabsent at 3 months, but at 3.5–4 months nuclearanisoploidy, apoptotic bodies, hepatocellular mito-sis and a few inflammatory microfoci dispersedthroughout the lobular parenchyma were present.

TableThe pathological response of the livers of Cambridge a

Liver copper con-

centration (?g/g) Liver changes in Cambridge sheeps

!300 (Controls) Hepatocytes: normalKupffer cells: activated (C)

HSCs: quiescent400–550 Hepatocytes: mitochondria, matrix granular

lysosomes, C; SER, proliferation and dilatatioRER, parallel arrays

Kupffer cells: activatedCHSCs: quiescent

800–900 Not examined

1000–1400 Hepatocytes: mitochondria, matrix granularitswelling C; lysosomes, C; SER, swelling C to CC

disaggregation C; apoptosis, CC; necrosis

Kupffer cells: activated C

HSCs: quiescent

SER, smooth endoplasmic reticulum; RER, rough endoplasmic reticulCC, moderate; CCC, marked.

The Kupffer cells occasionally contained brownpigment and were stained positively for copper byrhodanine.

Electron microscopy. With a moderate liver copperconcentration of 400–500 mg/g hepatocytechanges varied. Dilated smooth endoplasmic reti-culum (SER) cisternae were present in somehepatocytes; mitochondria were occasionally mildlyswollen with increased matrical granularity, whichtended to obscure the cristae. Rough endoplasmicreticulum (RER) was normal and organized intoparallel arrays. Electron-dense lysosomes werepresent, but not in great numbers. Kupffer cellsoccasionally contained electron-dense particles butfor the most part appeared inactive. HSCs, whichwere sparsely dispersed throughout the lobules andquiescent, consisted of ovoid cells containing oneor two lipid (retinol) droplets at the nuclear pole.

At a liver copper concentration of 1000 mg/gand over, abundant apoptosis and apoptotic bodieswere identified, both free within the sinusoids(Fig. 1) and phagocytized within hepatic parench-ymal cells, some still viable and others undergoingdissolution (Fig. 2). The lesions were not confinedto any one zone. Hepatocytes undergoing necrosiswere identified by the breakdown of the plasmamembrane and degeneration of cytoplasmic

1nd North Ronaldsay sheep to excesss dietary copper

Liver changes in North Ronaldsay sheep

Hepatocytes: normalKupffer cells: activated (C)

HSCs: quiescentity;n C;

Hepatocytes (periportal zone): mitochondria, swellingand balloon degeneration with rupture CC; SER,

proliferation and dilatation C; RER,disaggregation (C); lysosmes, C

Kupffer cells: activated CCHSCs: activated C to CC, collagen synthesis

Hepatocytes (panlobular): mitochondria, (i) balloondegeneration with rupture CC to CCC, (ii) matrical

condensation (pyknosis) CC; peroxizomes, CC;lysosomes, C; SER and RER as above

Kupffer cells: activated CC

HSCs: activated CC, collagen synthesisy and

; RER,, C

Hepatocytes (panlobular): mitochondria, (i) balloondegeneration with rupture C, (ii) pyknosis C, (iii)

hypertrophy and pleomorphism CC to CCC; SER,CC; RER, disaggregation and degranulation C toCCC; apoptosis, (C); necrosis (periportal), C to

CC/CCC

Kupffer cells: activated CC to CCC

HSCs: activated CC, collagen synthesis

um; HSCs, hepatic stellate cells. (C) to C, negligible to minimal;

Fig. 1. Cambridge sheep liver (Cu 1017 mg/g). Apoptotichepatocyte (A) in sinusoid showing surface protuber-ances (blebbing), marked condensation of cytoplasmiccontents with organelle and nuclear remnants.Residual body (R) and inactive Kupffer cell (K). EM.Bar, 3 mm.


organelles within autophagic vacuoles (cytolyso-somes; Fig. 2). Residual bodies containing bothfloccular and electron-dense undegraded materialwere identified (Fig. 1 and 2). Nuclear degenera-tive changes consisted of peripheral chromatincondensation and pyknosis.

