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The effect of excess dietary copper on the liver and kidney of the male rat
J. (:OMP.
PATH.
lI)80.
\'oL.
THE EFFECT THE LIVER
‘2 17
90.
OF EXCESS AND KIDNEY
S.
DIETARY OF THE
COPPER MALE
ON RAT
HAYJVOOD
lki,artment oJ‘ l,iterimq Pathology, Universi!r I$ Liwrpxd, P.O. Box 147. Liuerpool L69 3B.Y 1NTRODCC:TIOS
Liver injury in the rat as a result of cumulative copper poisoning is frequentl) referred to in the literature but seldom described. Wolffe (1960), after injecting a solution of copper salts daily by the intrapcritoneal route for approximately 8 months, reported the existence of a chronic hepatitis with fibrosis. Wiederanders, Evans and Wasdahl (1968)) observed hepatitis without fibrosis after the parenteral administration of copper sulphate for a similar period. The kidney is generally disregarded as a site of pathological change whell copper is ingested over long periods since it is not considered important in the regu tation of copper under normal circumstances (Underwood, 197 1). Wolf%* 11960), however, observed degenerative changes in the proximal convoluted tubules of the kidneys in rats accompanying the hepatic changes. He also recorded similar changes in the kidneys of human patients suffering from Wilson’s disease, a hereditary disorder of copper metabolism (Wolffe, 1964l. There is also evidence that copper poisoning in sheep is associated with renal changes in the proximal convoluted tubules (Gopinath, Hall and Howell, 1974\. However, there appears to be no account of the development of either ththepatic. or renal lesions and their significance. The rat is an interesting species in that it is less susceptible to cumulativt copper poisoning than certain species such as the sheep and the pig I:Underwood, 1971). .L\ study of the development of copper poisoning in the rat, in addition to its intrinsic interest, might throw light on more fundamental mec.hanisms of toxity and tolerance. 4 preliminary investigation (Haywood, 1979) has shown that liver damage c.an be consistently produced in young male rats after 4 weeks on a diet contailling 2000 mg per kg of copper in decided to extend feeding trials over a into-rvals to follow the accumulation of relate. this to the development of lesions MATERIALS
AND
the form of copper sulphatr. It was longer period, sampling the group at copper in the liver and kidneys and in these organs. LIETHODS
>lalc weanling rats of uniform age and weight were randomly allocated to 9 separately caged groups, each consisting of 4 animals. Groups 1 to 6 were fed Spillers expanded powdered diet to which 2000 mg per kg copper had been added in the form of copper sulphate. Groups 7 to 9 received the unsupplemented diet 002 I
9975/8O/lT202
17 1 16 .sOt2.00/0
(‘. 1980
.\mtlen~ic
Press Inc.
(I,ondon)
Lirnitrtl
218
5. 1fr\~\VOOI~
and served as controls. ‘The food was fed ad lib fro111 hopper5 a11cl waler wa\ lr(~.l\ available. The diet was analyscd fi)r SC and Vit E*at the beginning and t,nd 01‘ t/l;, experiment and the content was found to be adequate. Rats on tht, t’oppt’l’ supplcmcnted diets were killed, from groups 1 to 6 at intervala of 1, 2. 3. 6. 0 at1t1 15 weeks, respectively. Two control animals were killed at the same tin1c.s. The animals were exsanguinated under ether anaesthesia and the blood wab I(‘rained. The livers and kidneys were removed and weighed. Slices from the left, right and median Inbcs were fixed in IO per ant li,rtnalitl. The liver was further divided into its right and left components and frozen at -70 ‘C:. The liver was so designated according to its vascular areas (Himsworth. 1947), whcrcby the left and omental lobes receive blood in the portal stream, mainlL from the gastrosplenic vein and the right and median lobes from the mesent&. vein. Subsequently, the copper content of the right and left components of rath liver was analyzed. Triplicate samples (0.5 to 1.0 gj were digested in “,%&tar” grade nitric, perchloric and sulphuric acids (3: 2: I ) and their copper content measured in an EEL 140 Atomic Absorption Spectrophotomcter. All glassware used in copper estimations was soaked in concentrated HClL for 24 h and rinsed several times in deionized water before use. Tissue was cut with a clean stainlcsh steel scalpel blade and transferred into polythenc containers with polythenc forceps which had been cleaned in acid. After routine processing, paraffin sections were stained with haematoxylin and eosin and rubeanic acid for the demonstration 01 copper and with Gomori’s reticulin stain counterstained with Van Gieson. The kidneys were frozen at -70 ‘C: after blocks had been taken for histology and subsequently analysed for their copper content by the methods described for the liver. RESULTS
Pathological Findings The Liver Grosschanges The livers were smaller than the controls but the ratio to body weight remained unaltered. The appearance was unchanged until week 6. At this time 3 of the 4 animals had livers that showed marked changes in the right and median lobes. The affected lobes contained peripheral areas of necrotic tissue which were variable in size, pale, swollen and sharply demarcated from adjacent tissue, sometimes by a zone of hyperaemia (Fig. 1). By week 9 pale areas were still visible on the right and median lobes but were not so clearly demarcated and by week 15 the livers were apparently normal, except for fine scarring which was seen on the ventral surface of some of these lobes.
Histological changes Week 1. All livers appeared normal. Copper was not present in sections stained with rubeanic acid. Week 2. All livers showed hypertrophy of parenchymal cells in the periportal zones. The change was not uniform and frequently groups of cells appeared * Se Assay-Ministry of Agriculture, Fisheries and Food, V.I. Centre, Crown Street. Liverpool. Se content of diet at beginning and end of experiment 0.342 mg per kg and 0.325 mg per kg respectively. Vit. E Assav-Roche Products Ltd.. Dunstable. L-tocoph&ol content at beginning and end of experiment 7 21 mg per kg and 18 mg prr respectively (approximately = 30 1. U Vit. E per kg).
kg
COPPER
ON
THE
LIVER
AND
KIDNEY
Fig. I. liver of rat after G weeks on copper supplemented affected liver contains pale. elevated. well dcmnrcated lobes ( r ) (approx. natural size).
