Methods. Activity of genes was estimated by semiquantitative RT-PCR-analysis. The copper transporting genes (CTR1, АТР7А, АТР7В, APP), extracellular cuproenzymes (Cp, GPI-Cp), and intracellular cuproenzymes (SOD1 and COX (Cox4i1)) were analyzed. Relative level of Cp, CTR1, and SOD1 proteins were estimated by immunoblotting analysis. Enzymatic activity of Cp and SOD1 were measured by “in-gel” assay. Cp concentration was fixed by rocket immunoelectrophoresis. Subcellular fractions were isolated by equilibrium ultracentrifugation. Mitochondrial intermembranous space was obtained using swelling-shrinking-freezing-thaw procedure. Concentrations of Cu, Fe, Zn and Ag in tissues and fluids were measured by atomic absorptive spectrometry Data are expressed as means ±S.D. Statistical comparisons were made using Student’s t-test. Differences were considered significant at P<0.05.
Results. Copper concentration was measured in brain of 5-, 10-, 20-, 30-, and 120-day-old rats. Each group contained 5 animals. The data shown, that during this time copper concentration rises in cortex, cerebellum, and hippocampus; doesn’t change in hypophysis, and reduces in hypothalamus. Cu-transporting gene profiles also had been changed during development. So ATP7A has been expressing in brain of newborns mainly, and its expression level increases in adult brain. Alternatively, ATP7B gene is much more active in brain of adult animals. A splice-form mRNA of secretory Cp is typical for brain departments in newborns mainly. On the contrary Cp-mRNA isoform coding GPI-Cp is prevalent for brain of adult rats. Also during development the concentration of the intracellular cuproenzymes increases in all brain regions. Thus copper metabolism in brain of newborn rats is different with it in adult ones as well as copper metabolism in liver. Perhaps, it could be definite as embryonic type of copper metabolism in brain.
It is known that consuming of Ag salts with feeder results in drop of copper concentration in plasma, and simultaneously silver accumulates in liver. Using this model we shown that Cp, CTR1, ATP7A/B genes expression are don’t change in liver of Ag-rats. In brain, silver is accumulated by hypothalamus-hypophysis system only. Here copper concentration decreases, and iron increases. Zinc concentration doesn’t change. Cu-transporting genes activity is decreased in Ag-rats brain, but expression of Cu-enzymes genes doesn’t change.
In next experiments the model “Dam-rat treated with AgCl feeds the newborns during 10 days” was used (Ag(10)-rats). It was shown that Ag is transferred with milk into gastro-intestinal tract of newborns, absorbed, and accumulated by liver. In liver of Ag(10)-rats silver distributes within the same compartments which accumulate Cu, exactly. Besides these animals have low level of Cp oxidative activity in blood serum, but expression profile of Cp gene and copper transporting genes weren’t changed in liver of them. In their brain, Cp gene activity is suppressed. Expression of intracellular cuproenzymes didn’t change.
We found that concentration of copper and zinc is decreased in MDAT-rats within all investigated regions of brain, excluding choroids plexus. Expression of Cu-transporting genes is decreased in the brain of MDAT animals as well as Cu-enzymes expression. In the same time expression of examined genes doesn’t change in liver.
When copper turnover is disturbance (Ag- and MDAT-rats), apo-SOD1 passes to mitochondria, and turns into holo-enzyme herein. The same process described for yeast, where mitochondria maintain a pool of copper.
It is well known that tumor growth is accompanied by blood Cp level increasing. But data on nature of this phenomenon are absent. However it could be suggest that tumor growth could be regulated by copper status in organism. To carry out the approaches to check this assumption we studied copper transporting genes expression in ApcMin-mice. It was shown that the growing of adenomas entails the increasing of expression level of Cu-transporting genes (CTR1, ATP7B, and Cp) and activation of ATP7A gene in liver. At the same time CTR1 expression increases for about 30% in non-malignant intestines and near up to 2 times in adenomas in Min-mice.
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