- Email: [email protected]
Studies with copper calcium E.D.T.A.
J.
corn.
PATH.
1971.
VOL.
STUDIES
279
81.
WITH
ACUTE
COPPER
TOXICITY
CALCIUM
IN HOUSED
E.D.T.A.
SHEEP
BY
J.
ISHMAEL+,
C.
GOPINATH
and J. McC.
HOWELL
Department of Veterinary Paihology, University of Livcrfkmt
INTRODUCTION
The copper calcium complex of ethylene d&nine tetra acetic acid (Cu Ca EDTA) raises liver copper levels when administered to sheep by subcutaneous injection (Camargo, Lee and Dewey, 1962). When given to ewes midway through pregnancy the compound is effective in preventing the development of swayback (Hemingway, MacPherson and Ritchie, 1970). In several flocks, however, a number of ewes have died within a short time of administration of the compound (Ishmael, Howell and Treeby, 1969; Wiener and Field, 1970). The occurrence of these deaths appears to be sporadic and the incidence within individual flocks may vary considerably. Flocks that have suffered losses in one season do not necessarily lose further animals if the compound is given the following year. There is some evidence to suggest that younger animals may be more susceptible (Ishmael et al., 1969.) A group of lesions, consistently found in the dead ewes, is centrilobular necrosis and haemorrhage of the liver, excess fluid in the serous cavities, oedema of the lungs, epicardial and subendocardial haemorrhages, haemorrhage into the alimentary tract and subcutaneous haemorrhage at the injection site (Ishmael, Treeby and Howell, 1970). This communication records the results of an experiment designed to observe the effects of administering high doses of Cu Ca EDTA to young housed sheep and to study changes in tissue copper levels, and the effects on the liver and various blood constituents following the administration of this compound. MATERIALS
AND METHODS
Twelve yearling Swaledale sheep, which ranged in weight from 16 to 32 kg., were used. They were housed for 2 to 8 weeks before the administration of Cu Ca EDTA and were fed a diet of flaked maize and crushed oats with hay and water ad lib. Copper analysis. Blood samples were collected for copper analysis prior to injeetion and at one and, in most cases, six and twelve hours after injection. Further samples were collected daily. The blood was taken from the jugular vein into heparinised copper-free polythene bottles through a 16 b.w.g. stainless steel needle. Analysis was performed on duplicate samples of whole blood and plasma by a modification of the method of Eden and Green (1940). Samples of liver and kidney were taken at post-mortem examination with stainless steel instruments and stored in polystyrene pots at -2OOC. Analysis of these tissues was performed by atomic absorption spectroscopy after digestion with a mixture of sulphuric, perchloric and nitric acids. + Present addres:
WiImslow, Ghenhin.
Toxicology
Department,
Geigy (U.K.)
Ltd., Stamford L&c,
Altrincham
Road,
280
CRYOSTAT
COPPER
CALCIUM
E.D.T.A.
IN SHEEP
Haematology. Blood was collected into bottles containing dipotassium E.D.T.A. and examined as described by Dacie and Lewis (1966) : packed cell volumes with Wintrobe haematocrit tubes, red and white cell counts with an improved Neubauer haemocytometer and haemoglobin by the cyanmethaemoglobin method. Blood films were stained by Leishman for differential white cell counts and cell morphology and supravitally with brilliant cresyl blue to demonstrate reticulocytes and Heinz bodies. Histopathology. Tissues were fixed in 10 per cent. formalin and embedded in paraffin wax. Sections were cut at 4 p and stained routinely by haematoxylin and eosin. Selected sections were stained by periodic acid-Schiff (P.A.S.) with or without digestion with diastase, Perls’ Prussian blue, rubeanic acid for copper, reticulinVan Gieson and Heidenhain’s iron haematoxylin. Frozen sections were stained by Oil red 0 to demonstrate fat. The methods used were as described by Pearse (1960). Bacteriology. Bacteriological examinations were carried out on all animals which died and intestinal contents were examined for enterotoxins by mouse inoculation, Collection of biopsy samples. Samples of liver were obtained by needle biopsy one week before and at 48 hours and at two weeks after the injections. Each sample, cleared of blood and briefly drained on blotting paper, was then divided into two pieces, one of which was fixed in 10 per cent. formalin and the other was immersed for not less than 30 seconds in iso-pentane, cooled to -68OC. with solid carbon Paraffin dioxide and kept at - 70°C. before sectioning in a cryostat at -18OC. sections were prepared from the formalin fixed sample as described above. Enzyme histochemistry. Cryostat sections of the liver biopsy samples (8 p thick) were mounted on glass slides and examined for adenosine triphosphatase (A. T. Pase) (Wachstein and Meisel, 1957), non- specific esterase, succinic tetrazolium reductase, glumatic dehydrogenase (Pearse, 1960) and alkaline phosphatase (Burstone, 1962). Serum enzymes and bilirubin. The sheep were bled on three occasions, before and at daily intervals for the first 4 days after the injections. Further samples were collected at weekly intervals. The blood samples were allowed to clot and the separated serum was stored at -2OOC. until analysed. Total bilirubin was measured by Dangerfield and Finlayson’s (1953) method, glutamateoxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) by the methods described by Reitman and Frankel (1957), glutamate dehydrogenase (GD) the technique of Ford and Boyd (1962), sorbitol dehydrogenase (SD) as described by Ford (1967) and arginase by the method of Cornelius and Freedland (1962).