IS
TIIE
KA1
diet compared with control ,<:I. ‘I’~Ic. areas of necrosis in right ancl nlryli:tv,
especially prominent with enlarged nuclei and homogeneous intensely stained cytoplasm. Occasionally such foci were associated with a cellular response. (topper was present in the outer zones of the lobules in rubeanic acid stained sections. IVeek 3. Inflammatory foci had now become clearly established but \vert~ still limited to the periportal zone (Fig. 2a). The lesions consisted of small aggregates of hypertrophied hyperchromatic parenchymal cells some of which showed signs of necrosis. These parenchymal cells had excited a cellular inflammatory response of polymorphonuclear neutrophil leucocytes and mononuclear cells (Figs Za, b). The changes were generally more common in the right and median lobes. Marked deposition of copper was present in rubeanic acid stained sections in which it appeared in particulate form in hepatocytes in the outer zones 01 the lobules; pericanalicular in distribution (Fig. 3). Week 6. Marked changes were present in the livers of all animals. Thcsc. however, varied in their severity and age both between individuals and within each liver. In all cases changes were much more severe in the right and median. lobes than the left lobe. Even within a particular lobe there was arkedm variation in response so that a section of the lobe frequently prrscnted a mosaic appearance. The broad bands of necrotic cells which were present in the outer zones linked up portal canals and frequently extended more drcply into the lohulr.
220
s. 11.4Y\v00D
Often whole lobules were necrotic (Fig. 4) and quite rxteusive portions of’li\,cb1 showed massive necrosis. The necrotic tissue stimulated a marked ~ellul;~r inAammatory reaction consisting of polymorpho-nuclear neutrophil Irucoc~-cc,s and mononuclear cells. In addition, there was bile duct hypcrplasia and some. attempted regeneration of still viable cells (Fig. 5). Severely affected regions showed a loss of rubeanic-positive material from ~IIC parenchymal cells and its appearance in Kupffer cells and wandering phagocytes (Fig. 6). This severe necrotizing lesion was the most prominent finding at this time and it was frequently visible to the naked eye. In some other areas necrotic tissue had become heavily invaded by macrophages. Marked regencrative activity on the part of surviving cells was apparent in such regions.
fig.
2. (a) Liver of rat after 3 weeks on copper supplemented zone. HE. >: 230. (b) Same liver as above. Inflammatory parenchymal cells. many of which arc disintegrating, HE. x375.
diet.
Inflammatory focus consisting and associated
focus in pcriportal of hypertropbietl c-ellular responsr.
COPPER
ON
THE
I.IVER
AiXD
,‘iq:. :;. l.lver of rat after 3 weeks on copper supplemented z,r:udes in hepatic cells. Rubeanic acid ;. 7’20.
big. 1. l.iver of‘ rat after 6 weeks on copper matory response. HE. x 234.
supplemented
KIDNEY
ht.
diet.
1X
THE
Pericanalicular
Necrosis
$7’) -- 1
RAT
of whole
deposition
lobule
o!’ coppr~
a11tl ;tlfl.,~,l-
Il’eek 9. Healing was very active at this time. Regeneration of parenchymal tissue was well established, individual cells were normal in size with plentiful giycogen. Bands of connective tissue surrounded and dissected lobules causing a considerable amount of architectural distortion in some areas (Fig. 7). These bands were in part composed of condensations of reticulin but also contained thicker strands of collagen which stained red with Van Gieson. Bile duct hyperplasia accompanied the fibroplasia. Necrosis was limited to a cuff of cells in the periportal zone and the cellular response had subsided but was still present.
S)‘,O h-w
5.
I1.\Y\\‘OC~U
Copprr had largely disappeared li~ni rubcauic. acid atainvd .scx.tiollr. I+‘eeX- 1.5. All livers showed advanced hraling thougIl tlicw \vas still (~~id(~tt~~c~ or architectural distortion in the right and median lol~cs. Tlic c1issc‘c.ti itx conncctivc tissue bands appcarcd to lla\.c undcrgonc a degree of‘condcnsatio~~ and maturation, resulting in a fine difhsc fibrosis ill some ;LL‘W?S1Pig. 8 . Bile duct h).pcrplasia was still present, though not x’cry marked, and uc-crcxic remnants lverc seen in portal areas consisting of cosinophilic Iiyalinc lxxlic3. occasionally containing nuclear material Fig. !I;.
COPPER
ON
THE
LIVER
,431)
KII)NE:l’
IN
THE
RAT
9’)‘; --.
~ldne_~l changes ese were
to a greenish
discoloration
in some animals
of \vc y=kri.
‘o,qical changes
Ili.str Ii strat
limited
!eX-I to 2. All the kidneys appeared ~1 in sections stained with rubcanic
normal. acid.
Copper
could not he de mon-
Fig. 9. Liver of rat and necrosis.
Fig.
after HE.
1.5 weeks x 282.
on copper
supplcmcnted
diet.
Residual
portal
rractiw
10. Kidney of rat after 3 weeks on copper supplemented diet. Proximal tubules containing cytoplasmic droplets and extrusion of droplet-containing cells and their desquamation HE. x614.
tilxwis
intrai ’ I.
Week 3 (Fig. 10). Small eosinophilic droplets were present in the cytoplasm of the proximal convoluted tubules. Extrusion of droplet-containing cells, their detachment and desquamation into the lumen of the tubules was common. There was some variation in nuclear size in the epithelial cells of the proximal convoluted tubules and the presence of mitotic figures indicated proliferative activity. Copper could not be demonstrated in rubeanic acid stained sections.
