Intravenously administered tetra-thiomolybdate and the removal of copper from the liver of copper-loaded sheep

J. (:omp.

Path.

1989 1’111. 101

Intravenously Administered Tetra-thiomolybdate and the Removal of Copper from the Liver Copper-loaded Sheep *J. S. Kumaratilake School of‘ I’eterinay

and

Studies, Murdoch

7J. McC.

lJniversi[y, Murdoch. Australia Summary

of

Howell Western Australia

6150,

Eighteen ewes divided into two groups were dosed orally with CuSO, in order IO induce chronic Cu toxicity. Copper dosing was stopped at the first rise of serum acid phosphatase activity in sheep of group 1 and on the first day of haemolysis in sheep of group 2. Tetra-thiomolybdate was administered intravenously to five group 1 sheep (group 1B) and to group 2 from the c.essation of Cu dosing. Following thiomolybdate administration, in groups IB and 2, there was a reduction in the concentration of Cu in the liver and liver fractions, the Ilumber and size of electron-dense lysosomes in particulate liver fractions, the T;olume density and the mean volume of electron-dense lysosomes in hepatocytes and the number of necrotic cells in the liver. Thiomolybdate appeared to remove Cu from the lysosomes and the cytosol of Cu-loaded liver cells. However, neither the total specific activity of acid phosphatase in liver homogenate and liver fractions nor the numerical density of electron-dense lysosomes in hepatocytes decreased significantly. This may be due to the I)roduction of new lysosomes in the liver cells. Furthermore, following I hiomolybdate administration, MO concentration in the liver and liver iiactions increased indicating that MO of thiomolybdate was entering liver culls. The percentage distribution of Cu and MO in the liver fractions was similar. This may suggest that MO is bound to Cu and that they remain logether with each fraction. The decrease in Cu concentration may indicate I hat the liver retains its ability to excrete copper via bile.

Introduction copper (Cu) poisoning in sheep is associated with accumulation of Cu, predominantly in the liver (Howell, 1978’1.A reduction in the liver Cu content has been reported in sheep following the ingestion of diets containing added molybdenum (MO) and sulphate (SO,) !(Suttle, 1977; Bremner and Young, 1978) or after the intravenous (IV) administration of thiomolybdate (T&1 ) (Gooneratne, Howell and Gawthorne, 1981a). However, the mechanisms b!, whic,h the Cu is removed from the liver cells are poorly understood. Chronic

The

present

experiment

was

performed

to investigate

the changes

in the

intracellular distribution of Cu in the livers of Cu-loaded sheep following the IV administration of TM. Subcellular fractionation, ultrastructural, morphometric and histopathological methods were used.

178

J. S. Kumaratilake Materials

and J. McC. Howell and

Methods

,4nimals and Treatments Eighteen Merino ewes aged 12 months were separated into two groups, with IO and 8 sheep in groups 1 and 2, respectively. All animals were dosed orally with a 2 per cent solution of CUSO,.SH.~O at the rate of 10 ml per kg body weight on 5 days of the week. Elevation of serum acid phosphatase (AP) activity was used as an indicator of the onset of liver damage (Kumaratilake, Howell and Gooneratne, 1981). Copper dosing ceased at the first rise of serum AP activity in sheep of group 1 and on the first day of haemolysis in sheep of group 2. A week prior to Cu dosing, liver biopsy samples were obtained by laparotomy from all group 1 sheep and six group 2 sheep. Futher liver biopsy samples were obtained from all sheep of group 1 at the first rise of serum AP activity and from four sheep of group ‘2 on the first day of haemolysis. These tissues were used for a subcellular fractionation study to investigate the intracellular distribution of Cu in the liver of sheep with increasing Cu loading. The findings of this study are presented in a previous paper (Kumaratilake and Howell, 1989). At the first rise of serum AP activity, five sheep from group 1 were separated (group 1A) and were killed 11 weeks later without TM administration. The other five group 1 sheep (group 1B) were given twice weekly 50 l.tg of TM IV (as 1 ml of a 5 per cent solution of TM in 0.9 per cent sterile saline). These injections were given for 11 weeks from the first rise of serum AP activity. The sheep were then killed. Group 2 sheep were given TM IV at the rate of 100 pg TM as 2 ml of the 5 per cent solution. The injections were given on the first day of haemolysis and at 24 h intervals, with a maximum of three doses during haemolysis, followed by 50 mg (1 ml of the 5 per cent solution) twice weekly for 11 weeks. They were then killed. Liver samples were obtained from all sheep at the time of killing. Serum AP and plasma sorbitol dehydrogenase (SD) activities were determined in all sheep at frequent intervals during Cu dosing and subsequently, in sheep of groups 1B and 2 before and 24 h after TM injections. In sheep of group 1 A, the time of collection coincided with the pre-injection samples from sheep of groups 1B and 2. Homogenization

and

Subcellular

Fractionation

Liver samples obtained at the time of killing were taken into chilled 14°C) 0.25 M sucrose, washed free of blood, drained on a filter paper approximately 1.0 g of liver was removed for determination of Cu. The rest of the liver was scraped with the blunt edge of a scalpel blade on a perspex plate and the pulp was forced through a 16-mesh stainless steel gauze (fitted to the nozzle end of a 20 ml nylon syringe] into a pre-weighed glass tissue grinder tube (Potter Elvehjem 30 ml, Wheaton Scientific. USA) and reweighed. This was designated liver pulp (LP). The LP was homogenized and the liver homogenate (LH) was separated by subcellular fractionation into nuclear \ N) , heavy mitochondrial (MH), light mitochondrial (ML) (lysosomal), microsomal MI) and cytosolic (CY) fractions. The method for homogenization and subcellular fractionation was as described by Kumaratilake and Howell ( 1989). The composition of liver fractions was determined by light and electron microscopy. The movement of’ lysosomes between liver fractions was monitored by estimating the total specific activitv of AP in these fractions. Copper atld Molybdenum