RESULTS
The from copper in the sheep
sheep were given subcutaneous injections of Cu Ca EDTA in doses ranging body weight. The weights of the sheep, the dose of which they received, the time of death and the amounts of copper found livers and kidneys at post-mortem examination are given in Table 1. Five (26, 87, 61, 64 and 67) which received from 3 to 5 mg. Cu/kg. body weight,
l-6 to 5 mg. Cu/kg.
were found dead on the morning following the injection while two (65 and 66) which received 5 and 4 mg. Cu/kg. respectively, lived for 3 days. The latter animals had haemolysis and there was a progressive fall in their packed cell volumes, haemoglobin and red cell counts (Fig. 1) Heinz bodies were first seen in the red cells of both these sheep 24 hours after the injection of Cu Ca EDTA. The number of cells affected increased until in the last samples collected before death, more than 50 per cent. of the erythrocytes contained Heinz bodies (Fig. 6). The haemolysis was accompanied by neutrophilia (Fig. 2). The only significant changes noted in the
J. ISHMAEL
281
et al.
'. b
2.0 -
‘Q *-.-a
1.0 00
6
--*-.__
- IO -0
I2 18 I 24 I 30 , 36 . 42, 46I 54 I 60 , 66’ Time in hours efter injection of CuCe EDTA
Fig. 1. Changes in the P.C.V., red cell count and haemoglobm level in sheep 66 following of 90 mg. Cu. as Cu Ca EDTA.
0
the injection
,
0
6
Fig. 2. Changes in the neutrophil Cu Ca EDTA.
12 18 24 30 36 42 48 54 60 66 ?2 Time in hours after injection of Cu Ca UITA count in a sheep undergoing
haemolysis following
the injection
of
282
CRYOSTAT
COPPER
CALCIUM TABLE
DETAILS
Sheep number
64
:f
5
z
21 * Killed
7 Killed
4 weeks after injection
IN SHEEP
1
EXPERIMENTAL
Total doseof Cu as Cu Ca ED TA in mg.
Dose of Cu in mg./kg.
Weight in kg.
OF THE
E.D.T.A.
SHEEP
Time of death after Cu Ca EDTA administration
Tissue Cu level in fi.fi.m. dry weight at post-mortem examination --__ Kidmy Li0er
Survived* Survived * Survived * Less than 24 hrs. Survived?
452.4 454-6 532.6 505.7 679.5
LessSurvivedt than 24 hrs. Less than 3 davs24 hrs.
303.1 676.1 I .084.5 779.2
Less than’24 hrs. 3 days Less than 24 hrs.
’437.8 823.2 331.7
13.7 16-l 18.5 121.9 42.4 145.2 118.0 100.2 52.1 203.8 82.3 187.7
5 days after injection.
blood picture of the 5 sheep which died within 24 hours of injection were in sheep 26 and 87 where the neutrophil count increased from 2,480 and 1,250 per cu. mm. immediately before the injection, to 10,050 and 5,400 cu. mm. respectively 6 hours afterwards. Five sheep, which received either 1.6 mg. (85 and 88) 2 mg. (86) or 3 mg. (63 and 60) of &,/kg. body weight, survived. Sheep 63 and 60 appeared dull 24 hours after injection. Their appetite was depressed and remained so until they were killed after 5 days.
.’
8
5
VI’
0 0’
0
\
\
\
-- - - Received 65mg Cu as EDTA ‘1 \
\
\
\
\
**‘-“‘.‘*‘a’~‘.’ ’ - ’ ’ ’ 24 36 12 Time in hours after
Fig. 3. Changes in the neutrophil
\
\
\
Received 50 mg Cu as EDTA
\
’ * ’ a ’ 8 ’ 60 72 84 40 injection of Cu Ca EDTA
count in 2 sheep following
the injection
J 96 of Cu Ca EDTA.
J.
.
ISHMAEL
t?t d.
283
284
CRYOSTAT
COPPER
CALCIUM
E.D.T.A.
IN SHEEP
Little change was noted in the clinical appearance of sheep 85, 86 and 88. In all 5 surviving animals there was an increase in the numbers of circulating neutrophils and this was most pronounced in those given 3 mg. &/kg. (Fig. 3).
Blood Copper Levels The whole blood and plasma copper levels of the 12 sheep (Table 2) were raised one hour after the administration of Cu Ca EDTA and this was most marked in those given the higher doses. The values tended to reach a peak within the first 12 hours and thereafter declined. The red cell copper levels were calculated from the whole blood and plasina copper levels and the packed cell volumes for sheep 65 and 66. The copper content of the red cells was markedly raised 24 hours after injection and at this time haemolysis was occurring.
Serum Constituents There was a massive release of liver specific enzymes into the serum following Cu Ca EDTA administration. In sheep 63 and 60 which received 3 mg. Cu/kg. levels of S.D., G-D., arginase and GOT rose sharply within 24 hours to very high concentrations and remained high for about 72 hours. Four days after dosing there was a slight reduction in levels of S.D. and GOT. and a marked
ARG.
600
t 308 [ G.O.T.
0' -72
4 -46 in hours -24 Time
Fig. 4. Changes in the serwn enzyme activity 3 mg. G/kg. body weight.
Y2
24
46
72
36
of a sheep given Cu Ca EDTA at a &xc equivalent
to
J.
ISHMAEL
et al.
285
ot Ill‘O”
ARG.
01234 t Fig. 5. Changes in the serum enzyme activity 2 mg. &/kg. body weight.