COPPER
ON THE
LI\‘ER
AXD
KIDNEY
IS TI1E
RAT
2%
Week 6. Changes within the proximal convoluted tubules had now become marked although they varied in severity between individuals. The cytoplasmic droplets were now larger and more numerous and had generally assumed the appearance of greenish globules (Fig. 1 laj. Rubeanic acid stained preparations revealed the presence of copper ill botlr particulate and droplet form, the latter resembling the gIohules apparrnt ~II the HE-stained sections (Fig. 11 b). Some kidneys showed extensive desquamation of the epithelial cells of tlrc. proximal convoluted tubules, the lumens of which were frequently oblitcratcd I)v necrotic debris. Regenerative activity was a prominent feature amongst surviving epithelial cells in these areas; mitotic figures were common and tlrc
Fig. 1 I. (a) Kidney of rat after 6 weeks numerous and large. HE. x 614. f + \. Ruhennic acid x 720.
on copper supplemented diet. Intracytoplasmic droplets (b) Same as above. Copper-positive granules and droplrts
Fig.
COPPER
? IT 0, P f 0 G+
OS THE
LIVER
AND
KIDNEY
IX TIIE
RAT
““7 --
I 3000 2000 11 Time m weeks
nuclei varied considerably in size, being frequently enlarged and biiarr(. I Figs 12a,h,c). The remainder of the nephron, with the exception of :I \w)’ kw isolated necrotic cells, appeared to be unaffected. llkek 9. Regeneration was still active in some of the proximal convoluted tubules although, for the most part, they appeared to bc reconstituted ((Fig. 1:i 1.
+&@imental ;
2I
3 4 .5 G
3 6 9 15
Cbntro1 79
l-15
14086+_ 792.9 &
315.2 403.1
I6634 877.3
‘398.1 rlr 468.3 3359.7 +- 1145.3 2537.3+ 541.2 2143..5+_ 415.7
7+
G9 439.31 4
11.54,2+ 708..5 &
L’720.2? 4904 3454. I 7 1073.5 2ti23~4 I G2G4 YO264 +_ 1466
17.7rf:3.3
202 1.9 3’,fj,5,‘, 24.5 1 ,I CViO4i
176*.5.1
unsuitable is the
for number
analysis ofrats
+ i43.0 + ,‘,70.‘1 F 527.2 g 7 I!l,.T
17.8+2.4
S.D. -~: Standard Deviation. * The number of animals sampled at any time I‘or the cspcrimental groups groups 2. The control values are given as the- o\wxll rnc’an of 12 samples with time.
s.n. := Standard deviation. * Group 5 kidneys were spoilage. t The number in brackets
“Gl.2 39 I 4
due in the
was 4 and fix the cwntrol sinw thcay did not charlgr
to accidental group.
Eosinophilic droplets were very noticeable within the tubules but had become smaller and most had lost their greenish appearance. Copper could still bc demonstrated in both particulate and droplet form in rubeanic acid stained sections. Week 1.5. Reconstitution of the proximal convoluted tubules was apparently complete although some nuclear heterogeneity and the occasional mitotic figure indicated a low level of regenerative activity. Small eosinophilic droplets were still numerous within the cytoplasm of the proximal tubular cells and extrusion and detachment of droplet filled cells was a prominent feature. Little particulate copper could be demonstrated in rubeanic acid stained preparations,
COPPER
ON
THE
LIVER
AND
KIDNEY
IN
THE
RAT
‘)‘,() --.
Copper Analysis The her (Table 1) Total liver copper content rose rapidly during the early period to reach a maximum at week 6, after which it fell and had begun to level out at week 15. The right and median lobes contained more copper than the left lobes iu most instances but the difference was not significant at any sampling timt. P .. 0.05j. The Kip’ntys (Table 2) The copper concentration of the experimental group was significantI) rose to greater than the control group at 1 week i P -; O-05) and thereafter ;lttain a maximum concentration at 6 weeks, falling somewhat in succcrding weeks. In Fig. 14 the liver and kidney copper concentrations are compared. DISCUSSION
The pattern of events in the liver of cumulative copper toxicity in the. rat c.onstructed from the histological changes and analysis of liver copper content may he divided into 3 phases. In the first phase, copper accumulated in the liver with little initial effect but, as the copper content rose, there were incrcasccl signs of cellular disruption, until the copper c.ontcnt reached a maximum and the second phase was abruptly precipitated. This critical phase, or crisis, rvah associated with a severe necrosis. It was followed by a decline in liver copper until a condition approaching a steady state was achieved, the liver mranwhiI(~ showed evidence of repair and regeneration. The animal by now had beconic. tolrrant. The pathological changes and sequence of events can be compared wit h those described in chronic copper poisoning in sheep by Ishmael, Gopinat t L and Howell (1971). The morphological changes observed in the liver in the cumulative phase were similar, in both cases, except for the distribution of tllc. lesions which, in the sheep, were predominantly centrilobular whereas in rhrh ral they were periportal and coincided with the concentration gradirnt 01 copper. When liver copper values reached a critical level, around 3000 ppm in both species, necrosis became marked and copper was released from dving c.ctls and taken up by phagocytes. Hacmolysis, which was a consistent ft.atutx. in sheep, was not detected in rats and the period following the crisis occurrcbtl in rats without any intervening deaths. or any obvious signs of clinical discasc. However, in the sheep that survived the hacmolytic crisis pathological changes associated with healing were seen and as such Lverc essentially similar to those. observed in rats in this study and by Wolffe t 1960). Xs to whether these changes represrntrd a chronic hepatitis or were the forerunner to complete healing and resolution can not be answered and must be the aim of future investigation. The difference in response of the left lobes from the right and median lotus was an interesting feature and worth consideration. Local variations of copper distribution within the liver are known to exist (Brck, 1956) ; however diffcrcntial copper analysis in this study showed no significant difference betbvecn riglit and left parts of the liver. A more detailed analysis of copper content w+thin strictly localized regions of the liver is being undertaken. Massive liver necrosis
230
s.