Content

Liver, LH and liver fractions were acid digested and the Cu content atomic absorption spectrophotometry with a Varian Techtron instrument. In the same acid digest, the MO content of liver, LH were determined by the method of Bingley ( 1963). The Cu and MO expressed as mg per kg liver wet weight, while those of LH and expressed as mg per kg LP wet weight.

was determined by (Model 1200) and liver fractions content of liver are liver fractions are

Cu

and

1 75,

IV Thiomolybdate

Acid t’hosphatase Activity Acid phosphatase activity of serum, LH and liver fractions was determined by the method described by Walter and Schutt (1974) after adjusting the pH of the buffered substrate to 4.5 (Shannon, Quin and Courtice, 1977). Triton X-100 (0.1 per cent w for v) was added to LH and liver fractions before the assay in order to determine the total activity and the results are expressed as the specific activity (mole per min per kg protein). Plasmn Sorbitol Detydrogenase Activicv Sorbitol (1967’.

‘dehydrogenase

activity

of plasma

was determined

by the method

of Ford

Proteiw The protein content of LH and liver fractions was determined Rosebrough, Farr and Randall (1951). Crystalline bovine Chemical Co.) was used as the standard.

as described by Lowry, serum albumin (Sigma

Histopathology Small pieces of liver samples were fixed in 10 per cent buffered (neutral’! formaldehyde, embedded in paraffin wax and 5-pm-thick sections were stained hy harm;) toxylin and eosin (HE) and examined by light microscopy. In the liver sections, the lobules studied by light microscopy were examined as centrilohular, midlobular and periportal zones. In each section, degenerating and necrotic hepatocytes within the three zones of three or four lobules were counted. The counting was carried out independently by the two authors and the average of the two counts (i.e. the mean count per zone per lobule) was used.

Small pieces of liver and pellets of particular liver fractions were fixed for 2 h at 4°C in 5 per cent phosphate-huffered glutaraldehyde (pH 7.3 1. post-fixed for 1.5 h at 4°C in I>altor,‘s chrome osmic acid, dehydrated through a graded series of ethanol and clc~arecl iii propylene oxide. Thereafter, these tissues were infiltrated with epon 812. fhllowing a transitional stage in 40 per cent epon in propylene oxide, and were embedded in fresh epon. Thick sections (1 pm) were cut, stained with toluidine blue and used for the selection of areas for the preparation of ultrathin sections. The ultrathin sections (silver-gold) were collected on plain 200 or 300 mesh Cu grids. stained with uranyl acetate and lead citrate, carbon coated and examined at 80 k\’ in a Philips 302 electron microscope.

hlorphometric analysis was carried out only on the liver samples obtained from group 2 sheep on the first day of haemolysis and after 11 weeks of TM administration. Thr changes in volume density (Vv), numerical density (NV) and mean volume i 1”) of &-loaded hepatocyte lysosomes were studied. Six epon-embedded liver blocks were selected from each liver sample (i.e. 24 blocks for the four sheep on the first day of haemolysis and 24 blocks for the four sheep following TM administration). The areas for ultrathin sectioning were selected from these blocks, hut no attempt was made to classif+, the blocks according to their position in the liver lobule. From each ultrathin section, five electron micrographs were obtained from randomly selected sites at a mpgnitication of 7100. At frequent intervals, the magnification of the electron microscope was checked with a replica grating (Blazer0 Union). At 7 100 magnification

180

J. S. Kumaratilake

and

J. McC.

Howell

of’ the electron microscope, the real magnification was 7200. For point ~.ounting. electron micrographs approximately 24.6 X 17.2 cm in size with approximate final magnification of 19 992 were used. In each micrograph, the dimensions and the final magnification were determined. For point counting of these micrographs, a transparent square lattice with 8 horizontal and I l vertical lines, i.e. 88 points fallirlg on the micrograph was used. In each electron micrograph, the number of points overlying electron-dense lysosomes and cytoplasm were counted and the Vv I fraction of points enclosed within each structure) was determined. In addition, the number of electron-dense iysosomes per electron micrograph was counted. By combining thcsc counts with the values of Vv of lysosomes, the NV (number per unit volume of cytoplasm) and V of lysosomes were calculated (Loud, 1968; \%‘eibel, Staublie, Cnagi and Hess, 1969). Cytoplasmic mass was used as the reference volume. Pwparation

qf Tetra-thiomolgbdate

( TM)