7 Time in
14
21
28
days
of a sheep given Cu Ca EDTA
at a dose equivalent
to
reduction in the levels of arginase and G.D. (Fig. 4). In contrast to the above, the rise in concentration of G.P.T. was very slight. Serum bilirubin levels were increased 24 hours after dosing and reached the highest level, 5 mg. per cent. in sheep 63, at 48 hours : thereafter, the levels declined. The pattern of enzyme release was similar in sheep 85,88 and 86, but the amounts released were not as high. The serum levels of SD. and G.D. showed very sudden and high elevations reaching peaks within 48 hours and then a gradual fall (Fig. 5). The G.O.T. and arginase levels rose but much less. (Fig. 5). Seven days after dosing the levels of G.O.T., arginase and G.D. were within the pre-dosing range whereas SD. levels were still elevated. The latter fluctuated, but remained high throughout the period of observation. (Fig. 5). Little alteration in bilirubin levels was seen in 85 and 86, but in sheep 88 the bilirubin level was raised at 24 hours and reached the highest value 3 days after dosing. The level was normal again one week after injection. Alterations in the levels of G.P.T. during the course of the experiment were negligible in these 3 animals. Liver Biopsies Histopathology. The liver biopsies taken at 48 hours showed marked centrilobular necrosis and in sheep 63, 60 and 88 the mid zonal areas were necrotic also (Fig. 7). Small numbers of neutrophils were infiltrating the necrotic tissue and there was centrilobular congestion and haemorrhage. The parenchymal cells
286
CRYOSTAT
COPPER
CALCIUM
E.D.T.A.
IN
SHEEP
at the edge of the necrotic areas were ballooned and had lightly stained cytoplasm (Fig. 7). Sections stained with rubeanic acid showed fine green particles in the cytoplasm. In the samples taken two weeks after injection from 85, 86 and 88 the necrotic tissue had been replaced by irregular cords of new parenchymal cells which were larger than normal : they had a vacuolated cytoplasm and in some there was nuclear enlargement. Small numbers of neutrophils were present in the sinusoids and there were several swollen Kupffer cells containing brown granular pigment. This pigment was P.A.S. positive, diastase resistant and stained with rubeanic acid, but not with Perls’ method. Small foci of fibroblast proliferation were noted in the portal areas and within the lobules. In sections stained with reticulin-Van Gieson an increase in the number and thickness of reticulin fibres was seen. Histochemistry Succinic tetrazolium reductase. The biopsy samples removed 48 hours after the administration of Cu Ca EDTA from sheep 63, 60, 88 and 85 showed slight overall reduction and marked centrilobular depletion of enzyme activity. Centrilobular depletion was limited to isolated cells in sheep 86. Glu,tamic dehydrogenase. At 48 hours there was complete absence of enzyme activity in the centrilobular areas in 63, 60, 88 and 85 (Fig. 8). Normal activity was confined to only a few periportal cells and a few isolated cells in the midzonal regions, In 86 there was central reduction and isolated cell depletion. In 85, 86 and 88 activity had returned to normal by two weeks. Non-specific esterase. The 48 hour samples showed overall reduction in activity which was most marked in the centrilobular areas (Fig. 9). Several centrilobular parenchymal cells showed opaque, black irregular deposits. These changes were least severe in sheep 86. Activity had returned to normal by two weeks. Alkaline phosphatase. With the exception of sheep 86 in which activity was weak, at 48 hours there was a widespread increase in alkaline phosphatase deposits. The centrilobular parenchymal cells showed increased diffuse cytoplasmic deposits and activity was intense in the Kupffer cells. There was a normal distribution of activity two weeks after injection. Adenosine triphosphatase. The 48 hour samples showed loss in canalicular activity from the centrilobular areas and weak irregular activity in the mid zonal regions. The periportal liver cells contained moderate to well marked canalicular deposits. The mid zonal and central cells showed an increase in diffuse cytvplasmic deposits. (Fig. 10). Activity had returned to normal by two weeks. Post Mortem Findings These were similar in the 7 animals which died. There was excessive fluid in the pleural, pericardial and peritoneal cavities, which was blood stained in sheep 65 and 66. All sheep had oedema of the lungs and in the 5 which died in less than 24 hours there were subendvcardial haemvrrhages which were particularly prominent in the left ventricle. The livers appeared dark and mottled, and there was vedema and haemorrhage of the gall bladder wall in sheep 87. The kidneys
J. KunfaL
et al.
287
were congested. There was slight congestion of the abomasal and duodenal mucosa in 64 and 67, and in 87 the contents of the duodenum were haemorrhagic and the mucosa was inflamed. Sheep 65 and 66 had haemoglobinuria. Significant organisms were not isolated by aerobic and anerobic cultures from the dead animals and examination for enterotoxins proved negative. The liver was mottled in sheep 60 and 63 which were killed after five days, but post-mortem examination of 85,86 and 88, which were killed after four weeks, did not reveal abnormalities apart from small healed biopsy scars on the diaphragmatic surface of the liver. Histological Findings The livers of the 5 animals which died in less than 24 hours after the injection of Cu Ca EDTA showed marked congestion, haemorrhage, necrosis of the parenchymal cells and a polymorphonuclear leucocyte infiltration in the centres of the lobules. In sheep 65 and 66 which lived for 3 days and suffered a haemolytic crisis, there was centrilobular necrosis, but intense centrilobular congestion and haemorrhage was not seen. In the latter 2 animals the kidney tubules contained granular casts, and in places the lining epithelial cells were necrotic and there was some neutrophil infiltration. The spleens were engorged and contained large amounts of haemosiderin. In sections of liver from 63 and 60 much of the necrotic tissue had been partially replaced by new parenchymal cells. These appeared to be rapidly dividing and many showed mitotic figures. There were several swollen Kupffer cells containing brown granular pigment in the centrilobular areas. The pigment was P.A.S. positive, diastase resistant and gave a positive reaction with rubeanic acid, but not with Perls’ stain. In both these sheep many of the kidney tubules contained granular casts. Histological examination of sheep 85,86 and 88 revealed that the liver changes were essentially the same as in the biopsy samples at two weeks after injection and that the swollen pigmented Kupffer cells were still prominent in the centrilobular areas. A small number of hyaline casts were noted in the kidney tubules. DISCUSSION
The usual form of copper toxicity encountered in sheep is chronic copper poisoning, which is character&d by the accumulation of large amounts of copper in the liver over a period of weeks or months and eventually culminates in severe haemolysis and the development of jaundice (Todd, 1969). There are numerous reports of this type of poisoning in sheep, but there are few of toxicity associated with the parenteral administration of copper compounds in the treatment or prevention of copper deficiency syndromes. Harvey and Sutherland (1953) investigated the effects of several copper compounds administered by subcutaneous or intramuscular injection. They found that copper versenate at a dose equivalent to 80 mg. Cu killed 5 out of 6 copper sufficient Merino sheep. Copper glycinate at a dose equivalent to 100 mg. Cu killed 3 out of 11 sheep. One of these had acute hepatitis and enteritis and another one showed liver damage. Cunningham (1957) recorded that 3 out of 100 yearling sheep given the cupric-bis-hydroxyquinoline 5-7 disulphonic acid salt of tetra diethylamine died after injection and in 2 of these there was “some possibiity of copper poisoning”. Allcroft, Buntain
288
CRYOSTAT
COPPER
CALCIUM
E.D.T.A.