IIAY\VO01~
can be induced in rats by feeding diets low in Se and;or \‘it. F: t)ut I/U. n~:i,icr~ distribution of lesions occurs in the left lobes, in contrast to the present obs(.r\.;ttions. h,‘Ioreover SC and Vit. E concentrations in the diet of our rats \v(‘t’(. adequate. It is possible that reduced blood supply may have bran at I~.;IsI partly responsible; local variations in vascular supply have bren rc~c~ordetl (Himsworth, 1947) and have been attributed to the pressure of surrountlillg viscera. The sequence of events in the kidney followed a similar patter11 to those which occurred in the liver. Early on, copper accumulated with little efyect, although after week 3 eosinophilic droplets appeared in the cytoplasm of the proximal convoluted tubules. By week 6, however, copper concentrations had reached their peak; this was associated with severe desquamation and necrosis of cells of the proximal convoluted tubules. This was followed by a phase of tubular regeneration leading to recovery, at the end of which there was a slight decline* in total kidney copper content. The nature of the eosinophilic droplets is uncertain. When they first appearctl they could not be stained for copper but later some of the larger droplets ot globules were obviously copper-positive, although the majority still did not take up the copper stain. They were structurally dissimilar from the particulate. or granular copper which appeared in the proximal convoluted tubules around this time. Observations from the literature do little to clarify the issue. Wolfit (1960), commented upon the small refractile yellow droplets which appeared in the proximal convoluted tubules of copper-poisoned rats and were copperpositive with rubeanic acid. Gopinath ek al. (1974) described eosinophilic intracytoplasmic granules in the proximal convoluted tubules of sheep kidneys in chronic copper poisoning, some of which stained with rubeanic acid. It is probable that the eosinophilic droplets contain excretory or secretory products of some kind; the pattern of their accumulation and release is similar to that seen in a holocrine gland. It is possible that they include coppercontaining substances (of a type not stained by rubeanic acid), probabl) proteinaceous, possibly caeruloplasmin absorbed from the blood and excreted into the tubular lumen and thence into the urine. It is also likely that the granular copper which can be demonstrated represents lysosomc-bound copper, as is the case in the liver. Rubeanic acid appears to stain copper in this form preferentially. The nature of these structures is currently under investigation. Desquamation of the proximal tubular epithelium was a marked feature in the crisis phase and has been reported previously by Wolffe (1960). It is interesting that the necrotic debris did not stain for copper despite the fact that viable cells contained stainable copper in appreciable quantities. The mechanism of toxicity in the liver and kidney is not yet understood although Lindquist (1968) suggested that, at least in the liver, it was the result of lysosomal damage. McNary ( 1963)) with ultrastructural techniques, showed that the copper in hepatocytes was located in membrane-bound bodies bearing a strong resemblance to lysosomes. Goldfischer and Sterntieb (1968), demonstrated that peri-canalicular particulate copper in copper loaded rats was associated with high acid phosphatasc activity, from which they concluded
COPPER
ON
THE
LIVER
AND
KIDNES
IN
‘TIIE
RAT
23
I
that it was localized within lysosomes. This, together with the evidence from this study of the accumulation and subsequent disappearance of particulate, copper from damaged cells, supports Lindquist’s hypothesis although furthe investigation is necessary before it can be accepted as conclusive. One of the most interesting findings from this study has bern the rcco\cr! of the liver and kidney and the development of tolerance by the rat to coppc>r. The phenomenon of tolerance in metal intoxication is obscure. Luckey a11tl Venugopal (1975), defined tolerance as “the ability to endure the continur(l administration of a toxicant”. This ability must invol\.c an increased facility to excrete a potentially toxic cation or to detoxify it. Toxicity is nssociatcd \\illl the presence of particulate (lysosomal) copper in the hcpatocytes and prol)al)l!the c,ells of the proximal convoluted tubules. Tolcrancc is associated \\.ith t III. disappearance of this particulate copper. It is possible that it has been cscrc*tc.d. although the part played by the lysosomes in I)ile storage and excretiotl i\ unknown (Popper, Schaffner, Rubin, Barka and Parronctto, 1963). .Utt,rnatively, copper may be stored in a diflerent and non-toxic fbrtn. ‘1%~ SIII)cellular localization of copper in relation to toxicitv and tolerance is c*strcmt.l\ important and is currently the subject ofinvcstigat;oll. Finally, the role of the kidney in copper regulation appears to be importalll. :Although normally the liver is principally concerned with the storage alltl c.xcretion of copper, in copper loading the kidney probably plays a significant role in maintaining homeostasis and contributing to the overall devclopmcnt of‘ tolerance by the animal. SUMMARY
Ciumulative copper poisoning was induced in rats by feeding a diet COIItaining 2000 mg per kg copper as copper sulphate for 15 weeks. Animals wer(’ killed at intervals and their livers and kidneys examined histologically and analysed for their copper content. Subsequent events occurred in three phases, cumulative, crisis and recovery. Thai early period was associated with the gradual accumulation of copper ill the liver and kidney and some signs of cellular disturbance; it culminated in ;I crisis characterized by maximal liver and kidney copper concentrations and severe cellular disruption. The final phase was one of regeneration and healin,y in which the animal demonstrated its tolerance to copper. It was concluded that long term copper dosing elicits a similar response in both the kidney and the liver of rats and that adaptation to copper takes place, in I)oth these organs, probably as a result of increased excretion and dif?‘ercnt modes of storage, which, in turn, contributes to the development of tolerant-ck to (sopper. ACKNOWLEDGMENTS
1 wish to thank Professor D. L. Hughes and Dr D. ,411an for their encouragemcnr and advicca, Mrs M. W. Harling and Mr R. Comerford for technical assistance and Mrs P. Jenkins for the photography, also, Mr 1). F. Gibbons of M.A.F.F. Veterinary Investigation Centre, Liverpool for Se assay. and Mr M. E. Putnam of Rochr Products Ltd., for Vit. E assay.
KEFEKENCES
Beck, A. B. (1956). The copper content of the liver and blood of some vcrtebratc.\. Australian Journal of
lIlqy
14th, 19791
PATH.
lI)80.