Thiomolybdate was prepared as described by Tridot and Bernard (1962) and tetraTM was identified as the predominant component from the absorption spectra with peaks at 241, 316 and 467 nm (Aymonino, Ranade and Muller, 1969). The harvested crystals of TM were dissolved in 0.9 per cent sterile saline to make a 5 per cent solution (50 mg per ml) which was stored at - 20°C as 5 ml aliquots. Before use, the solutions were thawed at room temperature, centrifuged and the supernate was used for IV administration. Analysis oJ‘ Data The values of Cu, MO, total specific activity of AP in liver, LH and liver fractions from individual liver samples were averaged per group and the standard error of the mean (SE) was calculated. The values of Vv, NV and V of lysosomes obtained from the 30 micrographs of each liver sample were averaged to give values for individual animals. The values for individual animals were then averaged for the group and the SE calculated. One-way analysis of variance was used for the calculation of probability values (Steel and Torrie, 1960).

Results Animals

The details of the sheep and their treatments are given in Table 1. The results of a subcellular fractionation study on the liver of these sheep have previously been reported (Kumaratilake and Howell, 1989). Copper

Copper content of liver, LH and liver fractions and the percentage of Cu in liver fractions in sheep of groups 1, 1A, 1B and 2 are given in Table 2. In sheep of all groups, at the end of the 1 l-week period after the cessation of Cu dosing, the amounts of Cu in liver, LH and liver fractions were reduced. However, the Cu content of liver, LH and liver fractions of sheep of group 1A at this time was not significantly lessthan that observed at the first rise of serum AP activity in sheep of group 1. In groups 1B and 2, the Cu content of liver, LH and liver fractions following TM administration was significantly lower than that observed in groups 1 and 2 at the first rise of serum AP activity and on the first day of haemolysis, respectively.

Cu

and

IV Thiomolybdate

181

Molybdenum content of liver, LH and liver fractions and the percentage of MO in liver fractions in groups 1B and 2, given in Table 3, show that after TM administration the MO content of liver, LH and liver fractions increased. Furthermore, following TM administration, the percentage of MO observed in liver fractions was similar to that of Cu in the respective fractions (Tables 2 and 3). Acid Phosfihatase Activity in Liver Fractions The total specific activities of AP in LH and liver fractions are presented in Table 4.. In group IA, at the end of the 1 l-week period after the cessation of Cu dosing and in group 2 following TM administration, no significant changes in tcjtal specific activity of AP in LH and liver fractions were observed. However, in group IB following TM administration, significant increases in the total specific activity of AP in N, MH and ML fractions occurred.

Composition of Liver Fractions Light microscopic examination showed that the N fractions consisted mainly of nuclei, but red blood cells and a few unbroken cells were also present. Electronmicroscopical examinations revealed the presence of tissue debris, a feu mitochondria and electron-dense lysosomes (Fig. 1; see also Kumaratilake and Howell, 1989). All MH and ML fractions were composed mainly of mitochondria and lysosomes, respectively, but these organelles were present in both fractions. The majority of the lysosomes in the MH fractions and some in the ML lkactions were electron-dense (Figs 2 and 3). The electron-dense lysosomes in N, MH and ML fractions obtained from group 1 sheep at the first rise of‘ serum AP activity and group 2 on the first day of haemolysis were large and numerous. The highest number was found in group 2. The predominant change seen in the N, MH and ML fractions obtained from sheep of all groups I 1 wcaeks after the cessation Cu dosing or haemolysis was a reduction in the number and size of electron-dense lysosomes, which was marked in sheep of groups 1B and 2 that had received TM (Figs 1, 2 and 3). Microsomal fractions were homogenous and composed mainly of endoplasmic reticulum, but also contained glycogen granules. This fraction was not affected by TM administratil:)n.

The \‘v, NV and V of electron-dense hepatocyte lysosomes, liver Cu and total specific activity of AP in the LH of group 2 before and after TM administration are presented in Table 5. The Vv, NV and V of electron-dense hepatocyte lysosomes were reduced following TM administration, but only the reductions obserl-ed in Vv and V were statistically significant.

treatments

I I()(1

1 their

15’34

Table and

1100 --

of sheep

:(;.I,5 27.30

Details

Cu and

IV

Thiomolybdate

LH

.___~ Liver

.~

of liver,

liver

.~ __

-

1014.15 f 86.94

367.45 f 50.35

265.23 f 29.75

1073.49 f 95.55

Amount

82966 + 83.32

31.78 f I.51

per cent

35.55 3x3.19

-

per cent

G2 n=4

Gl N=lO

AlIKWn1 __-___813.76 * 74.77

~__

On the 1st dq of haemolysis

--

‘41 the 1st rise of AP

.- -

liver fractions 1, lA, IB and

228.85 f 63.96 *NS

597.80 f 113.28 *NS

594.57 f 104.29 *NS

Amount

G IA n=3

38.10 f 5.54 *NS

per cent

II weeks after 1st rise of AP without TM

-___

copper mean f SE (liner-mg

109.51 f21.98 *P
302.7 1 f 49.46 *p
305.78 f 48.25 * P < 0.005

G IB n=3

with TM

Amount

II weeks

35.35 f I.21 *NS

per cent

___~ after 1st rise of BP

copper

per kg LP wuu’)

total

in all

103.07 k27.11 **P
31867 f 58.43 **Pio~ool

_~___Amount .~__-~__31865 161.32 **P
G2 n=4 __

30.75 f 2$2 **TVs

-_ per cent

11 weks of/u the cessafion oJ hatmo!vstc wrth 7.44

___-

of the

LH and lioer fraction-mg

as a percentage administration

per kg uw:

Table 2 and the copper in the liver fractions 2 with and without thiomolybdate

and a.c a percuniagr LT~the total offraction

homogenate and fractions in groups

~~‘oppur umtml in absolute &IPJ

content

hw. 1-H and Itlo jiniti0n.r

-

Copper

28.64 f 3.95

172.39 f 18.32

MI

CY

WM = w.ct wright, Ll’= LH = liver homogenate; lkartion; I’= probability:

20.63 f I.30

3.61 f0.51

15.66 f I.16

28.33 * I.19

101~65f0.94

182.10 f 14.52

49.31 f 5.87

156.97 f 16.28

272.78 i 37.37

18.31 f2.74

4.75 f 0.28

15.24 f 0.83

26. I7 et I 92

99.63 f 1.18

96.68 f27.11 *NS

15.82 f 5.05 *NS

94.07 f 19.95 *NS

157.55 f ‘348 *x3

15.63 f 1.90 *IN!3

2.57 f 0.40 *NS

16.05 f 2.60 *NS

27.65 + 4.23 *NS

101~16f0~73

17.36 f 5.91 * P < 0.005

5.58 f 1.20 *PC 0.025

67.91 f 8.56 *p
106.18 i i7.3i *r < 0.05

5.36 f 0.93 P < 0.005

1.80 f0.23 *NS

22.67 f 2.66 *P < 0.025

34.82 i i ,S.i *p
101.24f

24.17 f4.61 **P
8.80 f 1.24 **P
76.07 f 8.54 **p
110.44 f 1940 **pio.o1

1.52

7.46 f 0.40 **p
2.80 f 0.24 **p
24.60 f 2.47 **p<0.01

34.39 f 0.46 **p
libcr pulp; St = standard error of the mran; AI’= serum acid phosphatase activity; CG= group; n = number of sheep: TM = thiomolybdatr; N = nuclrar fraction: MH = heavy mitochundrial fraction; ML= light mirochondrial fraction; MI = microsomal fraction; CY =cytosolic NS = not significant~ * = compared to values at the first risr of serum AP activity: ** = compared to values on the first day of haemolysis.

100.54 f I .26

128.79 rt 12.99

ML

Kecovrry per crnl Mean zk SE

238.66 *2i-3.f

MH

3 .%

s 5’

F

186

J. S. Kumaratilake

Molybdenum liver fractions

content of liver, as a percentage before

hfo!vbdenum

Lmer. LH and liuer JkzlmL~

Isl rise oJ‘;lP

ualues

Li\,er

pm cent .-____--

IN

MH

CY

Recovery per cent McanfSE

the molybdenum in groups 1B and

Content

per

the IAI rw

reulh

TM

II weeks cessation

in 2

cent

-

*

-

-..

Content

prr

a&~

the

oj haemo!vm with 7.W

G II3 n=3

ML

MI

weeks ctffer

c2 II = 4

042 f0.17 *

fractions and in all fractions administration

OJ.AP

0.93

0.82 f 0.62

liver

II

On the ISI dqv q/ haemolvsiJ

* LH

Howell

and a., n prrcentage of the total mo!vbdenum I,, nllfiactron., per k~< u~w: LH and liuer,fractions-mg per kg)

G 1B n=3

Content

J. McC.

Table 3 liver homogenate and of the total molybdenum and after thiomolybdate

contenl as absolutr mean f SE (L&--q

Al lhr

and

c; 2 II = -1 cent

Content

87.25 f 16.00

f

79.79 f 12.28

97.23 f IO.29

per cent

107.47 13.33

27.85 f 5.29

34.36 + 2.02

30.94 f 5.04

31.98 f 2.69

26.95 f 4.29

33.67 f 0.56

32.25 f3.14

33.77 zk 0.52

17.47 f 3.58

21.70 f 1.90

21.43 f 2.32

22.54 f 2.03

2.22 f 0.39

2.78 f0.18

3.34 f 0.23

3.60 f 0.43

5.86 f 0.74

7.49 f 0.88

7.73 f 0.86

8.12 f0.73

100~74f5~01

98.60f

ww=wet weight; LP=liver pulp; SEEstandard error of the mean; AP=serum acid phosphatase G = group; ” = “umber of sheep; TM = thiomolybdate; LH = liver homogenate; N = nuclear MH= heavy mitochondrial fraction; ML=light mitochondrial fraction; MI = microsomal fraction; low the detection limit of the method.

1.14

activity; fraction; * = be-

Histopathology The mean counts for the degenerating and necrotic hepatocytes in the liver samples from groups 1A, 1B and 2 are presented in Table 6. In the samples obtained at the first rise of serum AP activity or on the first day of haemolysis, the predominant histopathological change was the presence of individual, degenerating and necrotic hepatocytes in all three zones of the liver lobules. This was more marked in group 2 and the majority of the necrotic cells was seen in the centrilobular zones. In group 1A samples taken 11 weeks after the cessation of Cu dosing, an increase in the number of degenerating and necrotic hepatocytes were seen in the centrilobular and midlobular zones. Only occasional necrotic hepatocytes were observed in groups IB and 2 after TM administration.