IN
SHEEP
J. ISHMAEL
et
d.
289
SUMMARY
Twelve housed yearling Swaledale sheep of normal copper status were given single subcutaneous injections of Cu Ca EDTA at doses ranging from 1.6 to 5 mg. Cu/kg. body weight. Two out of four given 3 mg. Cu/kg. and five given higher doses died within three days. Two had a haemolytic crisis. In all animals there was a rise in whole blood and plasma copper levels within one hour of injection. Liver biopsy samples taken 48 hours after injection from the 5 surviving animals showed hepatic centrilobular necrosis associated with a depletion of succinic
ACKNOWLEDGMENTS
The authors are indebted to Professor D. L. Hughes for encouragement and advice, to Mrs. M. W. Harling and Miss P. Landon for technical assistance and Mr. G. Weston for the photomicrographs. REFERENCES
Allcroft, R., Buntain, D., and Rowlands, W. T. (1965). Iret. Rec., 77, 634. Burstone, M. S. (1962). in Enzyme Histochemistry and Its Applicatzon in the Study of Neoplusms, p. 275, Academic Press; New York and London. Cam;gci y. z de A., Lee, H. J., and Dewey, D. W. (1962). Proc. Aust. Sot. anim. Corneius;‘C: E.; and Freedland, R. A. (1962). Cornell Vet., 52, 344. Cunningham, I. J. (1957). New Zealand vet. J., 5, 9. Dacie, J. V., and Lewis, SM. (1966). Practical Haematology, 3rd edition, J. & A. Churchill; London. Dangerfield, W. G., and Finlayson, R. (1953). J. clin. Path., 6, 173. Eden, A., and Green, H. H. (1940). Biochem. J., 34, 1202. Ford, E. J. H. (1967). f. camp. Path., 77, 405. Ford, E. J. H., and Boyd, J. W., (1962). J. Path. Bacf., 83, 39. Harvey, J. M., and Sutherland, A. K. (1953). Aust. uet. J., 29, 261. Hemingway, R. G., MacPherson, A., and Ritchie, N. S. (1970). In Truce Element Metabolism in Animals, Ed. Mills, C. F., p. 264, E. & S. Livingstone; Edinburgh and London. Ishmael, J., Howell, J McC., and Treeby, P. J. (1969). Tret. Rec., 85, 205. Ishmael, J, Treeby, P. J., and Howell, J. McC. (1970). In Truce Element Metabolism in Antmals, Ed. Mills, C. F., p. 268, E & S Livingstone; Edinburgh and London. Pearse, A. G. E. (1960). Histochemistry, Theoretical and Applied, 2nd edition, J. & A. Churchill; London. Reitman, S., and Frankel, S. (1957). Amer. J. clin. Path., 28, 56.
290
CRYOSTAT
COPPER
C:hLCIUM
E.D.T.A.
IN
SHEEP
Thorpe, E., Gopinath, C., Jones, R. S., and Ford, E. J. H. (1968). J. Path., 97, 241. Todd, J. R. (1969). Proc. Nutr. SOL, 28, 189. Wachstein, M., and Meisel, E., (1957). Amer. J. clin. Path., 27, 13. Wiener, G., and Field, A. C. (1970). In Trace Element Merabolism in Animals, Ed Mills, C. F., p. 92, Livingstone; Edinburgh and London. [Received
/or publication.
july
10th. 19701
J.
ISHMAEL
et al.
Fig. 6. Blood film from a sheep undergoing haemolysisfoll;w$; cells contain Heinz bodies. Brilliant cresyl blue
the injection of Cu Ca EDTA. Many
Fig. 7. Liver biopsy from a sheep taken 48 hours after the injection of Cu Ca EDTA at a dose equivalent to 3 mg. &/kg. body weight. The parenchymal cells in the central and mid zonal areas are necrotic. The cells at the periphery of the lobule have a ballooned appearance and very lightly stained cytoplasm. H & E x 155.
CRYOSTAT COPPER CALCIUM E.D.T.A.
IN SHEEP
Fig. 8. Liver biopsy from a sheep 48 hours after the injection of Cu Ca EDTA. There is complete absenceof glutamic dehydrogenaseactivity in the centrilobular arca\. \ 70. Fig. 9. Liver biopsy from a shcrp 48 hours aftrr thr injection of Cu Ca EDTA. Thrrr is an ovrrall rrduction in non-qxcific rstcrasc activity which is mo\t markrd in thr crntrilobular away. 1 70. Fig. 10. I.i\cr biopsy from a shrrp 48 hour aftrr the injection of Cu Ca EDT.\. There is loss of canalirular AT. Paw acti\ ity from tbr cmtrilobular arca and 1,cak 11I cqul,lt activity in thr mid zonal region. \ 70.
corn.