Some Kupffer cells contained electron-densematerial; they appeared unreactive and were not

Fig. 2. Cambridge sheep liver (Cu 1017 mg/g). Viablehepatocyes juxtaposed to necrotic hepatocytes(arrows) showing dissolution of plasma membraneand cytoplasmic organelles. Apoptotic body (A),residual body (R), concentric whorled cytolysosome(C). EM. Bar, 5 mm.

engaged in phagocytosis of apoptotic bodies(Fig. 2). HSCs were uniformly quiescent.

North Ronaldsay Sheep

Liver copper concentrations. Two sheep that receiveda Cu-supplemented diet and were killed 1 monthafter the commencement of the experiment hadliver copper concentrations (mg/g) of 528.0 and364.8; at 2 months there was little change (491.5and 543.9). Subsequently, however, liver copperincreased strikingly (879.9 and 899.5 at 4 monthsand 993.9 and 1489.9 at 6 months). The results insheep receiving the non-supplemented diet werehighly variable (295.8 and 337.3 at 1 month; 187.8and 421.8 at 2 months; 539.6 and 426.3 at 6months), in contrast to equivalent values inCambridge sheep (Fig. 3).

Liver pathology. The results are given in Table 1.

Light microscopy. At a moderate copper overload of400–550 mg/g, changes were restricted to nuclearanisoploidy. At higher Cu concentrations of 800–900 mg/g, hepatic parenchymal swelling was

Fig. 3. Copper content in liver of normal and copper-loadedNorth Ronaldsay sheep. Samples from the perfuseddorsal and ventral lobes of control (open symbols) orcopper-loaded (closed symbols) sheep. The results aresuperimposed on previously published values (Simpsonet al., 2004) for normal liver copper values in Cambridgesheep (shaded).


noticeable in periportal zones (zone 1) of liverlobules, in which hepatocytes had a rarified andoften vacuolated appearance. Kupffer cells wereplump and prominent and often contained rhoda-nine-positive granules. At Cu concentrations ofapproximately 1000 mg/g and over, there wasevidence of irreversible damage in many periportalhepatocytes (20%), with increased cytoplasmiceosinophilia and karyolysis. Mild inflammatorychanges were represented by polymorphonuclearor mononuclear microfoci and a low grade generalincrease in polymorphs.

Histochemically, Cu was seen to be present in afine granular form (at the limit of the lightmicroscope resolution). Excess Cu, unlike that incopper-supplemented Cambridge sheep, accumu-lated particularly in periportal lobular zones.

Fig. 5. North Ronaldsay sheep liver (Cu 492 mg/g). Balloonedand ruptured mitochondrion within periportal hepato-cyte, including concentric whorled mitochondrialmembrane (arrow). EM. Bar, 1 mm.

Electron microscopy. The livers of control sheep withhepatic Cu concentrations of !300 mg/g did notdiffer from their Cambridge counterparts, consist-ing of unremarkable hepatocytes and Kupffer cells,and quiescent HSCs.

At moderate copper loading (400–550 mg/g),however, marked changes were identified inRonaldsay livers, in contrast to those of theequivalent Cambridge sheep. These changesaffected hepatocytes and mesenchymal cells(Kupffer cells and HSCs).

The hepatocytes occupying the periportal lobu-lar zone (zone 1) were mildly distended with

Fig. 4. North Ronaldsay sheep liver (Cu 544 mg/g). Distressedmitochondria within periportal hepatocyte showingballooning and rupture of limiting membranes(arrows). Electron-dense lysosome (L). EM. Bar, 1 mm.

dilated SER. Their mitochondria were swollenwith increased matrical granularity or radialclumping and blunting of cristae, and ballooningof mitochondria with rupture of mitochondrialmembranes was common (Fig. 4). Rupturedmitochondria often contained concentric mem-brane whorls within their ballooned portions(Fig. 5), the apparent prelude to cytolysosomeformation (Fig. 6). SER was abundant and RER wasmainly arranged in parallel arrays. By contrast,hepatocytes from the intermediate and periacinarlobular zones (zones 2 and 3) contained

Fig. 6. North Ronaldsay sheep liver (Cu 1400 mg/g). Cytoly-sosme within hepatocyte; whorled membranes enclos-ing degraded organelle (arrow). EM. Bar, 1 mm.