Cu

and

IV Thiomolybdate

188

J. S. Kumaratilake

Sekm

‘4cid Phosphatase

and Plasma

and

Sorbitol

J. McC.

Howell

Detydrogenase

A4ctiuitie.r

The serum AP and plasma SD activities in groups IA, 1B and 2 are presented in Tables 7 and 8, respectively. In group IA, elevations of plasma SD and serum AP activities were observed at different times during the 1 l-week period after the cessation of Cu dosing. Similarly, in groups 1B and 2, elevations of plasma SD activities were observed at different times during the 11 weeks of TM administration, but elevations of serum AP activities were not observed after the 17th and 12th injections of TM, respectively.

Fig.

2.

Elrrtron micrugraphs of heavy mitochondrial fractions (2050 X g ) obtained from a sheep in group 2, (:I: 011 thv lirst day of hacmolysi~ and 1.b I I wrcks after thp ~~rasation of hacmolysis and the commencement of administration of thiomolybdatc. Note the rrduction in thr six of electron-dense Iysosomes jarrow! following thiomolybdatc administration. LTrarryl acetate and Icad citrate x 5640.

Fig.

3.

Electron micrographs of light mitochondrial fractions (24090 x g i obtained Corn a sheep in group 2, [ai on the first day of haemolysis and (b) I I weeks aftrr the cessation of haemolysis and the Note the reduction in the size of commencement of administration of thiomolybdatr. electron-dense lysosomes (arrow) following thiomolybdate administration. Ivranyl awtate and lead citrate x 5640.

Cu

and

IV Thiomolybdate

189

190 Mean

J. S. Kumaratilake

and

J. McC.

Table 5 density, numerical density and mean volume of electron-dense hepatocyte liver copper and the total specific activity of acid phosphatase in liver in sheep of group 2 before and after thiomolybdate administration

volume lysosomes; homogenate

of haemo
On the first dav

Lysosomes

f SE)

Volume density (mm’ per 100 mm’ cytoplasm)

6.47

f 0.8 1

1.34f0.30 P < 0.005

Numerical density (number per 100 mm’ cytoplasm)

4.43

f 0.56

2.43f0.77 NS

Liver (mg

SE=standard

volume (mm’)

Copper content per kg wet weight)

of the

mean;

TM

IMWlfSE)

1.51 f0.20

1073.49

Total specific activity of acid phosphatase (mol per min per kg protein) error

II weks after cessatron of haemo&is with TM administration

(Mean

Mean

Liver homogenate

Howell

0.60 f 0.06 P< O-005

f 95.55

318.65f61.32 P
0~0162f0~0014

= thiomolybdate;

P= probability;

0~0156f0~0015 NS

NS=

not significant

Discussion EIevation of plasma SD and serum AP activities and associated degeneration and necrosis of liver cells have been reported in chronic Cu-poisoned sheep 1971, 1972; Kumaratilake et al., 1981; (Ishmael, Gopinath and Howell, Kumaratilake, 1984; Howell and Kumaratilake, 1985). The numerous elevations of plasma SD and serum AP activities observed in group IA after the cessation of Cu dosing (Tables 8 and 7) indicate that necrosis of liver cells continued to occur during the ensuing 11 weeks. This is in agreement with the finding of an increase in the individual degenerating and necrotic hepatocytes in liver samples obtained from these sheep at the end of the experiment (Table 6). Loss of Cu from the liver and associated necrosis of liver cells has been observed in Cu-loaded rats (Haywood, 1980, 1985). The reduction of liver Cu seen in group 1A after the cessation of Cu dosing (Table 2) is probably the result of normal endogenous loss of Cu coupled with the loss from necrotic Culoaded hepatocytes. In sheep given TM (groups 1B and 2), numerous elevations of plasma SD activities were seen during TM administration, but elevations of serum AP activities were not observed after the 17th and 12th indicating that necrosis of liver cells was injections of TM respectively, minimal after these times. This is supported by the fact that liver cell necrosis was minimal in the samples obtained from these sheep following TM admini-

Cu

and

IV Thiomolybdate

IA

2

IB

IA

Group

I.958

40 I

416 4li 418 12-l

0.950 lb.893 1.267

409 422 423

2.908 (I.92 I 1.152 0.89”

P

I .562 *

0.749 I .037 I.036

0,806

0.748 0,806 1.296 0,778

P

I

activities activity (in

406 408

1,440 I.411 1.814

I.555 I ,440 1,411 I 440

416 417 418 424

405 41 I 420

111 thr 1st rise OJAP

Sheep number

Serum acid phosphatase acid phosphatase

‘I

.l

0.95U I.008 I.094

0.950 *

1.152 1,008 1,728

I.354

A

and

1.728 I.296 I.180 0~892

1’

I.094 1.354 I.843

0.864 *

1.756 0,950 1,612

I ,382

2.044 0,720 I.382 0.720

P

of groups group 1B)

IO

2

lA,

.-1

* * 1,065

0.864 *

1,641 0,806 1.555

I.094

rl

1,526 0,864 I.008 0,835

P

1.123 2.218 I.036

0.806 0.662

1.267 I.036 I .843

0.720

1.756 0.950 1.036 1.670

P

1B and 2 in after haemolysis

II

3

d

1.181 1.325 I.036

0,864 0.893

1,612 I.065 1,584

1,094

I.238 0.806 1.324 0.720

P

0.806 i .a43 0.576

0.518 1.123

1.728 1.152 1.468

1,180

I .382 1.210 2.044 2.390

P

I?