\'oL.
THE EFFECT THE LIVER
‘2 17
90.
OF EXCESS AND KIDNEY
S.
DIETARY OF THE
COPPER MALE
ON RAT
HAYJVOOD
lki,artment oJ‘ l,iterimq Pathology, Universi!r I$ Liwrpxd, P.O. Box 147. Liuerpool L69 3B.Y 1NTRODCC:TIOS
Liver injury in the rat as a result of cumulative copper poisoning is frequentl) referred to in the literature but seldom described. Wolffe (1960), after injecting a solution of copper salts daily by the intrapcritoneal route for approximately 8 months, reported the existence of a chronic hepatitis with fibrosis. Wiederanders, Evans and Wasdahl (1968)) observed hepatitis without fibrosis after the parenteral administration of copper sulphate for a similar period. The kidney is generally disregarded as a site of pathological change whell copper is ingested over long periods since it is not considered important in the regu tation of copper under normal circumstances (Underwood, 197 1). Wolf%* 11960), however, observed degenerative changes in the proximal convoluted tubules of the kidneys in rats accompanying the hepatic changes. He also recorded similar changes in the kidneys of human patients suffering from Wilson’s disease, a hereditary disorder of copper metabolism (Wolffe, 1964l. There is also evidence that copper poisoning in sheep is associated with renal changes in the proximal convoluted tubules (Gopinath, Hall and Howell, 1974\. However, there appears to be no account of the development of either ththepatic. or renal lesions and their significance. The rat is an interesting species in that it is less susceptible to cumulativt copper poisoning than certain species such as the sheep and the pig I:Underwood, 1971). .L\ study of the development of copper poisoning in the rat, in addition to its intrinsic interest, might throw light on more fundamental mec.hanisms of toxity and tolerance. 4 preliminary investigation (Haywood, 1979) has shown that liver damage c.an be consistently produced in young male rats after 4 weeks on a diet contailling 2000 mg per kg of copper in decided to extend feeding trials over a into-rvals to follow the accumulation of relate. this to the development of lesions MATERIALS
AND
the form of copper sulphatr. It was longer period, sampling the group at copper in the liver and kidneys and in these organs. LIETHODS
>lalc weanling rats of uniform age and weight were randomly allocated to 9 separately caged groups, each consisting of 4 animals. Groups 1 to 6 were fed Spillers expanded powdered diet to which 2000 mg per kg copper had been added in the form of copper sulphate. Groups 7 to 9 received the unsupplemented diet 002 I
9975/8O/lT202
17 1 16 .sOt2.00/0
(‘. 1980
.\mtlen~ic
Press Inc.
(I,ondon)
Lirnitrtl
218
5. 1fr\~\VOOI~
and served as controls. ‘The food was fed ad lib fro111 hopper5 a11cl waler wa\ lr(~.l\ available. The diet was analyscd fi)r SC and Vit E*at the beginning and t,nd 01‘ t/l;, experiment and the content was found to be adequate. Rats on tht, t’oppt’l’ supplcmcnted diets were killed, from groups 1 to 6 at intervala of 1, 2. 3. 6. 0 at1t1 15 weeks, respectively. Two control animals were killed at the same tin1c.s. The animals were exsanguinated under ether anaesthesia and the blood wab I(‘rained. The livers and kidneys were removed and weighed. Slices from the left, right and median Inbcs were fixed in IO per ant li,rtnalitl. The liver was further divided into its right and left components and frozen at -70 ‘C:. The liver was so designated according to its vascular areas (Himsworth. 1947), whcrcby the left and omental lobes receive blood in the portal stream, mainlL from the gastrosplenic vein and the right and median lobes from the mesent&. vein. Subsequently, the copper content of the right and left components of rath liver was analyzed. Triplicate samples (0.5 to 1.0 gj were digested in “,%&tar” grade nitric, perchloric and sulphuric acids (3: 2: I ) and their copper content measured in an EEL 140 Atomic Absorption Spectrophotomcter. All glassware used in copper estimations was soaked in concentrated HClL for 24 h and rinsed several times in deionized water before use. Tissue was cut with a clean stainlcsh steel scalpel blade and transferred into polythenc containers with polythenc forceps which had been cleaned in acid. After routine processing, paraffin sections were stained with haematoxylin and eosin and rubeanic acid for the demonstration 01 copper and with Gomori’s reticulin stain counterstained with Van Gieson. The kidneys were frozen at -70 ‘C: after blocks had been taken for histology and subsequently analysed for their copper content by the methods described for the liver. RESULTS
Pathological Findings The Liver Grosschanges The livers were smaller than the controls but the ratio to body weight remained unaltered. The appearance was unchanged until week 6. At this time 3 of the 4 animals had livers that showed marked changes in the right and median lobes. The affected lobes contained peripheral areas of necrotic tissue which were variable in size, pale, swollen and sharply demarcated from adjacent tissue, sometimes by a zone of hyperaemia (Fig. 1). By week 9 pale areas were still visible on the right and median lobes but were not so clearly demarcated and by week 15 the livers were apparently normal, except for fine scarring which was seen on the ventral surface of some of these lobes.
Histological changes Week 1. All livers appeared normal. Copper was not present in sections stained with rubeanic acid. Week 2. All livers showed hypertrophy of parenchymal cells in the periportal zones. The change was not uniform and frequently groups of cells appeared * Se Assay-Ministry of Agriculture, Fisheries and Food, V.I. Centre, Crown Street. Liverpool. Se content of diet at beginning and end of experiment 0.342 mg per kg and 0.325 mg per kg respectively. Vit. E Assav-Roche Products Ltd.. Dunstable. L-tocoph&ol content at beginning and end of experiment 7 21 mg per kg and 18 mg prr respectively (approximately = 30 1. U Vit. E per kg).
kg
COPPER
ON
THE
LIVER
AND
KIDNEY
Fig. I. liver of rat after G weeks on copper supplemented affected liver contains pale. elevated. well dcmnrcated lobes ( r ) (approx. natural size).