Fig. 8. North Ronaldsay sheep liver (Cu 544 mg/g). ActivatedHSC with collagen fibrils (C) penetrating betweenintercellular junctions of adjoining hepatocytes. Bilecanaliculus (bc). Lysosomes (L). Mitochondria withperipheral flocculent aggregates (m). EM. Bar, 2 mm.


mitochondria with mild swelling and increasedmatrical granularity.

Changes were also apparent in mesenchymalcells. Kupffer cells were prominent, with largeplump nuclei, and frequently contained electron-dense spheroidal bodies, identified as copper-containing lysosomes and consistent with therhodanine-positive granules seen under the lightmicroscope. About 20% of HSCs appeared acti-vated, i.e., while still possessing their identifyinglipid retinoid vacuole within the cytoplasm, theyalso displayed an intimate relation to extracellularcollagen fibrils, which appeared to be emanatingfrom their cytoplasm. The collagen fibrils either layin close apposition to HSCs within the space ofDisse bounding the sinusoids (Fig. 7) or hadpenetrated between the intercellular junctions ofhepatocytes (Fig. 8).

At higher copper loading (800–900 mg/g),ballooning and rupture of the mitochondria weresufficiently marked in periportal lobular hepato-cytes to cause considerable rarefaction of thecytoplasm (Fig. 9). Changes were now found inthe inner lobular zones (zones 2 and 3), mitochon-drial swelling, balloon degeneration and rupturebeing common; pyknotic mitochondria could alsobe identified, distinguished by intense matricalcondensation with electron-lucent cristae(Fig. 10a,b). Not all mitochondria, however, were

Fig. 7. North Ronaldsay sheep liver (Cu 544 mg/g). Activatedhepatic stellate cell (HSC) in perisinusoidal space,juxtaposed between hepatocytes. Collagen fibrils (C)in space of Disse. Retinol vacuole (R) within HSC. EM.Bar, 2 mm.

degenerate and some showed evidence of replica-tion in the form of a transecting membrane(Fig. 10b). Aggregates of spheroidal, homo-geneous, electron-dense microbodies or peroxi-zomes were seen in the vicinity of damagedmitochondria (Fig. 10a). Small numbers of elec-tron-dense bodies, unequivocally identified aslysosomes by the presence of single membrane,absence of cristae and inclusion of occasional fatdroplets (see Fig. 4), were present within hepato-cytes. Activation of Kupffer cells was characterizedby their increased size, prominent nuclei and

Fig. 9. North Ronaldsay sheep liver (Cu 880 mg/g). Periportalhepatocyte distended by vacuoles created by rupture ofgrossly ballooned mitochondria (M), including con-centric whorled membranes (C). EM. Bar, 2 mm.

Fig. 10a,b. North Ronaldsay sheep liver (Cu 880 mg/g). (a) Hepatocytes (intermediate zone) with distressed swollen mitochondriaexhibiting ballooning and rupture (Ms) and mitochondria with matrical condensation and dilated electron-lucentcristae (Mc), peroxizomes (P) and Kupffer cell (K) with electron-dense lysosomes. Hypertrophied and dilated SER. EM.Bar, 2 mm. (b) Pyknotic mitochondria with dilated electron-lucent cristae, contrasting with swollen mitochondria withgranular contents; also, a dividing mitochondrion with transecting membrane (arrow). EM. Bar, 1 mm.



electron-dense lysosomes (Fig. 10a). HepatocyteSER was hypertrophied, and RER, which showedmodest degranulation, was generally arranged inparallel arrays. Occasional lipid vacuoles wereidentified. Increased numbers of HSCs wereactivated, with associated collagen synthesis, butthese mesenchymal changes showed no regionalpredilection.