4

Dose number

:i

0,864 I.901 0.691

0,778 1,066

1.728 I.094 I.756

0,835

ri

I.468 2.304 I.094

0,749 0,979

2.246 1,324 I.872

1,411

1.786 0,720 * 2,793

P

I.440 I.642 I.296 0,835

P

in

1.7

5

.1

0,922 0.950 0.979 0.806

P

0,950

1.296

14

1.123

1,094 I.066

0.92 1 I.497

0.893

I.584 I .094 I .065 0.92 I

P

1.756

I .008 0.432

2.188 I.065 2.333

0.92 I

.4

6 .i

.I

1.152

1.267

0.778 0.922

I.065 I.699

I.728 I .065 1.267 0.979

P

7

the

1.5

I.094

I.526

1,152 0.69 I

0,864 I.670

0,806

1.444 0.922 0.864 1,267

P

from synchronized

0.806

administration group 1A was

qf 7.U

I. 1’. per I

qf 50 mginJectmu

Serum dP a&i&v

Table 7 to the time of thiomolybdate (in group 2). Blood sampling

.4

relation

.i

1,526

1.123

* 0.950

I.382 I .900

0,864

.-I

P

I.843 I.094 1.498 I.037

P

Iti

1411

1,094

2,765 I .008

I.584 1.631

1. I23

8

.I

.l

1.353

I.068

1.411 0.86-t

1,354 2.246

0.634

of serum times

1.238 I.065 0.979 0,748

first rise to these

F < &

5

4 5:

F a

I?

2 9 e 3 r;

4 v,

Cu

and

IV

Thiomolyhdate

I!-)3

e s

T

P

IA

G

Plasma

__-

22

5

401

405

154.6

78.7

~.--..

193

424

I.1

106.1 ~k8.5 (l-5)

(l-8)

31.0*

26.8 f 2.8 ( 1-22 + K)

65.7f2.5 (14)

(l-2)

35.7 f 0.9

144.9

acid

in

7@0f 9.2 (l-9)

activity serum

140.1

280.1

--

uycted

T-M

M,yy

ofAP

of At 1st rue

of

Number doses

dehydrogenase

418

417

416

Sheep number

sorbitol

lA,

54.7f5.1 (9~-13)

* (5)

65.9f4.6 (3-10)

(10)

.~~ 20.3

groups phosphatase

..~

(6)

33.8

(11)

43.5

58.0 (11)

Values

SD a&vi9

of thiomolybdate haemolysis (in

52.4 f 3.1 (22+K)

95.1 f 7.9 (7-17)

93.0 f 28.4 (12-17)

39.6 f 1.O (12-16)

(181

*

(18)

465.1

55.5 (17)

121)

178.7

(1%

(18) 120.3f9.2 ( 19-20)

53.1

(li’)

34.8

and at killing in brackets

oalues

administration, group 2)

(I. 11. per 1) mean. mean f SE or absolute

to the time 1B) and after

8

in relation to the dose number of TM injections The dose number of TM injections are given

Plasma

Table 2 in relation (in group

26.3 zk 3.2 (14-21)

1B and activity

the

221.1 122)

first

__..~ 32.2 f 2, I (20-22 + I(:

-.

from

iK\

122.2

.-

rise

of

s 5

A 3:

k

5 e c,

F

P, 5

Cu and

N

.