IS
TIIE
KA1
diet compared with control ,<:I. ‘I’~Ic. areas of necrosis in right ancl nlryli:tv,
especially prominent with enlarged nuclei and homogeneous intensely stained cytoplasm. Occasionally such foci were associated with a cellular response. (topper was present in the outer zones of the lobules in rubeanic acid stained sections. IVeek 3. Inflammatory foci had now become clearly established but \vert~ still limited to the periportal zone (Fig. 2a). The lesions consisted of small aggregates of hypertrophied hyperchromatic parenchymal cells some of which showed signs of necrosis. These parenchymal cells had excited a cellular inflammatory response of polymorphonuclear neutrophil leucocytes and mononuclear cells (Figs Za, b). The changes were generally more common in the right and median lobes. Marked deposition of copper was present in rubeanic acid stained sections in which it appeared in particulate form in hepatocytes in the outer zones 01 the lobules; pericanalicular in distribution (Fig. 3). Week 6. Marked changes were present in the livers of all animals. Thcsc. however, varied in their severity and age both between individuals and within each liver. In all cases changes were much more severe in the right and median. lobes than the left lobe. Even within a particular lobe there was arkedm variation in response so that a section of the lobe frequently prrscnted a mosaic appearance. The broad bands of necrotic cells which were present in the outer zones linked up portal canals and frequently extended more drcply into the lohulr.
220
s. 11.4Y\v00D
Often whole lobules were necrotic (Fig. 4) and quite rxteusive portions of’li\,cb1 showed massive necrosis. The necrotic tissue stimulated a marked ~ellul;~r inAammatory reaction consisting of polymorpho-nuclear neutrophil Irucoc~-cc,s and mononuclear cells. In addition, there was bile duct hypcrplasia and some. attempted regeneration of still viable cells (Fig. 5). Severely affected regions showed a loss of rubeanic-positive material from ~IIC parenchymal cells and its appearance in Kupffer cells and wandering phagocytes (Fig. 6). This severe necrotizing lesion was the most prominent finding at this time and it was frequently visible to the naked eye. In some other areas necrotic tissue had become heavily invaded by macrophages. Marked regencrative activity on the part of surviving cells was apparent in such regions.
fig.
2. (a) Liver of rat after 3 weeks on copper supplemented zone. HE. >: 230. (b) Same liver as above. Inflammatory parenchymal cells. many of which arc disintegrating, HE. x375.
diet.
Inflammatory focus consisting and associated
focus in pcriportal of hypertropbietl c-ellular responsr.
COPPER
ON
THE
I.IVER
AiXD
,‘iq:. :;. l.lver of rat after 3 weeks on copper supplemented z,r:udes in hepatic cells. Rubeanic acid ;. 7’20.
big. 1. l.iver of‘ rat after 6 weeks on copper matory response. HE. x 234.
supplemented
KIDNEY
ht.
diet.
1X
THE
Pericanalicular
Necrosis
$7’) -- 1
RAT
of whole
deposition
lobule
o!’ coppr~
a11tl ;tlfl.,~,l-
Il’eek 9. Healing was very active at this time. Regeneration of parenchymal tissue was well established, individual cells were normal in size with plentiful giycogen. Bands of connective tissue surrounded and dissected lobules causing a considerable amount of architectural distortion in some areas (Fig. 7). These bands were in part composed of condensations of reticulin but also contained thicker strands of collagen which stained red with Van Gieson. Bile duct hyperplasia accompanied the fibroplasia. Necrosis was limited to a cuff of cells in the periportal zone and the cellular response had subsided but was still present.
S)‘,O h-w
5.
I1.\Y\\‘OC~U
Copprr had largely disappeared li~ni rubcauic. acid atainvd .scx.tiollr. I+‘eeX- 1.5. All livers showed advanced hraling thougIl tlicw \vas still (~~id(~tt~~c~ or architectural distortion in the right and median lol~cs. Tlic c1issc‘c.ti itx conncctivc tissue bands appcarcd to lla\.c undcrgonc a degree of‘condcnsatio~~ and maturation, resulting in a fine difhsc fibrosis ill some ;LL‘W?S1Pig. 8 . Bile duct h).pcrplasia was still present, though not x’cry marked, and uc-crcxic remnants lverc seen in portal areas consisting of cosinophilic Iiyalinc lxxlic3. occasionally containing nuclear material Fig. !I;.
COPPER
ON
THE
LIVER
,431)
KII)NE:l’
IN
THE
RAT
9’)‘; --.
~ldne_~l changes ese were
to a greenish
discoloration
in some animals
of \vc y=kri.
‘o,qical changes
Ili.str Ii strat
limited
!eX-I to 2. All the kidneys appeared ~1 in sections stained with rubcanic
normal. acid.
Copper
could not he de mon-
Fig. 9. Liver of rat and necrosis.
Fig.
after HE.
1.5 weeks x 282.
on copper
supplcmcnted
diet.
Residual
portal
rractiw
10. Kidney of rat after 3 weeks on copper supplemented diet. Proximal tubules containing cytoplasmic droplets and extrusion of droplet-containing cells and their desquamation HE. x614.
tilxwis
intrai ’ I.
Week 3 (Fig. 10). Small eosinophilic droplets were present in the cytoplasm of the proximal convoluted tubules. Extrusion of droplet-containing cells, their detachment and desquamation into the lumen of the tubules was common. There was some variation in nuclear size in the epithelial cells of the proximal convoluted tubules and the presence of mitotic figures indicated proliferative activity. Copper could not be demonstrated in rubeanic acid stained sections.