At a high liver Cu concentration (1000 mg/g) theswollen and ruptured hepatocyte mitochondriahad become less evident and at a concentrationof 1400 mg/g could no longer be identified. Bycontrast, hepatocellular mitochondria showedincreased numbers (hyperplasia), hypertrophy(megamitochondria) and marked pleomorphism,often with bizarre forms. These altered mitochon-dria exhibited either a uniform matrix withrecognizable cristae or a matrix with flocculentdensities; alternatively, some mitochondria werepyknotic with an electron-dense matrix containingelectron-lucent structures identified as dilatedcristae (Fig. 11 and 12a,b). The SER was stilldilated, but compaction with increased homogen-eity was present in some hepatocytes and the RERshowed some disaggregation and degranulation(Fig. 12a). Once again electron-dense bodies,unequivocally lysosomes, were relatively few innumber. The nuclei of many periportal hepatocyteswere often noticeably enlarged (polyploidy) andcontained more than one nucleolus; the chromatinshowed a tendency to dispersal.

Fig. 12a,b. North Ronaldsay sheep liver (Cu 1400 mg/g). (a)Hepatocyte (periportal zone) with enlargednucleus showing dispersal of chromatin. Cytoplasmcontains large numbers of hypertrophied, pleo-morphic mitochondria, some of which showintense matrical condensation (arrows), whileothers show flocculent densities. SER showsincreased homogeneity and granularity. RERshows some disaggregation and degranulation.EM. Bar, 2 mm. (b) Pleomorphic hepatocytemitochondrion with matrical flocculent densities,electron-lucent crista (C) and double limitingmembrane (arrow). EM. Bar, 0.5 mm.

Fig. 11. North Ronaldsay sheep liver (Cu 1400 mg/g).Hepatocyte (periportal zone), enlarged (polyploid)nucleus with two nucleoli; also, numerous enlarged,frequently pleomorphic mitochondria, some ofwhich exhibit matrical condensation (pyknosis)(arrows). SER abundant and dilated. EM. Bar, 2 mm.

Fig. 13a,b. North Ronaldsay sheep liver (Cu 1400 mg/g). (a)Necrotic hepatocyte (periportal zone) with centralnuclear chromatolysis, peripheral margination ofextant chromatin and nucleolus. Mitochondria(Mc) are pleomorphic with many hypertrophiedbizarre forms and show nearly uniform matricalcondensation. Smooth endoplasmic reticulum(SER) uniformly homogeneous and finely gran-ular. Rough endoplasmic reticulum (RER) showsmarked disaggregation and degranulation. Golgiapparatus (G) is dilated. EM. Bar, 2 mm. (b)Pyknotic hypertrophied hepatocyte mitochon-drion with intense matrical condensation anddilated electron-lucent cristae. Note discontinuityof nuclear outer membrane (arrow). EM. Bar,0.5 mm.


Necrosis, particularly in the periportal zones ofliver lobules, was now a striking feature, greatlyoutweighing apoptosis. This contrasted with thesituation in Cambridge sheep counterparts. Hep-atocytes undergoing necrosis were identified bynuclear chromatolysis (Fig. 12a), frequently culmi-nating in a complete central depletion of chroma-tin, leaving a residual rim with nucleolarmargination (“signet ring” appearance)(Fig. 13a). Mitochondria increasingly were of thepyknotic variety with marked dilatation of cristae(Fig. 13b). In addition, the outer nuclear mem-brane was incomplete or missing (Fig. 13b). Thecytoplasm had become condensed, with a uni-formly homogeneous and finely granular SER(Fig. 12a and 13a); the RER was disaggregatedand degranulated (Fig. 13a).

Kupffer cells were much in evidence, beinghypertrophied and containing electron-dense lyso-somes. HSCs were frequently enlarged, withcopious cytoplasm and obvious stellate pro-longations that contained numerous fine filaments.The majority of the HSCs showed evidence ofactivation with some collagen synthesis; however,retention of the retinoid droplet, often reduced insize, suggested that full phenotypic transformationhad not yet taken place.