IV

Thiomolybdate

196

J. S. Kumaratilake

and

J. McC.

Howell

The reductions in liver Cu concentrations in sheep of groups 1B and 2 following TM administration were 62 and 70 per cent, respectively, more thari twice the reduction in liver Cu seen in group 1A (27 per cent) which did not receive TM. The greater proportion of the Cu removed from the livers of group IB and 2 sheep could, therefore, be directly attributed to TM administration. In groups 1 and 2 during Cu loading, the Cu content increased in the lysosomes and cytosol of hepatocytes. This was associated with a marked increase in the number and size of electron-dense lysosomes that sedimented with the N and MH fractions and with a significant increase in the total specific activity of AP in these fractions (Kumaratilake and Howell, 1989). Following TM administration, the concentration of Cu in all liver fractions decreased (Table 2). In particulate liver fractions (N, MH and ML), this was associated with a marked reduction in the number and size of electron-dense lysosomes that sedimented with these fractions (Figs 1, 2 and 3). Furthermore, following TM administration, the Vv and V of electron-dense hepatocyte lysosomes significantly decreased (Table 5). These findings indicate that the observed reduction in liver Cu following TM administration was associated with the loss of Cu from the lysosomes. A similar reduction was found in the cytosol (Table 2). Kumaratilake and Howell (1987) showed that when liver samples obtained from groups 1A, 1B and 2 were stained for Cu histochemically, there was a reduction in the number and the size of positively stained granules in hepatocyte cytoplasm in the animals which had received TM. The intracytoplasmic granules stained positively for Cu in liver cells have been identified as lysosomes (Goldfischer and Moskal, 1966; Goldfischer, 1967; Jones, Gooneratne and Howell, 1984). These findings together with those presented here indicate that there was a reduction in the number and size of Cu-loaded lysosomes in the liver cells of group 1B and 2 sheep following TM administration. Therefore, following TM administration, a decrease in the total specific activity of AP a marker enzyme for lysosomes (Ghadially, 1982) should have occurred in LH and liver fractions. However, neither the total specific activity of AP in LH and liver fractions of sheep in groups 1B and 2 nor the Nv (number per mm3 cytoplasm) of electron-dense hepatocyte lysosomes in group 2 decreased significantly following TM administration (Tables 4 and 5). It may be that TM administration is followed by a reduction in the number of large Cu-loaded lysosomes and a simultaneous production of new, small, acid hydrolase-rich lysosomes. Furthermore, in groups 1B and 2 following TM administration, markedly swollen Kupffer cells packed with granules staining positively for Cu remained in the lobules, and there was a marked increase in the portal triads of macrophages with a similar appearance (Kumaratilake and Howell, 1987). The lysosomes of these cells may also have contributed in part to the total specific activity of AP observed in LH and liver fractions following TM administration (Table 4). The increase in the concentration of MO in CY and particulate liver fractions after TM administration (Table 3) indicates the entry of MO from TM into the liver cells. Neither the mechanism of entry of MO nor the form in

Cu and IV Thiomolybdate

197

which it enters is certain. Molybdenum might enter liver cells independently as TM or as TM-protein complexes or bound to Cu as Cu-MO complexes or as a combination of the above. The similarity of the percentage distribution of Cu and Mo in the liver fractions from groups 1B and 2 following TM administration (Tables 2 and 3) may indicate that MO in liver cells is bound to Cu and that they remain together in each fraction. In particulate liver fractions, Cu is in the lysosomes (Kumaratilake and Howell, 1989). The MO in these fractions may also be in the lysosomes; presumably as Cu-MO protein complexes. The mechanisms by which Cu is removed from the lysosomes and cytosol ot liver cells following TM administration are poorly understood. Either one or a combination of the following mechanisms may act: (1 1 Following TM administration, there is an increase in the concentration of Cu in plasma (El-Gallad, Bremner and Mills, 1977; Gooneratne, Howell and Gawthorne, 1981 b; Kumaratilake, 1984). Much of this is insoluble in trichloroacetic acid (El-Gallad et al., 1977; Gooneratne et al., 1981b) and appears to be complexed with MO and protein (Smith and Wright, 1975a,b). This complex may not be available physiologically and may not enter liver cells (Smith and Wright, 1975a,b). Thus, the accumulation of Cu by liver cells may be impeded. but the excretion of Cu from the cells via bile is not, hence there will be a net loss of Cu from the liver. (2 I Thiomolybdate may enter liver cells and complex with Cu in the cytosol to produce macromolecules (Cu-MO protein complexes) which arc biologically unavailable (Smith and Wright, 1975a,b), and hence arc taken up by lysosomes to be excreted in bile. Gooneratne, Christensen, Chaplin and Trent (1985) have shown that administration of TM to sheep is followed by an increase in the excretion of Cu in the bile. In group 2> five of the seven sheep given 100 mg doses of TM during haemolysis recovered. Furthermore, in three of the four sheep in group 1B and four of the five sheep in group 2 given 50 pg doses of TM, the liver Cu concentration decreased dramatically. These sheep did not develop haemolysis during TM administration (Tables 1 and 2). Therefore, IV administration of 100 mg doses of TM during haemolysis and 50 mg doses to Cu-loaded sheep appears to be effective in the treatment and prevention, respectively, of chronic, Cu poisoning. The number of animals used in this experiment was small, hut our tinding regarding the preventive effect of a 50 mg dose confirms those of Gooneratne et al. (1981a). The method has also been used successfully to prevent naturally occurring chronic copper poisoning in sheep (Humphries. Mills, Greig, Roberts, Inglis and Halliday, 1986).

Acknowledgments I’he authors thank Dr S.R. Gooneratne and Dr J. M. Gawthorne for valuable discussions, Dr J. L. Hill for the assistance in molvbdenum determinations and Mr X,1. (iate\, Mr M. D. McKenzie and Mr D. N. C:aviile for technical assistance. Financial support from the Australian Research Grants Scheme and the Wool Research Trust Funtl is gratefully acknowledged.