PATH.
1971.
VOL.
STUDIES
279
81.
WITH
ACUTE
COPPER
TOXICITY
CALCIUM
IN HOUSED
E.D.T.A.
SHEEP
BY
J.
ISHMAEL+,
C.
GOPINATH
and J. McC.
HOWELL
Department of Veterinary Paihology, University of Livcrfkmt
INTRODUCTION
The copper calcium complex of ethylene d&nine tetra acetic acid (Cu Ca EDTA) raises liver copper levels when administered to sheep by subcutaneous injection (Camargo, Lee and Dewey, 1962). When given to ewes midway through pregnancy the compound is effective in preventing the development of swayback (Hemingway, MacPherson and Ritchie, 1970). In several flocks, however, a number of ewes have died within a short time of administration of the compound (Ishmael, Howell and Treeby, 1969; Wiener and Field, 1970). The occurrence of these deaths appears to be sporadic and the incidence within individual flocks may vary considerably. Flocks that have suffered losses in one season do not necessarily lose further animals if the compound is given the following year. There is some evidence to suggest that younger animals may be more susceptible (Ishmael et al., 1969.) A group of lesions, consistently found in the dead ewes, is centrilobular necrosis and haemorrhage of the liver, excess fluid in the serous cavities, oedema of the lungs, epicardial and subendocardial haemorrhages, haemorrhage into the alimentary tract and subcutaneous haemorrhage at the injection site (Ishmael, Treeby and Howell, 1970). This communication records the results of an experiment designed to observe the effects of administering high doses of Cu Ca EDTA to young housed sheep and to study changes in tissue copper levels, and the effects on the liver and various blood constituents following the administration of this compound. MATERIALS
AND METHODS
Twelve yearling Swaledale sheep, which ranged in weight from 16 to 32 kg., were used. They were housed for 2 to 8 weeks before the administration of Cu Ca EDTA and were fed a diet of flaked maize and crushed oats with hay and water ad lib. Copper analysis. Blood samples were collected for copper analysis prior to injeetion and at one and, in most cases, six and twelve hours after injection. Further samples were collected daily. The blood was taken from the jugular vein into heparinised copper-free polythene bottles through a 16 b.w.g. stainless steel needle. Analysis was performed on duplicate samples of whole blood and plasma by a modification of the method of Eden and Green (1940). Samples of liver and kidney were taken at post-mortem examination with stainless steel instruments and stored in polystyrene pots at -2OOC. Analysis of these tissues was performed by atomic absorption spectroscopy after digestion with a mixture of sulphuric, perchloric and nitric acids. + Present addres:
WiImslow, Ghenhin.
Toxicology
Department,
Geigy (U.K.)
Ltd., Stamford L&c,
Altrincham
Road,
280
CRYOSTAT
COPPER
CALCIUM
E.D.T.A.
IN SHEEP
Haematology. Blood was collected into bottles containing dipotassium E.D.T.A. and examined as described by Dacie and Lewis (1966) : packed cell volumes with Wintrobe haematocrit tubes, red and white cell counts with an improved Neubauer haemocytometer and haemoglobin by the cyanmethaemoglobin method. Blood films were stained by Leishman for differential white cell counts and cell morphology and supravitally with brilliant cresyl blue to demonstrate reticulocytes and Heinz bodies. Histopathology. Tissues were fixed in 10 per cent. formalin and embedded in paraffin wax. Sections were cut at 4 p and stained routinely by haematoxylin and eosin. Selected sections were stained by periodic acid-Schiff (P.A.S.) with or without digestion with diastase, Perls’ Prussian blue, rubeanic acid for copper, reticulinVan Gieson and Heidenhain’s iron haematoxylin. Frozen sections were stained by Oil red 0 to demonstrate fat. The methods used were as described by Pearse (1960). Bacteriology. Bacteriological examinations were carried out on all animals which died and intestinal contents were examined for enterotoxins by mouse inoculation, Collection of biopsy samples. Samples of liver were obtained by needle biopsy one week before and at 48 hours and at two weeks after the injections. Each sample, cleared of blood and briefly drained on blotting paper, was then divided into two pieces, one of which was fixed in 10 per cent. formalin and the other was immersed for not less than 30 seconds in iso-pentane, cooled to -68OC. with solid carbon Paraffin dioxide and kept at - 70°C. before sectioning in a cryostat at -18OC. sections were prepared from the formalin fixed sample as described above. Enzyme histochemistry. Cryostat sections of the liver biopsy samples (8 p thick) were mounted on glass slides and examined for adenosine triphosphatase (A. T. Pase) (Wachstein and Meisel, 1957), non- specific esterase, succinic tetrazolium reductase, glumatic dehydrogenase (Pearse, 1960) and alkaline phosphatase (Burstone, 1962). Serum enzymes and bilirubin. The sheep were bled on three occasions, before and at daily intervals for the first 4 days after the injections. Further samples were collected at weekly intervals. The blood samples were allowed to clot and the separated serum was stored at -2OOC. until analysed. Total bilirubin was measured by Dangerfield and Finlayson’s (1953) method, glutamateoxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) by the methods described by Reitman and Frankel (1957), glutamate dehydrogenase (GD) the technique of Ford and Boyd (1962), sorbitol dehydrogenase (SD) as described by Ford (1967) and arginase by the method of Cornelius and Freedland (1962).