Proteomics

Fig. 14 shows a 3dimensional representation of aprotein identified by MALDI-ToF MS and peptidemass fingerprinting as cytosolic NADPC-dependentisocitrate dehydrogenase (IDH). In Cambridgesheep (Simpson et al., 2004), IDH was induced inresponse to a Cu-supplemented diet and waspresent in the livers of sheep with both moderate(Fig. 14) and high Cu concentrations but was notdetected in the livers of control Cambridge sheep(Fig. 14). In contrast, IDH was present in the liversboth control and copper-supplemented NorthRonaldsay sheep (Fig. 14). Moreover Cu-loadedNorth Ronaldsay sheep, unlike Cambridge sheep,showed a 4–5 fold increase in mitochondrialthioredoxin-dependent peroxide reductase (anti-oxidant protein-1) (Fig. 15). In addition, cathepsinD was increased (Fig. 16) in 2D gels derived fromcytosolic liver samples of North Ronaldsay sheepgiven high doses of copper.

Discussion

Both the Cambridge and North Ronaldsay sheepaccumulated excessive liver Cu concentrations(1000 mg/g) from similar basal levels within the

Fig. 14. The expression of cytosolic NADPC-dependent isocitrate dehydrogenase in Cambridge and North Ronaldsay sheep withmoderate Cu-overload. The region of the 2D gel encompassing a protein identified as cytosolic NADPC-dependentisocitrate dehydrogenase by MALDI-ToF MS and peptide mass fingerprinting is shown for two Cu-loaded Cambridge(Camb) and North Ronaldsay (NR) sheep and their control counterparts (arrows). The copper concentration in eachindividual liver (mean of dorsal and ventral lobes), determined by ICP-MS, is indicated.


observation period of 4–6 months. However, the Cudose level administered to the North Ronaldsays(15 mg/kg) was only one-tenth of that given to theCambridge sheep. Moreover, the unsupplementedNorth Ronaldsay sheep, unlike their Cambridgecounterparts, continued to accumulate copper ondiets containing as little as 6 mg/kg. This reaf-firmed the propensity of the North Ronaldsaysheep to accumulate potentially toxic

Fig. 15. The expression of thioredoxin-dependent peroxide redsheep. The regions of the 2D gels encompassing a proteMALDI-ToF MS and peptide mass fingerprinting is shownThe copper concentration in each individual liver (mean o

concentrations of copper when removed fromtheir Cu-impoverished habitat.

Early in the trial, moderate copper loading(400–550 mg/g) provoked marked changes in thelivers of the North Ronaldsay but not Cambridgesheep. Severe damage was incurred by the hepato-cyte mitochondria of the North Ronaldsay sheep,accompanied by reactive changes in local mesench-ymal cells, including HSC activation and collagen

uctase in control and copper-supplemented North Ronaldsayin identified as thioredoxin-dependent peroxide reductase byfor control and Cu-challenged North Ronaldsay sheep (arrows).f dorsal and ventral lobes), determined by ICP-MS, is indicated.

Fig. 16. The expression of cathepsin D in control and copper-supplemented North Ronaldsay sheep. The regions of the 2D gelsencompassing a protein identified as cathepsin D by MALDI-ToF MS and peptide mass fingerprinting is shown for controland copper-challenged North Ronaldsay sheep (arrows). The copper concentration in each individual liver (mean ofdorsal and ventral lobes), determined by ICP-MS, is indicated.


synthesis. Established differences in response wereexacerbated in copper overload (O1000 mg/g) inthat apoptosis preceded and overshadowed necro-sis in the Cambridge livers, whereas in the NorthRonaldsays necrosis was dominant. Laying down ofcollagen took place progressively along sinusoidsand between intercellular junctions, presaging thepericellular fibrosis that characterizes the estab-lished disease in North Ronaldsay sheep, and whichimpairs nutrient exchanges and disrupts intercel-lular communications (Haywood et al., 2001, 2004).

Copper poisoning has been studied in domesti-cated breeds of sheep, at the light microscope level(Ishmael et al., 1971; Dincer, 1994) and ultra-structurally (King and Bremner, 1979). The patho-logical changes in Cambridge sheep livers accordedwith these earlier reports. King and Bremner(1979) highlighted the incidence of apoptosisand established the importance of autophagy incontributing to cell death (necrosis). However,although limited expansion of the portal tracts byfibrous tissue was reported by these authors and byGopinath and Howell (1975), the state of the HSCsin copper toxicosis was not examined.