COPPER
ON THE
LI\‘ER
AXD
KIDNEY
IS TI1E
RAT
2%
Week 6. Changes within the proximal convoluted tubules had now become marked although they varied in severity between individuals. The cytoplasmic droplets were now larger and more numerous and had generally assumed the appearance of greenish globules (Fig. 1 laj. Rubeanic acid stained preparations revealed the presence of copper ill botlr particulate and droplet form, the latter resembling the gIohules apparrnt ~II the HE-stained sections (Fig. 11 b). Some kidneys showed extensive desquamation of the epithelial cells of tlrc. proximal convoluted tubules, the lumens of which were frequently oblitcratcd I)v necrotic debris. Regenerative activity was a prominent feature amongst surviving epithelial cells in these areas; mitotic figures were common and tlrc
Fig. 1 I. (a) Kidney of rat after 6 weeks numerous and large. HE. x 614. f + \. Ruhennic acid x 720.
on copper supplemented diet. Intracytoplasmic droplets (b) Same as above. Copper-positive granules and droplrts
Fig.
COPPER
? IT 0, P f 0 G+
OS THE
LIVER
AND
KIDNEY
IX TIIE
RAT
““7 --
I 3000 2000 11 Time m weeks
nuclei varied considerably in size, being frequently enlarged and biiarr(. I Figs 12a,h,c). The remainder of the nephron, with the exception of :I \w)’ kw isolated necrotic cells, appeared to be unaffected. llkek 9. Regeneration was still active in some of the proximal convoluted tubules although, for the most part, they appeared to bc reconstituted ((Fig. 1:i 1.
+&@imental ;
2I
3 4 .5 G
3 6 9 15
Cbntro1 79
l-15
14086+_ 792.9 &
315.2 403.1
I6634 877.3
‘398.1 rlr 468.3 3359.7 +- 1145.3 2537.3+ 541.2 2143..5+_ 415.7
7+
G9 439.31 4
11.54,2+ 708..5 &
L’720.2? 4904 3454. I 7 1073.5 2ti23~4 I G2G4 YO264 +_ 1466
17.7rf:3.3
202 1.9 3’,fj,5,‘, 24.5 1 ,I CViO4i
176*.5.1
unsuitable is the
for number
analysis ofrats
+ i43.0 + ,‘,70.‘1 F 527.2 g 7 I!l,.T
17.8+2.4
S.D. -~: Standard Deviation. * The number of animals sampled at any time I‘or the cspcrimental groups groups 2. The control values are given as the- o\wxll rnc’an of 12 samples with time.
s.n. := Standard deviation. * Group 5 kidneys were spoilage. t The number in brackets
“Gl.2 39 I 4
due in the
was 4 and fix the cwntrol sinw thcay did not charlgr
to accidental group.
Eosinophilic droplets were very noticeable within the tubules but had become smaller and most had lost their greenish appearance. Copper could still bc demonstrated in both particulate and droplet form in rubeanic acid stained sections. Week 1.5. Reconstitution of the proximal convoluted tubules was apparently complete although some nuclear heterogeneity and the occasional mitotic figure indicated a low level of regenerative activity. Small eosinophilic droplets were still numerous within the cytoplasm of the proximal tubular cells and extrusion and detachment of droplet filled cells was a prominent feature. Little particulate copper could be demonstrated in rubeanic acid stained preparations,
COPPER
ON
THE
LIVER
AND
KIDNEY
IN
THE
RAT
‘)‘,() --.
Copper Analysis The her (Table 1) Total liver copper content rose rapidly during the early period to reach a maximum at week 6, after which it fell and had begun to level out at week 15. The right and median lobes contained more copper than the left lobes iu most instances but the difference was not significant at any sampling timt. P .. 0.05j. The Kip’ntys (Table 2) The copper concentration of the experimental group was significantI) rose to greater than the control group at 1 week i P -; O-05) and thereafter ;lttain a maximum concentration at 6 weeks, falling somewhat in succcrding weeks. In Fig. 14 the liver and kidney copper concentrations are compared. DISCUSSION
The pattern of events in the liver of cumulative copper toxicity in the. rat c.onstructed from the histological changes and analysis of liver copper content may he divided into 3 phases. In the first phase, copper accumulated in the liver with little initial effect but, as the copper content rose, there were incrcasccl signs of cellular disruption, until the copper c.ontcnt reached a maximum and the second phase was abruptly precipitated. This critical phase, or crisis, rvah associated with a severe necrosis. It was followed by a decline in liver copper until a condition approaching a steady state was achieved, the liver mranwhiI(~ showed evidence of repair and regeneration. The animal by now had beconic. tolrrant. The pathological changes and sequence of events can be compared wit h those described in chronic copper poisoning in sheep by Ishmael, Gopinat t L and Howell (1971). The morphological changes observed in the liver in the cumulative phase were similar, in both cases, except for the distribution of tllc. lesions which, in the sheep, were predominantly centrilobular whereas in rhrh ral they were periportal and coincided with the concentration gradirnt 01 copper. When liver copper values reached a critical level, around 3000 ppm in both species, necrosis became marked and copper was released from dving c.ctls and taken up by phagocytes. Hacmolysis, which was a consistent ft.atutx. in sheep, was not detected in rats and the period following the crisis occurrcbtl in rats without any intervening deaths. or any obvious signs of clinical discasc. However, in the sheep that survived the hacmolytic crisis pathological changes associated with healing were seen and as such Lverc essentially similar to those. observed in rats in this study and by Wolffe t 1960). Xs to whether these changes represrntrd a chronic hepatitis or were the forerunner to complete healing and resolution can not be answered and must be the aim of future investigation. The difference in response of the left lobes from the right and median lotus was an interesting feature and worth consideration. Local variations of copper distribution within the liver are known to exist (Brck, 1956) ; however diffcrcntial copper analysis in this study showed no significant difference betbvecn riglit and left parts of the liver. A more detailed analysis of copper content w+thin strictly localized regions of the liver is being undertaken. Massive liver necrosis
230
s.
