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

Eighteen ewes divided into two groups were dosed orally with CuSO4 in order to 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 cessation of Cu dosing. Following thiomolybdate administration, in groups 1B and 2, there was a reduction in the concentration of Cu in the liver and liver fractions, the number and size of electron-dense lysosomes in particulate liver fractions, the volume 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 production of new lysosomes in the liver cells. Furthermore, following thiomolybdate administration, Mo concentration in the liver and liver fractions increased indicating that Mo of thiomolybdate was entering liver cells. 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 together with each fraction. The decrease in Cu concentration may indicate that the liver retains its ability to excrete copper via bile

Effect of intravenously administered tetrathiomolybdate on plasma copper concentrations of Copper-loaded sheep

Chronic copper toxicity was induced in 14 ewes in two groups by oral dosing with CuSO4. Copper dosing was stopped in sheep of groups 1 and 2 at the first rise of serum acid phosphatase activity and on the first day of haemolysis, respectively. Thiomolybdate was administered intravenously (i.v.) to sheep of group 2 at the rate of 100 mg on the first day of haemolysis and at 24-h intervals, with a maximum of 3 doses during haemolysis. Thiomolybdate was also given intravenously at a dose of 50 mg twice weekly for 11 weeks to four sheep of group 1 after the cessation of copper dosing (group 1B) and to five sheep of group 2 at the end of haemolysis. Plasma copper concentration was determined before and 24 h after each injection of 50 mg thiomolybdate and "elevations" of plasma copper concentration were seen after each injection of thiomolybdate. The differences between plasma copper concentrations observed before and after each thiomolybdate injection for doses 1 to 11 were significantly higher than those seen for doses 12 to 22. Following thiomolybdate administration, the copper content of the liver of sheep in groups 1B and 2 was reduced much more than in sheep of group 1A, in which copper dosing also ceased but which did not receive thiomolybdate. It was concluded that the high plasma copper response to thiomolybdate doses 1 to 11 was due to an influx of copper into the bloodstream from the heavily copper-loaded liver cells. The lower plasma copper response during the latter part of thiomolybdate administration was due to a gradual reduction in the amount of copper entering the bloodstream from the liver cells, as these cells became depleted of copper. Some of this copper may become part of the glomerular filtrate and be taken up by the cells of the proximal convoluted tubules of the kidney or may be excreted in the urine

Effects of intravenously administered tetra-thiomolybdate on the distribution of copper in the liver and kidney of copper loaded sheep: a histochemical study

The effects of intravenously administered thiomolybdate on the liver and kidney of copper loaded sheep were studied using 16 ewes in three groups. Copper, iron and molybdenum concentrations were determined by spectrophotometry and the distribution of copper in the liver and kidney was studied histochemically. Following thiomolybdate administration, the concentration of copper in the liver was reduced, that of molybdenum increased and the concentration of copper and molybdenum in the kidney increased. The reduction of copper concentration in the liver was associated with reductions in the number and size of granules in hepatocytes which stained positively for copper and in the number of Kupffer cells containing positively staining granules. The decrease in the amount of copper in hepatocytes appeared to be greater than that in Kupffer cells. This effect was greatest in the centrilobular zones and least in the periportal zones. The increased concentration of copper and molybdenum in kidney was associated with an increase in the number and size of granules staining positively for copper in the epithelial cells of the proximal convoluted tubules which suggested an uptake of copper-molybdenum complexes by the lysosomes of these cells

Intracellular distribution of copper in the liver of copper-loaded sheep--a subcellular fractionation study