Ishmael et al. (1971) reported initial depositionof histochemical copper in centrilobular zone 3 incopper-loaded Clun Forest lambs, a findingrepeated in Merino sheep by Kumarilatike andHowell (1986). However, King and Bremner (1979)reported the converse in copper-loaded Finn-Dorset!Suffolk ewes. The findings from our ownstudies have been equally conflicting. Thus, copper

deposition in the Cambridge trial followed thepattern established by Ishmael et al. (1971),whereas excess copper accumulation in the NorthRonaldsays had more in common with the findingsof King and Bremner (1979); certainly the onsetand severity of lesions were particularly marked inthe periportal zones.

Variations in the microanatomical distributionof lesions in copper-storage diseases were summar-ized by Fuentealba et al. (1989b), who found somerelation with intralobular copper retention. Gen-erally, there would seem to be a relationship tometabolic zonation of liver function along avascular gradient of oxygen tension, which ishighest periportally.

The histochemical demonstration of copper,however, has limited usefulness. At the cellularlevel, X-ray electron microanalysis is one possiblealternative (Fuentealba et al., 1989a; Haywood et al.,1996).

Copper, which is a transition metal that can existin more than one oxidation state, is highly reactiveand participates in many enzyme-induced electrontransfer reactions. However, if present in excess itreadily catalyses the conversion of hydrogenperoxide to the hydroxyl radical, which causeslipid peroxidation of cell membranes, cleavage ofnucleic acids and chemical modification of pro-teins. To counteract this, antioxidant systems haveevolved, e.g., the glutathione system, specificintramitochondrial antoxidant systems, and cata-lase within the peroxizomes.


Induction of cytosolic IDH was indicative of anoxidant challenge ameliorated by upregulation ofglutathione synthetase and glutathioneS-transferase mu in response to moderate copper-dosing in Cambridge sheep. At higher copperoverload only IDH was detectable and oxidativestress-induced injury was apparent (Simpson et al.,2004). In North Ronaldsay sheep, IDH (controllingthe delivery of glutathione precursors) wasexpressed in the livers of both control andcopper-supplemented sheep, in contrast to theCambridge sheep, in which IDH was induced solelyin response to copper-supplementation. Thisimplies that the susceptibility to oxidative stressgreater in the North Ronaldsay breed.

Furthermore the disparities with regard to celldeath between the two breeds at high copperoverload suggest that oxidative stress is of sufficientmagnitude in North Ronaldsay sheep to inhibitcaspase-induced apoptosis; in addition, the preva-lence of hepatic peroxisomes indicates heightenedoxidative stress.

The severe mitochondrial distress culminatingin membrane rupture recorded in the Cu-loadedNorth Ronaldsay sheep liver (copper 400–900 mg/g) in this study reaffirms the high suscep-tibility of this breed. Such severe injury is unknownin other copper-associated diseases in man andanimals. The disruption of mitochondrial mem-brane integrity was supported by the increase inmitochondrial thioredoxin-dependent peroxidereductase in 2D gels derived from cytosolic liversamples of Cu-overloaded North Ronaldsay sheep.This reductase is a component of the mitochon-drial peroxide-destroying system, responsive tooxidant challenge and not expected to be foundoutside the mitochondria (Rabilloud et al., 2001).

A second distinct type of mitochondrial damageidentified was matrical condensation, which variedfrom flocculent deposits of electron-dense materialto complete mitochondrial matrical electron-den-sity. These pyknotic mitochondria were oftenbizarre in shape.