RESULTS
The from copper in the sheep
sheep were given subcutaneous injections of Cu Ca EDTA in doses ranging body weight. The weights of the sheep, the dose of which they received, the time of death and the amounts of copper found livers and kidneys at post-mortem examination are given in Table 1. Five (26, 87, 61, 64 and 67) which received from 3 to 5 mg. Cu/kg. body weight,
l-6 to 5 mg. Cu/kg.
were found dead on the morning following the injection while two (65 and 66) which received 5 and 4 mg. Cu/kg. respectively, lived for 3 days. The latter animals had haemolysis and there was a progressive fall in their packed cell volumes, haemoglobin and red cell counts (Fig. 1) Heinz bodies were first seen in the red cells of both these sheep 24 hours after the injection of Cu Ca EDTA. The number of cells affected increased until in the last samples collected before death, more than 50 per cent. of the erythrocytes contained Heinz bodies (Fig. 6). The haemolysis was accompanied by neutrophilia (Fig. 2). The only significant changes noted in the
J. ISHMAEL
281
et al.
'. b
2.0 -
‘Q *-.-a
1.0 00
6
--*-.__
- IO -0
I2 18 I 24 I 30 , 36 . 42, 46I 54 I 60 , 66’ Time in hours efter injection of CuCe EDTA
Fig. 1. Changes in the P.C.V., red cell count and haemoglobm level in sheep 66 following of 90 mg. Cu. as Cu Ca EDTA.
0
the injection
,
0
6
Fig. 2. Changes in the neutrophil Cu Ca EDTA.
12 18 24 30 36 42 48 54 60 66 ?2 Time in hours after injection of Cu Ca UITA count in a sheep undergoing
haemolysis following
the injection
of
282
CRYOSTAT
COPPER
CALCIUM TABLE
DETAILS
Sheep number
64
:f
5
z
21 * Killed
7 Killed
4 weeks after injection
IN SHEEP
1
EXPERIMENTAL
Total doseof Cu as Cu Ca ED TA in mg.
Dose of Cu in mg./kg.
Weight in kg.
OF THE
E.D.T.A.
SHEEP
Time of death after Cu Ca EDTA administration
Tissue Cu level in fi.fi.m. dry weight at post-mortem examination --__ Kidmy Li0er
Survived* Survived * Survived * Less than 24 hrs. Survived?
452.4 454-6 532.6 505.7 679.5
LessSurvivedt than 24 hrs. Less than 3 davs24 hrs.
303.1 676.1 I .084.5 779.2
Less than’24 hrs. 3 days Less than 24 hrs.
’437.8 823.2 331.7
13.7 16-l 18.5 121.9 42.4 145.2 118.0 100.2 52.1 203.8 82.3 187.7
5 days after injection.
blood picture of the 5 sheep which died within 24 hours of injection were in sheep 26 and 87 where the neutrophil count increased from 2,480 and 1,250 per cu. mm. immediately before the injection, to 10,050 and 5,400 cu. mm. respectively 6 hours afterwards. Five sheep, which received either 1.6 mg. (85 and 88) 2 mg. (86) or 3 mg. (63 and 60) of &,/kg. body weight, survived. Sheep 63 and 60 appeared dull 24 hours after injection. Their appetite was depressed and remained so until they were killed after 5 days.
.’
8
5
VI’
0 0’
0
\
\
\
-- - - Received 65mg Cu as EDTA ‘1 \
\
\
\
\
**‘-“‘.‘*‘a’~‘.’ ’ - ’ ’ ’ 24 36 12 Time in hours after
Fig. 3. Changes in the neutrophil
\
\
\
Received 50 mg Cu as EDTA
\
’ * ’ a ’ 8 ’ 60 72 84 40 injection of Cu Ca EDTA
count in 2 sheep following
the injection
J 96 of Cu Ca EDTA.
J.
.
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Little change was noted in the clinical appearance of sheep 85, 86 and 88. In all 5 surviving animals there was an increase in the numbers of circulating neutrophils and this was most pronounced in those given 3 mg. &/kg. (Fig. 3).
Blood Copper Levels The whole blood and plasma copper levels of the 12 sheep (Table 2) were raised one hour after the administration of Cu Ca EDTA and this was most marked in those given the higher doses. The values tended to reach a peak within the first 12 hours and thereafter declined. The red cell copper levels were calculated from the whole blood and plasina copper levels and the packed cell volumes for sheep 65 and 66. The copper content of the red cells was markedly raised 24 hours after injection and at this time haemolysis was occurring.
Serum Constituents There was a massive release of liver specific enzymes into the serum following Cu Ca EDTA administration. In sheep 63 and 60 which received 3 mg. Cu/kg. levels of S.D., G-D., arginase and GOT rose sharply within 24 hours to very high concentrations and remained high for about 72 hours. Four days after dosing there was a slight reduction in levels of S.D. and GOT. and a marked
ARG.
600
t 308 [ G.O.T.
0' -72
4 -46 in hours -24 Time
Fig. 4. Changes in the serwn enzyme activity 3 mg. G/kg. body weight.
Y2
24
46
72
36
of a sheep given Cu Ca EDTA at a &xc equivalent
to
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285
ot Ill‘O”
ARG.
01234 t Fig. 5. Changes in the serum enzyme activity 2 mg. &/kg. body weight.