Eighteen ewes in two groups were dosed orally with CuSO4 to induce chronic Cu toxicity. Copper dosing was stopped at the first rise of acid AP activity in the serum in group 1 sheep and on the first day of haemolysis in group 2 sheep. Liver samples were obtained 1 week prior to the start of Cu dosing, at the first rise of acid phosphatase (AP) activity in serum and on the first day of haemolysis. These liver samples were homogenized and were separated into nuclear (N), heavy mitochondrial (MH), light mitochondrial (ML), microsomal (MI) and cytosolic (CY) fractions by centrifugation. The Cu concentration and specific activities of AP were determined in the liver, LH and subcellular fractions. The composition of the fractions was studied by light and electron microscopy. In the predosing biopsies, the concentration and percentage of Cu and the total specific activity of AP were highest in the ML fractions. With increasing Cu loading, the concentration of Cu in all fractions increased; the percentage of Cu increased in the N and MH fractions, decreased in the ML and MI fractions and was maintained at a constant level in the CY fractions. The total specific activities of AP in LH, N, MH, MI and CY fractions were increased and the activity was highest in the MH fraction. The results indicate that the increase in the concentration of Cu in liver cells was predominantly in lysosomes and cytosol. Furthermore, it is suggested that the necrosis of isolated hepatocytes observed in chronic Cu-poisoned sheep may be due to a saturation of the uptake of Cu into the lysosomal system of the cell, leading to the accumulation of toxic levels of Cu in the cytosol

Lysosomes in the pathogenesis of liver injury in chronic copper poisoned sheep: an ultrastructural and morphometric study

Chronic copper poisoning was induced in sheep by oral dosing with CuSO4. The distribution of copper between hepatocytes was unequal and, with increasing liver copper concentration, isolated hepatocytes packed with electron-dense lysosomes were seen. These cells underwent degeneration and necrosis. During the pre-haemolytic period, the concentration of Cu in the liver increased and the volume density, numerical density and mean volume of hepatocyte lysosomes increased in a linear fashion, indicating that there was proliferation as well as increase in the size of lysosomes. However, in animals killed during haemolysis, the numerical density had decreased but the volume density was little changed which indicates that lysosomal production may have diminished. It is postulated that the necrosis of hepatocytes packed with electron-dense lysosomes may be due to the accumulation of toxic amounts of copper in the cytosol, resulting from a reduced uptake of copper into the lysosomal system of these cells, and that the susceptibility of liver cells to Cu-induced damage may be increased if lysosome production is diminished

Subcellular distribution of copper in the kidneys of normal, copper-poisoned, and thiomolybdate-treated sheep

Twenty-seven sheep given either copper (Cu) and/or tetrathiomolybdate (TM) were used to study the subcellular distribution of Cu within the kidney and to monitor the location of lysosomes within the subcellular fractions using acid phosphatase (AP) as a marker enzyme. Copper dosing alone increased the Cu content in the liver and the kidneys. The administration of intravenous TM prevented the development of chronic copper poisoning (CCP) in sheep, reduced the rate of accumulation of Cu in the liver of Cu-dosed animals, but increased the Cu content of kidneys in both the control and Cu-dosed sheep. The total amount of Cu that accumulated in the kidneys of sheep given TM appears to depend on several factors: a) liver Cu concentration, b) Cu intake, and c) dosage of TM. Thus, the highest Cu concentration was found in the kidneys of sheep that continued to receive Cu orally at the same time as they were given TM. The intracellular distribution of Cu and AP in the kidneys showed that in the control sheep given neither Cu or TM, the highest proportion of Cu was in the cytosol fraction, and the highest specific activity of AP was in the light mitochondrial (lysosomal) fraction. Dosing with Cu markedly increased the Cu concentration and greatly elevated the total activity of AP in the heavier fractions, i.e., the nuclear (N) and heavy mitochondrial (MH). Thus, the increase in Cu observed in the N and MH fractions was not caused by an accumulation of Cu by nuclei and mitochondria, but was due to an accumulation of Cu by lysosomes that sedimented with the heavier fractions. The intracellular distribution of Cu in the kidneys of TM-treated sheep was similar to that seen in Cu-loaded sheep. Although Cu accumulated readily in the kidneys of animals receiving TM, kidney function tests showed neither glomerular nor tubular functional impairment