Mitochondrial oxidant injury has been reportedin Cu-overloaded rats (Sokol et al., 1993), Wilson’sdisease and Bedlington terrier copper toxicosis(Sokol et al., 1994). Accompanying morphologicaldamage varies considerably, and in both rats(Fuentealba et al., 1989a) and Bedlington terriers(Haywood et al., 1996), it is at best muted,consisting of mild swelling and increased matrixgranularity with a few matrical crystallineinclusions. Such damage resembles that recordedin the Cambridge sheep in this study. Mitochon-drial abnormalities have been recorded in the early

stages of Wilson’s disease (Sternlieb, 1992) andLong-Evans cinnamon (LEC) coloured rats, thegenetic homologue of Wilson’s disease (Sternlieb etal., 1995). Such changes bear a striking resem-blance to the matrical condensation found inNorth Ronaldsay hepatic mitochondria in thisstudy.

Ghadially (1977) found that mitochondrialinvolution or dissolution broadly took one of twoforms, namely, either swelling (balloon degener-ation) or condensation of the matrix with increasedelectron-density, termed mitochondrial pyknosis.Both forms were identified in Ronaldsay coppertoxicosis (RCT) in this study, whereas in Wilson’sdisease and LEC rats mitochondrial condensationwas the most prevalent finding. Failure to transportcytosolic glutathione into mitochondria, is theexplanation for the mitochondrial oxidative injuryoccurring in alcoholic liver disease (Fernandez--Checa et al., 1997) and may have been important inour study. Alternatively, unregulated intramito-chondrial copper may provoke the generation ofOH in a Haber-Weiss Fenton type reaction. Copper,an integral component of cytochrome oxidaselocated in the intramitochondrial space andimported by a protein COX17 (Glerum et al.,1996) is tightly bound to this site and an unlikelycandidate. The yeast mitochondrion, however,contains a dynamic pool of non-protein boundcopper, responsive to cellular changes in copperconcentration and localized within the mitochon-drial matrix as a soluble, anionic, low molecularweight complex (Cobine et al., 2004). If such a poolis a feature of mammalian liver mitochondria, itmight explain the severe oxidant damage recordedin the North Ronaldsay sheep livers.

It is puzzling that the acute mitochondrialdamage recorded in the early phases of copperaccumulation in North Ronaldsay sheep subsidedfollowing persistent copper loading. In Wilson’sdisease, however, a parallel finding was reported bySternlieb (1992), who stated “paradoxically thesemitochondrial abnormalities tend to disappear..as the disease progresses to cirrhosis”. Possibly,there is an eventual adaptation (or selection) ofsurviving mitochondria to the adverse conditions, asuggestion supported by the pronounced mito-chondrial hypertrophy and pleomorphismexhibited.

Haywood et al. (2001, 2004) reported thatcopper-induced fibrosis in North Ronaldsay sheepresults from HSC activation, simulating the peri-cellular fibrosis that characterizes infantile coppertoxicosis. Activated HSCs provide the source ofmodified extracellular matrix (ECM), leading to


collagen fibril formation following liver injury.HSCs, which normally store vitamin A (retinol),change from their quiescent state to an activatedform, in which a sequence of responses occurs,leading to proliferation and fibrogenesis(Friedman and Eng, 2000). Initiation is provokedby reactive oxygen intermediates, amplified bydepletion of antioxidants, causing rapid changesin gene expression (collagen 1 gene) that renderthe cells responsive to cytokines. These, notablyTGF-b1 secreted by Kupffer cells and others,represent the dominant signal for ECM productionand fibrogenesis. In this study acute mitochondrialoxidant damage, Kupffer cell activation andcoincident HSC activation (affirmed by upregula-tion of cathepsin D precursor [Kristensen et al.,2000]), conforms to this line of reasoning.

In conclusion, the present investigation con-firms the idiosyncratic pathological response ofNorth Ronaldsay sheep to excess dietary copper.Furthermore, it extends knowledge of the putativepathogenesis, in respect of oxidant injury, in whichall the evidence points to mitochondrial vulner-ability as a major contributory factor. This may be aconsequence of failure of antioxidant deliverysystems or, alternatively, of increased productionof reactive oxygen species by uncontrolled mito-chondrial copper influx.

Acknowledgements

This work was supported by a research grant fromthe Wellcome Trust (ref 063730). Grants from theBiotechnology and Biological Science ResearchCouncil supported the purchase ofinstrumentation.

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Received;March 17th; 2004

Accepted ; February 7th; 2005

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