7 Time in
14
21
28
days
of a sheep given Cu Ca EDTA
at a dose equivalent
to
reduction in the levels of arginase and G.D. (Fig. 4). In contrast to the above, the rise in concentration of G.P.T. was very slight. Serum bilirubin levels were increased 24 hours after dosing and reached the highest level, 5 mg. per cent. in sheep 63, at 48 hours : thereafter, the levels declined. The pattern of enzyme release was similar in sheep 85,88 and 86, but the amounts released were not as high. The serum levels of SD. and G.D. showed very sudden and high elevations reaching peaks within 48 hours and then a gradual fall (Fig. 5). The G.O.T. and arginase levels rose but much less. (Fig. 5). Seven days after dosing the levels of G.O.T., arginase and G.D. were within the pre-dosing range whereas SD. levels were still elevated. The latter fluctuated, but remained high throughout the period of observation. (Fig. 5). Little alteration in bilirubin levels was seen in 85 and 86, but in sheep 88 the bilirubin level was raised at 24 hours and reached the highest value 3 days after dosing. The level was normal again one week after injection. Alterations in the levels of G.P.T. during the course of the experiment were negligible in these 3 animals. Liver Biopsies Histopathology. The liver biopsies taken at 48 hours showed marked centrilobular necrosis and in sheep 63, 60 and 88 the mid zonal areas were necrotic also (Fig. 7). Small numbers of neutrophils were infiltrating the necrotic tissue and there was centrilobular congestion and haemorrhage. The parenchymal cells
286
CRYOSTAT
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SHEEP
at the edge of the necrotic areas were ballooned and had lightly stained cytoplasm (Fig. 7). Sections stained with rubeanic acid showed fine green particles in the cytoplasm. In the samples taken two weeks after injection from 85, 86 and 88 the necrotic tissue had been replaced by irregular cords of new parenchymal cells which were larger than normal : they had a vacuolated cytoplasm and in some there was nuclear enlargement. Small numbers of neutrophils were present in the sinusoids and there were several swollen Kupffer cells containing brown granular pigment. This pigment was P.A.S. positive, diastase resistant and stained with rubeanic acid, but not with Perls’ method. Small foci of fibroblast proliferation were noted in the portal areas and within the lobules. In sections stained with reticulin-Van Gieson an increase in the number and thickness of reticulin fibres was seen. Histochemistry Succinic tetrazolium reductase. The biopsy samples removed 48 hours after the administration of Cu Ca EDTA from sheep 63, 60, 88 and 85 showed slight overall reduction and marked centrilobular depletion of enzyme activity. Centrilobular depletion was limited to isolated cells in sheep 86. Glu,tamic dehydrogenase. At 48 hours there was complete absence of enzyme activity in the centrilobular areas in 63, 60, 88 and 85 (Fig. 8). Normal activity was confined to only a few periportal cells and a few isolated cells in the midzonal regions, In 86 there was central reduction and isolated cell depletion. In 85, 86 and 88 activity had returned to normal by two weeks. Non-specific esterase. The 48 hour samples showed overall reduction in activity which was most marked in the centrilobular areas (Fig. 9). Several centrilobular parenchymal cells showed opaque, black irregular deposits. These changes were least severe in sheep 86. Activity had returned to normal by two weeks. Alkaline phosphatase. With the exception of sheep 86 in which activity was weak, at 48 hours there was a widespread increase in alkaline phosphatase deposits. The centrilobular parenchymal cells showed increased diffuse cytoplasmic deposits and activity was intense in the Kupffer cells. There was a normal distribution of activity two weeks after injection. Adenosine triphosphatase. The 48 hour samples showed loss in canalicular activity from the centrilobular areas and weak irregular activity in the mid zonal regions. The periportal liver cells contained moderate to well marked canalicular deposits. The mid zonal and central cells showed an increase in diffuse cytvplasmic deposits. (Fig. 10). Activity had returned to normal by two weeks. Post Mortem Findings These were similar in the 7 animals which died. There was excessive fluid in the pleural, pericardial and peritoneal cavities, which was blood stained in sheep 65 and 66. All sheep had oedema of the lungs and in the 5 which died in less than 24 hours there were subendvcardial haemvrrhages which were particularly prominent in the left ventricle. The livers appeared dark and mottled, and there was vedema and haemorrhage of the gall bladder wall in sheep 87. The kidneys
J. KunfaL
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were congested. There was slight congestion of the abomasal and duodenal mucosa in 64 and 67, and in 87 the contents of the duodenum were haemorrhagic and the mucosa was inflamed. Sheep 65 and 66 had haemoglobinuria. Significant organisms were not isolated by aerobic and anerobic cultures from the dead animals and examination for enterotoxins proved negative. The liver was mottled in sheep 60 and 63 which were killed after five days, but post-mortem examination of 85,86 and 88, which were killed after four weeks, did not reveal abnormalities apart from small healed biopsy scars on the diaphragmatic surface of the liver. Histological Findings The livers of the 5 animals which died in less than 24 hours after the injection of Cu Ca EDTA showed marked congestion, haemorrhage, necrosis of the parenchymal cells and a polymorphonuclear leucocyte infiltration in the centres of the lobules. In sheep 65 and 66 which lived for 3 days and suffered a haemolytic crisis, there was centrilobular necrosis, but intense centrilobular congestion and haemorrhage was not seen. In the latter 2 animals the kidney tubules contained granular casts, and in places the lining epithelial cells were