Morphometric, electron microscopic, biochemical and pathological investigation of liver of normal and copper poisoned sheep

Chronic copper poisoning was induced in sheep by repeated oral dosing with copper sulphate solution. Sheep were killed prior to, during and after cessation of haemolysis. The subcellular distribution of copper (Cu), zinc (Zn), iron (Fe), and the lysosomal marker enzymes acid phosphatase and 6-glucuronidase were investigated in the liver of all sheep. Copper loading increased the concentration of metals in all fractions. The proportion of Cu in the nuclear fraction continued to increase whilst that in the heavy mitochondrial fraction decreased. A significant increase in lysosomal enzymes along with electron microscopic studies on pellets of fractions indicated: (i) proliferation of lysosomes (ii) possible localisation of Cu in lysosomes (iii) sedimentation of denser lysosomes with the heavier nuclei. Correlated morphometric and ultrastructural studies of liver confirmed and extended these findings. Stereological analysis indicated that the most extensive proliferation of lysosomes occurred in the liver of the pre-haemolytic group of sheep while the liver of sheep killed during haemolysis had the largest and heaviest lysosomes. The action of lysosomal hydrolases and the deposition of Cu in nuclei were perhaps key factors responsible for the death of liver cells in sheep undergoing haemolysis. Biochemical studies on muscles of chronic Cu poisoned sheep confirmed that a sudden rise in the level of plasma creatine phosphokinase (CPK) occurred during haemolysis and the level returned to normal once the ii. "crisis" was over. This rise in CPK was not accompanied by an elevation of CPK in cerebrospinal fluid (CSF). An isoenzyme separation confirmed that the CPK was liberated from the muscle. Light microscopic studies failed to reveal consistent changes in muscle structure but ultrastructural changes, similar to those described as the earliest changes in vitamin E (Vit. E) deficiency, were seen in sheep killed during and after haemolysis. The elevations of CPK levels and mitochondrial changes were most marked in sheep that did not receive selenium (Se) and Vit. E supplements. It was concluded that a transient increase in the permeability of muscle membranes at haemolysis leading to CPK release may be brought about by a multitude of factors, such as: (i) decrease in Se and/or Vit. E in blood and tissues (ii) hypoxia and (iii) hypercupraemia. Copper, Zn and Fe levels were measured in the CSF of normal and Cu poisoned sheep. Copper values were within the normal range recorded for humans but Zn levels were 6 to 20 times and Fe levels were 20 to 30 times higher than values reported for humans. The amount of Cu, Zn and Fe in the CSF did not increase in sheep dosed with Cu and hence it is unlikely that the level of Cu in the CSF is related to the changes in the nervous system that have been reported in Cu poisoned sheep. A variety of biochemical and morphological criteria were evaluated in an investigation of the effects of intravenous administration of thiomolybdate (TM) in the treatment and prevention of chronic Cu poisoning in sheep. The results indicate that TM is: (i) 100% effective in preventing the development of chronic Cu poisoning and (ii) successful in treatment if administered in early haemolysis. Copper seems to be leached out of the liver, transported in the blood and excreted via the kidney. The changes in Cu content of plasma fractions and whole blood brought about by TM treatment gives more information on the mechanisms occurring at the time of haemolysis.