198

J. S. Kumaratilake

and

J. McC.

Howell

References

Aymonino, P. J., Ranade, A. C. and Muller, A. (1969). Evidence for the existence ot MoO,S*and WO,S’ions in aqueous solutions.
Cu and

IV

I O!~

Thiomolybdate

*Jones, H. B.. Gooneratne, S. R. and Howell, J. McC. ( 19841. N-ray microanalvsis of’ liver and kidney in copper loaded sheep with and without thiomoly~~datc~ administration. Research in L’eterinary Science, 37, 273 282. Kuntaratilake, J. S. (1984). Ph.D. ‘Thesis. PathogenesiJ ?fC’hronic C’opper Poisoning in LYhrep Western Australia. nnd the LTfect.\ q/‘ ‘Thiomolybdate. Murdoch LJniversity, Murdoch, Kumaratilake,,J. ,C. and Howell, J. McC. ( 1987 !. Effects of.itltra~(,nousl~ administered tetra-thiomolybdatr on the distribution of copper in the liver and kidney (11’ L\‘cirnc~t~. 42. copper loaded sheep: A histochemical study. Rewurch in 1Ztrrinary 15+161.

Kuntaratilake, .J. S. and Howell, J. McC. ( 1!)89!. Intracellular distribution ol‘coppt~r in the liver of’ copper loaded sheep: A s~tbct~llular firactiottatiott study. ~7ou~rrcl/ II/ (kmparc&r~ Patholopr 101, 161-I 76. S. R. ( 198 1 I. Blood c‘opp~r. Kuntaratilakc, ,J. S., Howell, J. MrC. and (;ootterattte, sorbitol dehydrogenase and acid phosphatasv in copper poisoning. Proceudin,q of/h/~ Fourth

International

~~~rnposium

on ‘Trace

Element

.2lrtaboli.vr~

in IIIon

and

:lnimal\.

,I.

Xl&.

.\c;tdtm\ 01 Howell, .J. M. Gawthorne and C. I,. IVhitr, Eds. :\ustrdiiltl Scirncc. CLtttberra, pp, 457-460. I ,C)UCl. .\. 1’. 119681. :2 quantitative stereological description of the ultrastruc.turt. 01‘ ttormal rat liver parrnchymal cells. ~7ownal of (.‘e// Biologv. 37, 27~-36. R. j. 19.51 8. Proteitt Ilow r),. 0. H.. Rosebrough. N. .J., E’arr. ;I. I,. and Randall, mc’;tsurr‘mc’nt with the fhlin phenol rraqnt. ~journol II/ Bioloqzc‘crl C.‘hrmi\t,-y. 193.

w--275.

. .\. I)., @in, J. LV. and Courtice, F. C. j 1977 I, I,~~aosomal enzyme at.ti\itic,!. itt sherp plasma and lymph. Rexearch in Cite,-innv L~‘~wn~~~. 22. 209%2 i5. Smith, B. S. \I’. and Wright. J. / 1975a). Effticts of‘s dietar) 110 on (:u mrtaboli~nt: I;,\,idrtitx, ii)r thr in~~ol~~cmrtit of MO in the abtiormal I~itiditig 01 C:it to pl;tWta pn~teitts. C,‘linico C’himico .-lcta, 62, 55- 63. Srnil h. K. S. \\‘. and LVright, .J. j 19751)). Chopper: molybdrttt~tn itltcraction: El1’rc.t ot clic,tarv tool\-bdrnum on the binding of‘t~opp~~ to places protrin~ in sheq). ,70~~m~i sI1;u1t1(ltl

I!/ C.*orr&rrttik

Strt.1.

Potholop.

8.5, 299-305.

K. (;.

(lnd Pro~.t~durtf\ I!/ .Slcrfi\/i~ 1. 1). and ‘I’orrir, ,J. H. , I 9(i( I ). t’rirrciplr\ ~It~(~~~;tw-HillBook Cornpan) Inc., Nr\\ York, ‘I‘orolltcJ. I.oIldoll. Suttlr.. .U. t:. 1977 ‘. Rrducing thr potential copptar toxicit\ of‘c.ortc.etttrate~ to stlt*rp t)\ t 111,

List’

01‘

7 (7 hnolq~.

tnol~bcltYlLllrl

;d

~lllphul~

suppltmIl1~.

Irrirrrctl

Ftwl

St im

I’

m/

2. 235-246.

‘l‘ridg)t, (G. and thiotuttgsticfltr~ hir~ntinr-run

Bernard, ,J. (:. I 1962 Idrtitificatioti et thiomolybdiqurs rn solution x1 iif’~i~i(‘. tiqpriccte,

34,

1 7%

t’t

filitatioti .-l~./tl &imii

(11.4 ions n .I( odtwit~f

19 1

C:. ( 19741. ;Icid and xlkalittr phosf)h;ttast* in serum. .\frthd\ II/ t:‘u.;tmcrlit. . lrrtllv.ciJ 2. E. H. L-. Brrgmeryc~r. Ed. .\cadtmic Press Inc.. NV\% \r’ork. S~II Ftxttciscc~. London, pp. 850-856. \\‘eil,rI, t<. R.. Staublie, IV., Gtt%gi, H. R. and Hrsh. b’. :\. ;196!)1. (:orrc~l;ttcxl rnc~rphcm~~tri~ and biochemical studies OII the liver (,vII. I. Llorphomctric. 1no(~~~1. \trrrologic mrthods. and normal morphometric dat;t fi)r t-at li\-rr. j’ourrrcll c!/ (.‘I// \\‘altt’r.

K. attd St,hiitt,

tiicdqq~~,

42.

ti8-9

I [ Ruceir~edJtir

publication,

i;ebrrrcly

2&h,

19881