necrotic and there was some neutrophil infiltration. The spleens were engorged and contained large amounts of haemosiderin. In sections of liver from 63 and 60 much of the necrotic tissue had been partially replaced by new parenchymal cells. These appeared to be rapidly dividing and many showed mitotic figures. There were several swollen Kupffer cells containing brown granular pigment in the centrilobular areas. The pigment was P.A.S. positive, diastase resistant and gave a positive reaction with rubeanic acid, but not with Perls’ stain. In both these sheep many of the kidney tubules contained granular casts. Histological examination of sheep 85,86 and 88 revealed that the liver changes were essentially the same as in the biopsy samples at two weeks after injection and that the swollen pigmented Kupffer cells were still prominent in the centrilobular areas. A small number of hyaline casts were noted in the kidney tubules. DISCUSSION
The usual form of copper toxicity encountered in sheep is chronic copper poisoning, which is character&d by the accumulation of large amounts of copper in the liver over a period of weeks or months and eventually culminates in severe haemolysis and the development of jaundice (Todd, 1969). There are numerous reports of this type of poisoning in sheep, but there are few of toxicity associated with the parenteral administration of copper compounds in the treatment or prevention of copper deficiency syndromes. Harvey and Sutherland (1953) investigated the effects of several copper compounds administered by subcutaneous or intramuscular injection. They found that copper versenate at a dose equivalent to 80 mg. Cu killed 5 out of 6 copper sufficient Merino sheep. Copper glycinate at a dose equivalent to 100 mg. Cu killed 3 out of 11 sheep. One of these had acute hepatitis and enteritis and another one showed liver damage. Cunningham (1957) recorded that 3 out of 100 yearling sheep given the cupric-bis-hydroxyquinoline 5-7 disulphonic acid salt of tetra diethylamine died after injection and in 2 of these there was “some possibiity of copper poisoning”. Allcroft, Buntain
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CRYOSTAT
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SUMMARY
Twelve housed yearling Swaledale sheep of normal copper status were given single subcutaneous injections of Cu Ca EDTA at doses ranging from 1.6 to 5 mg. Cu/kg. body weight. Two out of four given 3 mg. Cu/kg. and five given higher doses died within three days. Two had a haemolytic crisis. In all animals there was a rise in whole blood and plasma copper levels within one hour of injection. Liver biopsy samples taken 48 hours after injection from the 5 surviving animals showed hepatic centrilobular necrosis associated with a depletion of succinic
ACKNOWLEDGMENTS
The authors are indebted to Professor D. L. Hughes for encouragement and advice, to Mrs. M. W. Harling and Miss P. Landon for technical assistance and Mr. G. Weston for the photomicrographs. REFERENCES
Allcroft, R., Buntain, D., and Rowlands, W. T. (1965). Iret. Rec., 77, 634. Burstone, M. S. (1962). in Enzyme Histochemistry and Its Applicatzon in the Study of Neoplusms, p. 275, Academic Press; New York and London. Cam;gci y. z de A., Lee, H. J., and Dewey, D. W. (1962). Proc. Aust. Sot. anim. Corneius;‘C: E.; and Freedland, R. A. (1962). Cornell Vet., 52, 344. Cunningham, I. J. (1957). New Zealand vet. J., 5, 9. Dacie, J. V., and Lewis, SM. (1966). Practical Haematology, 3rd edition, J. & A. Churchill; London. Dangerfield, W. G., and Finlayson, R. (1953). J. clin. Path., 6, 173. Eden, A., and Green, H. H. (1940). Biochem. J., 34, 1202. Ford, E. J. H. (1967). f. camp. Path., 77, 405. Ford, E. J. H., and Boyd, J. W., (1962). J. Path. Bacf., 83, 39. Harvey, J. M., and Sutherland, A. K. (1953). Aust. uet. J., 29, 261. Hemingway, R. G., MacPherson, A., and Ritchie, N. S. (1970). In Truce Element Metabolism in Animals, Ed. Mills, C. F., p. 264, E. & S. Livingstone; Edinburgh and London. Ishmael, J., Howell, J McC., and Treeby, P. J. (1969). Tret. Rec., 85, 205. Ishmael, J, Treeby, P. J., and Howell, J. McC. (1970). In Truce Element Metabolism in Antmals, Ed. Mills, C. F., p. 268, E & S Livingstone; Edinburgh and London. Pearse, A. G. E. (1960). Histochemistry, Theoretical and Applied, 2nd edition, J. & A. Churchill; London. Reitman, S., and Frankel, S. (1957). Amer. J. clin. Path., 28, 56.
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Thorpe, E., Gopinath, C., Jones, R. S., and Ford, E. J. H. (1968). J. Path., 97, 241. Todd, J. R. (1969). Proc. Nutr. SOL, 28, 189. Wachstein, M., and Meisel, E., (1957). Amer. J. clin. Path., 27, 13. Wiener, G., and Field, A. C. (1970). In Trace Element Merabolism in Animals, Ed Mills, C. F., p. 92, Livingstone; Edinburgh and London. [Received
/or publication.
july
10th. 19701
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et al.
Fig. 6. Blood film from a sheep undergoing haemolysisfoll;w$; cells contain Heinz bodies. Brilliant cresyl blue
the injection of Cu Ca EDTA. Many
Fig. 7. Liver biopsy from a sheep taken 48 hours after the injection of Cu Ca EDTA at a dose equivalent to 3 mg. &/kg. body weight. The parenchymal cells in the central and mid zonal areas are necrotic. The cells at the periphery of the lobule have a ballooned appearance and very lightly stained cytoplasm. H & E x 155.
CRYOSTAT COPPER CALCIUM E.D.T.A.
IN SHEEP
Fig. 8. Liver biopsy from a sheep 48 hours after the injection of Cu Ca EDTA. There is complete absenceof glutamic dehydrogenaseactivity in the centrilobular arca\. \ 70. Fig. 9. Liver biopsy from a shcrp 48 hours aftrr thr injection of Cu Ca EDTA. Thrrr is an ovrrall rrduction in non-qxcific rstcrasc activity which is mo\t markrd in thr crntrilobular away. 1 70. Fig. 10. I.i\cr biopsy from a shrrp 48 hour aftrr the injection of Cu Ca EDT.\. There is loss of canalirular AT. Paw acti\ ity from tbr cmtrilobular arca and 1,cak 11I cqul,lt activity in thr mid zonal region. \ 70.