Some aspects of copper thioneins in chronic copper poisoning of sheep

This thesis reports on investigation of the roles of metallothionein and lysosomal enzymes in the pathogenesis of chronic Cu poisoning of sheep, with and without treatment by thiomolybdates. The changes in copper, zinc and molybdenum distribution were studied in the liver, bile, kidney and blood of chronic Cu poisoned sheep using histochemical, immunohistochemical and biochemical techniques including gel filtration and subcellular fractionation. Morphological changes, particularly in the liver and kidney were investigated using light and electron microscopy. The results of gel filtrations of liver indicated that metallothioneins were the main Cu binding protein in the cytosol and lysosomal rich mitochondrial fractions. Examination of kidneys showed that metallothionein was the most important Cu storage protein in that organ. Most of the Cu in the bile of chronic Cu poisoned sheep was bound to high molecular weight proteins. Small amounts of Cu thionein-like protein were detected in the bile just prior to and during haemolysis. It appears that thiomolybdate altered the binding of Cu so that it associated with more proteins than it normally does. Metallothionein mRNA determinations suggested a·correlation between liver Zn concentration and metallothionein mRNA. Increased intracellular Cu concentrations associated with either haemolysis or thiomolybdate treatment resulted in decreased Cu extractability. Thiomolybdate appeared to alter the binding of Cu by the formation of high molecular weight complexes most of which were insoluble. Studies with radiolabelled metallothionein showed that intravenously injected metallothionein was taken up at two sites - the kidney and the lung. Injected metallothionein did not appear to damage the tubular epithelial cells. Traces of metallothionein (Fraction III) appeared to be present in cell free lysates of erythrocytes taken from sheep in haemolysis though most of the Cu in the plasma and erythrocytes collected du'.ing the prehaemolytic and haemolytic phases of chronic Cu poisoning was bound to high molecular weight proteins. It is suggested that neither Cu thioneins nor haemoglobin, both of which are in the blood at haemolysis, cause serious damage to the kidney. However, both normal and iron loaded sheep kidneys were found to be an important site of uptake of a radiolabelled lysosomal enzyme preparation. The uptake of lysosomal enzymes may be an important causal factor in producing the clinically significant damage to sheep kidney which often occurs during the haemolytic phase of chronic Cu poisoning