IIAY\VO01~
can be induced in rats by feeding diets low in Se and;or \‘it. F: t)ut I/U. n~:i,icr~ distribution of lesions occurs in the left lobes, in contrast to the present obs(.r\.;ttions. h,‘Ioreover SC and Vit. E concentrations in the diet of our rats \v(‘t’(. adequate. It is possible that reduced blood supply may have bran at I~.;IsI partly responsible; local variations in vascular supply have bren rc~c~ordetl (Himsworth, 1947) and have been attributed to the pressure of surrountlillg viscera. The sequence of events in the kidney followed a similar patter11 to those which occurred in the liver. Early on, copper accumulated with little efyect, although after week 3 eosinophilic droplets appeared in the cytoplasm of the proximal convoluted tubules. By week 6, however, copper concentrations had reached their peak; this was associated with severe desquamation and necrosis of cells of the proximal convoluted tubules. This was followed by a phase of tubular regeneration leading to recovery, at the end of which there was a slight decline* in total kidney copper content. The nature of the eosinophilic droplets is uncertain. When they first appearctl they could not be stained for copper but later some of the larger droplets ot globules were obviously copper-positive, although the majority still did not take up the copper stain. They were structurally dissimilar from the particulate. or granular copper which appeared in the proximal convoluted tubules around this time. Observations from the literature do little to clarify the issue. Wolfit (1960), commented upon the small refractile yellow droplets which appeared in the proximal convoluted tubules of copper-poisoned rats and were copperpositive with rubeanic acid. Gopinath ek al. (1974) described eosinophilic intracytoplasmic granules in the proximal convoluted tubules of sheep kidneys in chronic copper poisoning, some of which stained with rubeanic acid. It is probable that the eosinophilic droplets contain excretory or secretory products of some kind; the pattern of their accumulation and release is similar to that seen in a holocrine gland. It is possible that they include coppercontaining substances (of a type not stained by rubeanic acid), probabl) proteinaceous, possibly caeruloplasmin absorbed from the blood and excreted into the tubular lumen and thence into the urine. It is also likely that the granular copper which can be demonstrated represents lysosomc-bound copper, as is the case in the liver. Rubeanic acid appears to stain copper in this form preferentially. The nature of these structures is currently under investigation. Desquamation of the proximal tubular epithelium was a marked feature in the crisis phase and has been reported previously by Wolffe (1960). It is interesting that the necrotic debris did not stain for copper despite the fact that viable cells contained stainable copper in appreciable quantities. The mechanism of toxicity in the liver and kidney is not yet understood although Lindquist (1968) suggested that, at least in the liver, it was the result of lysosomal damage. McNary ( 1963)) with ultrastructural techniques, showed that the copper in hepatocytes was located in membrane-bound bodies bearing a strong resemblance to lysosomes. Goldfischer and Sterntieb (1968), demonstrated that peri-canalicular particulate copper in copper loaded rats was associated with high acid phosphatasc activity, from which they concluded
COPPER
ON
THE
LIVER
AND
KIDNES
IN
‘TIIE
RAT
23
I
that it was localized within lysosomes. This, together with the evidence from this study of the accumulation and subsequent disappearance of particulate, copper from damaged cells, supports Lindquist’s hypothesis although furthe investigation is necessary before it can be accepted as conclusive. One of the most interesting findings from this study has bern the rcco\cr! of the liver and kidney and the development of tolerance by the rat to coppc>r. The phenomenon of tolerance in metal intoxication is obscure. Luckey a11tl Venugopal (1975), defined tolerance as “the ability to endure the continur(l administration of a toxicant”. This ability must invol\.c an increased facility to excrete a potentially toxic cation or to detoxify it. Toxicity is nssociatcd \\illl the presence of particulate (lysosomal) copper in the hcpatocytes and prol)al)l!the c,ells of the proximal convoluted tubules. Tolcrancc is associated \\.ith t III. disappearance of this particulate copper. It is possible that it has been cscrc*tc.d. although the part played by the lysosomes in I)ile storage and excretiotl i\ unknown (Popper, Schaffner, Rubin, Barka and Parronctto, 1963). .Utt,rnatively, copper may be stored in a diflerent and non-toxic fbrtn. ‘1%~ SIII)cellular localization of copper in relation to toxicitv and tolerance is c*strcmt.l\ important and is currently the subject ofinvcstigat;oll. Finally, the role of the kidney in copper regulation appears to be importalll. :Although normally the liver is principally concerned with the storage alltl c.xcretion of copper, in copper loading the kidney probably plays a significant role in maintaining homeostasis and contributing to the overall devclopmcnt of‘ tolerance by the animal. SUMMARY
Ciumulative copper poisoning was induced in rats by feeding a diet COIItaining 2000 mg per kg copper as copper sulphate for 15 weeks. Animals wer(’ killed at intervals and their livers and kidneys examined histologically and analysed for their copper content. Subsequent events occurred in three phases, cumulative, crisis and recovery. Thai early period was associated with the gradual accumulation of copper ill the liver and kidney and some signs of cellular disturbance; it culminated in ;I crisis characterized by maximal liver and kidney copper concentrations and severe cellular disruption. The final phase was one of regeneration and healin,y in which the animal demonstrated its tolerance to copper. It was concluded that long term copper dosing elicits a similar response in both the kidney and the liver of rats and that adaptation to copper takes place, in I)oth these organs, probably as a result of increased excretion and dif?‘ercnt modes of storage, which, in turn, contributes to the development of tolerant-ck to (sopper. ACKNOWLEDGMENTS
1 wish to thank Professor D. L. Hughes and Dr D. ,411an for their encouragemcnr and advicca, Mrs M. W. Harling and Mr R. Comerford for technical assistance and Mrs P. Jenkins for the photography, also, Mr 1). F. Gibbons of M.A.F.F. Veterinary Investigation Centre, Liverpool for Se assay. and Mr M. E. Putnam of Rochr Products Ltd., for Vit. E assay.
KEFEKENCES
Beck, A. B. (1956). The copper content of the liver and blood of some vcrtebratc.\. Australian Journal of
lIlqy
14th, 19791